Trizinc bis(orthophosphate)

Endpoint summary

Description of key information

Animal data

Oral: The repeated dose toxicity of the zinc category substances has been examined in a number of sub-chronic oral repeated dose toxictiy studies. The NOAEL for systemic effects of the zinc category substances was determined to be 25 mg Zn/kg bw/day, derived in an oral 90-day study with nano zinc oxide in rats.

Inhalation: The repeated dose inhalation toxicity of micro and nano-ZnO has been examined respectively in a subacute (28 days) and two subchronic (90 days) inhalation studies. The lowest NOAEC was determined to be 0.47 mg/m³ (target concentration: 0.5 mg/m³) for micro ZnO. For nano-ZnO the BMCL10 was determined to be 0.971 mg/m³.

Dermal: No adverse effects were observed in a 90-day repeated dose toxicity study via the dermal route with nano-ZnO.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not stated

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The following restrictions were noted: The applied doses were not analytically verified and the vehicle was not specified. Further, ophthalmological examination was conducted but results not reported. The test substance zinc oxide was modified by changing the surface charge, thus beside zinc oxide additional substances (L-serine and sodium citrate) were administered. A detailed clinical observation and the functional observation battery were not conducted. Historical control data and individual data were not provided.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21st September 1998

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

ZnO NPs used in this study were either positively (ZnO AE100(+)) or negatively (ZnO AE100(-)) charged. The surface charge was modified with coating reagents, citrate (for [–] charge) and L-serine (for [+] charge). For preparation of ZnO AE100(−), the sodium citrate (Sigma Aldrich, MO,USA, CAS No 6132-04-3) was dissolved in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Sigma Aldrich, CAS No 7365-45-9), which was set to pH 7 by using 1 M sodium carbonate (Na2CO3, Duksan Pure Chemical Co Ltd, Daejeon, Korea, CAS Nom497-19-8) solution.
For preparation of ZnOAE100(+), the L-serine (Sigma-Aldrich, CAS No 56–45–1) was dissolved in 20 mM HEPES buffer, which was set to pH 6 by using 1 M Na2CO3 solution. The final concentration of sodium citrate and L-serine was 1 w/v% in 20 mM HEPES buffer solution.

Before initiating this study, the physicochemical properties (including the primary particle size, morphology, hydrodynamic size, and zeta potential) of the two test articles were verified.*

*Kim K-M, Kim T-H, Kim H-M, et al. Colloidal behaviors of ZnO nanoparticles in various aqueous media. Toxicol Environ Health Sci. 2012;4(2):121–131.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Widely used in safety evaluations, including repeated-dose toxicity studies and recommended animal species in Organisation for Economic Co-operation and Development test guideline 408.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Inc. (Gyeonggi-do, Korea)
- Body weight ranges at dosing: Dose range finder: males: 183.8-204.9 g; females: 145.8-162.2 g
Main study: males: 181.2-196.1 g; females: 150.9-168.5 g
- Age (at purchase): six week
- Housing: Animals were housed in stainless wire cages (270 W × 500 D × 200 H mm), two animals per cage
- Diet: ad libitum, rodent feed sterilized with 2.0 Mrad radiation and ultraviolet radiation
- Water: ad libitum, water sterilized with 2.0 Mrad radiation and ultraviolet radiation
- Acclimation period: All animals were acclimated and quarantined for 8 days in an animal room of the health care institute.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-23.1
- Relative humidity (%): 42.6-54.5
- Air changes (per hr): ventilation frequency ten to 30 times per hour
- Photoperiod (hrs dark / hrs light): light hours from 8 am to 8 pm (150300 Lux)

Route of administration:

oral: gavage

Vehicle:

not specified

Details on oral exposure:

The dose formulations were prepared once a day throughout the study. The formulated test articles was freshly homogenized by vortexing before administration.
Dose range finder: The test articles were administered by oral gavage between 9 am and 12 midday.
Dose range finder and main study: The dose volume was 10 mL/kg for all groups.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Dose range finder: 14 days
Main study: 90 days (+14 days recovery period for 5/15 animals per sex per vehicle, control and high dose group)

Frequency of treatment:

daily

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

dose range finder (ZnO AE100(+))

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

dose range finder (ZnO AE100(-))

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

dose range finder (ZnO AE100(+))

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

dose range finder (ZnO AE100(-))

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

dose range finder (ZnO AE100(+))

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

dose range finder (ZnO AE100(-))

Dose / conc.:

31.25 mg/kg bw/day (nominal)

Remarks:

main study (ZnO AE100(+))

Dose / conc.:

31.25 mg/kg bw/day (nominal)

Remarks:

main study (ZnO AE100(-))

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

main study (ZnO AE100(+))

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

main study (ZnO AE100(-))

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

main study (ZnO AE100(+))

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

main study (ZnO AE100(-))

No. of animals per sex per dose:

Dose range finder: 5 rats of each sex per group (except negative control group which existed of two rats of each sex)
Main study: negative, vehicle control and high dose: 15 rats of each sex (5 rats of each sex for recovery group); mid and low dose: 10 rats of each sex (no recovery groups)

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose range finder: On the basis of the result of a preliminary acute oral pharmacokinetics absorption study, in which no significant effect was observed in the 2,000 mg/kg group, the high dose for the 14-day dose range finder study was set to 2,000 mg/kg body weight, and the middle and low doses were 1,000 mg/kg and 500 mg/kg body weight, respectively.
Main study: Significant effects were observed at 1,000 mg/kg and 2,000 mg/kg body weight in the 14-day repeated-dose study, and thus the high dose of this study was set to 500 mg/kg, and the middle and low doses were 125 mg/kg and 31.25 mg/kg body weight, respectively.
- Rationale for animal assignment: randomly distributed into groups of approximately equal initial mean body weights (both studies)
- Post-exposure recovery period in satellite groups: 14 days (vehicle, control and high dose animals)

Positive control:

not stated

Observations and examinations performed and frequency:

Dose range finder:
- Clinical signs and body weight were observed throughout the 14-day experimental period

Main study:
MORTALITY AND CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during the test, all animals were observed once daily after treatment for death.

CLINICAL SIGNS: Yes
- Time Schedule for examinations: during the test, all animals were observed once daily after treatment for clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: just before the first administration and once a week thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: daily after the starting date of the treatment
Consumption was calculated from the differences between the supplied amounts and the remaining amounts measured the next day.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily after the starting date of the treatment
Consumption was calculated from the differences between the supplied amounts and the remaining amounts measured the next day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before grouping and during the last week of the experiment
- Dose groups that were examined: negative and high-dose group only
The ocular fundus was observed through a fundus camera (Genesis; Kowa, Tokyo, Japan) after the pupil was dilated using a mydriatic drug (atropine [Ocu-tropine]; Samil Pharm Co, Seoul, Korea).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice
- Anaesthetic used for blood collection: Yes, blood was collected from the abdominal aorta under anesthesia with isoflurane.
- Animals fasted: Yes, overnight
- How many animals: 10 animals/group
- Parameters checked: total leukocyte and differential leukocyte (neutrophil, lymphocyte, monocyte, eosinophil, and basophil) counts, total erythrocyte count, hemoglobin concentration, hematocrit (Ht) level, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), reticulocyte, platelet, prothrombin time, and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice
- Animals fasted: Yes, overnight
- How many animals: 10 animals/group
- Parameters checked: total protein (TP) level, albumin (Alb) level, albumin/globulin ratio, total bilirubin level, alkaline phosphatase level, aspartate aminotransferase level, alanine aminotransferase, (ALT) level, creatinine level, blood urea nitrogen level, total cholesterol (T-Cho) level, triglyceride (TG) level, glucose (Glu) level, calcium (Ca) level, inorganic phosphorus (P) level, creatine kinase level, sodium level, potassium level, and chloride (Cl) level.

URINALYSIS: Yes (control and high dose group only, 5 animals of each sex/group)
- Time schedule for collection of urine: during the last week of the administration
- Metabolism cages used for collection of urine: Yes, by keeping animals in metabolic cages for 3 or 24 hours
- Animals fasted: Not specified
- Parameters checked:
3 hour urine: specific gravity; pH; leukocyte count; level of protein, glucose, ketone bodies, uroblilinogen, bilirubin, blood, and nitrite; and color. Microscopic examination of urinary sediments was performed for the following items: urine amount, red blood cell, white blood cell, epithelial cell, cast, and crystal in the fresh urine.
24 hour urine: the urine volume

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

Dose range finder:
- Gross findings were observed on the scheduled necropsy day

Main study:

Gross necropsy:
Necropsy examination was performed on moribund and dead animals, which were found immediately. After the blood samples were collected under deep anesthesia with isoflurane, the animal was killed by exsanguination. The external surface, all orifices, and all organs in the cranial cavity and thoracic and abdominal cavities and their contents were examined.

Organ weights:
The absolute and relative (organ-to-body-weight ratio) weights of the major organs were measured in all survivors when they were killed. The major organs included the liver, kidney, spleen, adrenal gland, testis, ovary, brain, pituitary gland, lung, heart, thymus, uterus, prostate, epididymis, and submaxillary gland.

Histopathology:
Internal organs from all animals were collected at necropsy and fixed in 10% neutral buffered formalin. The testis and epididymis were fixed in Bouin’s solution, and the eyes were fixed in Davidson’s solution. Histopathological examination was performed only in the tissues from negative control and high-dose groups. The internal organs examined were the liver, kidney, adrenal gland, heart, lung, pituitary gland, spleen, seminal vesicle, testis, ovary, epididymis, prostate gland, uterus, vagina, tongue, trachea, esophagus, thymus, thyroid gland, stomach, small and large intestine, urinary bladder, submandibular gland, eyeball, skin, pancreas, sternum, mammary gland, spinal cord, femur, mesenteric lymph node, and sciatic nerve.

Statistics:

Data on the body weights, feed and water consumption, hematological data, blood biochemical data, and organ weights were analyzed for homogeneity of variances using Levene’s test. One-way analysis of variance was performed to evaluate the significance of differences. If the variance was homogeneous and the significance of difference was confirmed, Scheffé’s multiple comparison test was performed as a post hoc test. If the variance was not homogeneous, the data were analyzed using Dunnett’s T3 test. Analysis of the data from recovery groups was performed using Student’s t-test. All analyses were performed using SPSS (version 19.0) software (IBM Corporation, Armonk, NY, USA).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation and white feces were sporadically observed after administration of both test articles. For example, in the ZnOAE100(−) group, salivation was sporadically observed in the male rats receiving 125 mg/kg on days 35, 63, and 65; in the male rats receiving 500 mg/kg from days 13–63; and in the female rats receiving 500 mg/kg from day 12 to day 87. In addition, in the ZnOAE100(+) group, salivation was observed in the male rats receiving 125 mg/kg on days 19, 48, and 74; in the male rats receiving 500 mg/kg on days 6–80; and in the female rats receiving 500 mg/kg on days 18–47. White feces were observed in the 500 mg/kg groups of both test articles from day 2 until treatment completion in both sexes. Fur loss and scarring were observed in some animals in the male and female groups receiving 500 mg/kg of ZnOAE100(−), male rats receiving 125 mg/kg of ZnOAE100(+), and rats of both sexes receiving 500 mg/kg of ZnOAE100(+).

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The MCV and MCH levels were significantly lower in male rats receiving 125 mg/kg ZnOAE100(−) than in negative control rats. Although the Hb, Hct (males only), MCV, MCH, PT (males only) and MCHC levels were significantly lower, the number of eosinophils was significantly greater in the male and female rats receiving 500 mg/kg of ZnOAE100(−) than in one or both control groups.

In addition, total erythrocyte count significantly increased in male rats receiving 500 mg/kg of ZnOAE100(−).

The number of eosinophils was significantly higher in males receiving 125 mg/kg of ZnOAE100(+) than in the control group (according to the author, not presented in the tabular overview).

Similar to the findings observed with ZnOAE100(−), ZnOAE100(+) significantly decreased the Hb, MCV, MCH, and MCHC levels in the males and females receiving 500 mg/kg compared with those in the control groups.

Compared with the control group, the group of male rats receiving 500 mg/kg ZnOAE100(+) showed significantly decreased Ht level and prothrombin time but significantly increased thrombocyte counts. The total white blood cell and thrombocyte counts were significantly increased in the female 500 mg/kg group compared with those in the control group.

During the post-treatment recovery period, total erythrocyte counts significantly increased, regardless of the surface charges of ZnO NPs, but monocyte counts (high dose females, ZnO-), MCV (not in high dose females ZnO+), and MCH significantly decreased in the 500 mg/kg groups in both sexes compared with those in the control groups. MCHC significantly decreased in high dose females treated with positively charged ZnO.

In addition, neutrophil counts and activated partial thromboplastin time significantly increased, but lymphocyte counts, Hb level, and MCHC significantly decreased in the male rats receiving 500 mg/kg of ZnOAE100(−) compared with those in the control groups.

For details please refer to the field "attached background material".

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Compared with the control groups, the group of male rats receiving 31.25 mg/kg of ZnOAE100(−) showed significantly decreased blood urea nitrogen levels (according to the author, not shown in the tabular overview) and those receiving 125 mg/kg showed significantly decreased Alb levels.

AP and P levels were significantly higher in the male group of rats receiving 500 mg/kg of ZnOAE100(−).

In the female 500 mg/kg ZnOAE100(−) group, the levels of TP, Alb, and Glu significantly decreased, but the Cl level significantly increased compared with those in the control groups.

Similarly, in the male 500 mg/kg group of ZnOAE100(+), the levels of TP, Alb, and Glu significantly decreased, but the P level significantly increased compared with those in the control groups.

In addition, compared with the control groups, the female 500 mg/kg group showed significantly decreased levels of TP, Alb, T-Cho, and Ca but significantly increased levels of AP and creatine kinase.

TP and Ca levels were significantly lower in the female 31.25 mg/kg group, and TP level was significantly lower in the female 125 mg/kg group than that in the control groups.

During the post-treatment recovery period of the group treated with ZnOAE100(−), the levels of aspartate aminotransferase, ALP, and TG significantly decreased in the male 500 mg/kg group.

In the female 500 mg/kg group, T-Cho and TG levels significantly decreased, but the AP level markedly increased compared with that in the control groups.

In addition, in the ZnOAE100(+) group, P level increased in the male 500 mg/kg group, and AP level and albumin/globulin ratio significantly increased in the female 500 mg/kg group compared with those in the control groups.


For details please refer to the field "attached background material".

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Compared with the control groups, the females in the recovery group receiving 500 mg/kg of ZnOAE100(−) showed an increase in the absolute weight of the submandibular gland (according to the author, data not shown).

Compared with the control groups, the group of females receiving 500 mg/kg of ZnOAE100(+) showed a significant increase in the absolute and relative weight of the liver.

The males in the 500 mg/kg group showed a significant increase in the relative but not absolute adrenal gland weight (according to the author, data not shown), and the absolute spleen weight decreased and the relative adrenal gland weight increased during the post-treatment recovery period.

For details please refer to the field "attached background material".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Light yellowish granule-shaped mass in the ventral prostate, reduced right testis, reduced and light brown color change and adhesion with surrounding tissue in the left kidney, enlarged right kidney, red color change in the margin of the right kidney, and dark red color change in the right lateral lobe of the liver were observed in males receiving 500 mg/kg of ZnOAE100(−). The smaller pituitary gland was observed in females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Regardless of the surface charge of ZnO NPs, squamous cell hyperplasia and vacuolation in nonglandular stomach, intracytoplasmic hyaline droplet, submucosal edema and inflammation, eosinophilic chief cell, and mucous cell hyperplasia in glandular stomach were observed in the 500 mg/kg groups of both sexes. Acinar cell apoptosis and chronic inflammation in the pancreas, retinal atrophy in the eye, and suppurative inflammation in the prostate gland were also observed in the 500 mg/kg groups of both sexes. As significant lesions in the stomach, pancreas, eye, and prostate gland were observed in the 500 mg/kg groups, these organs were also examined in the 31.25 mg/kg and 125 mg/kg groups. In the stomach, most lesions observed in the 500 mg/kg group were observed in the 31.25 mg/kg and 125 mg/kg groups and they exhibited dose dependency. The suppurative inflammatory lesion in the prostate gland, which was observed in the 500 mg/kg group, was also observed in the 31.25 mg/kg and 125 mg/kg groups. The lesion of retinal atrophy in the eye was observed in only one animal in the male 125 mg/kg group. However, in the pancreas, most lesions observed in the 500 mg/kg group were not observed in the 31.25 mg/kg and 125 mg/kg groups.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

DOSE RANGE FINDER
-the results of the dose range finding study is exclusively shown here and not above-

MORTALITY:
No moribund or dead animals were observed in the 14-day DRF study of ZnOAE100(−), but a dead animal was found in the group treated with ZnOAE100(+).

CLINICAL SIGNS:
The animals showed white feces.

BODY WEIGHT:
Loss of body weight regardless of the surface charge of 100 nm ZnO NPs.

NECROPSY:
Loss of corneal opacity regardless of the surface charge of 100 nm ZnO NPs.

HISTOPATHOLOGY:
Histopathological examination showed that the lesions of the stomach and spleen were related to the administration of both test articles.

MAIN STUDY

MORTALITY:
No deaths were observed in the animals treated with 100 nm ZnO NPs with either surface charge.

BODY WEIGHT:
No statistically significant differences were observed between the treated rats and their respective control groups in both sexes.

FOOD CONSUMPTION:
Food consumption was significantly increased in males receiving 31.25 mg/kg of ZnOAE100(−) at weeks 12 and 13; in males receiving 125 mg/kg of ZnOAE100(−) at weeks 1, 4, 5, 6, 7, 10 and 13; and in males receiving 500 mg/kg bw/day at weeks 2-13.
In females, feed consumption was increased in the 500 mg/kg group at weeks 1–8 and week 10 and at week 1 during the post-treatment recovery period.

Food consumption was significantly increased in males receiving 125 mg/kg of ZnOAE100(+) at weeks 5–9, 11, and 12, and those receiving 500 mg/kg of ZnOAE100(+) at weeks 3 and 5–13 and during both weeks of the post-treatment recovery period. In females, food consumption decreased in the 31.25 mg/kg group at week 7 and increased in the 125 mg/kg group at weeks 7 and 10 and in the 500 mg/kg group at weeks 2, 4, 7–11, and 13.

WATER CONSUMPTION:
Water consumption significantly increased in males receiving 125 mg/kg of ZnOAE100(−) at weeks 3, 8, 10, 11, 12, and 13 and in males receiving 500 mg/kg of ZnOAE100(−) at weeks 1, 2, 3, and 5, 8-11 and 13. Water consumption increased in females receiving 31.25 mg/kg and 125 mg/kg of ZnOAE100(−) at week 4 and in females receiving 500 mg/kg of ZnOAE100(−) at weeks 1, 3, 4, 8, and 9 and during both weeks of the post-treatment period.

Water consumption significantly increased in males receiving 125 mg/kg of ZnOAE100(+) at week 1, 4-7, 9, 10, 13, in males receiving 500 mg/kg at week 2-13 and in females receiving 500 mg/kg of ZnOAE100(+) at weeks 1–8, 10 and 14.

URINALYSIS:
No statistically significant changes were observed between the groups treated with ZnOAE100(−) and ZnOAE100(+) and the controls in both sexes (data not shown).

GROSS NECROPSY:
Multifocal light brown colored change was observed in both kidneys and multifocal dark red color change was also observed in the left kidney in the male negative control group. Reduced left seminal vesicle, multifocal black spots on the liver, light brown color change in the head of the spleen, and reduced size of the testis and epididymis were observed in the male vehicle control group.
The size of the left testis and the left thyroid gland was decreased in the male negative control group. Swollen and yellowish color change in the intestine, light brown color change in the kidney, adhesion to surrounding tissue in the left kidney, and red color change and enlarged submaxillary lymph node were observed (one each) in the female negative control group. In the male vehicle control, reduction in the size of the prostate gland and yellow color change in the right lateral lobe of the liver were observed (one each). In addition, adhesion to surrounding tissues in the spleen, dark red color change in the lung, and light brown color change in the left kidney were observed in one animal each in the female vehicle control group.
In the group receiving 31.25 mg/kg of ZnOAE100(−), reduced left seminal vesicle and light brown color change in the right kidney were observed in males and females, respectively. In males receiving 125 mg/kg, light yellowish granule-shaped masses were observed in the ventral prostate in three animals, light brown color change in the liver in two, reduced testis and epididymis in one, and reduced right seminal vesicle in one. In the female 125 mg/kg group, one animal had a light yellow color change in the stomach. The male 500 mg/kg group had light yellowish granule-shaped masses in the ventral prostate in 13, light brown color change in both kidneys in two, light brown color change in both adrenal glands in one, and cyst at the margin of the right kidney in one. One animal in the female 500 mg/kg group showed a red color change in the left lobe in the liver and a light brown color change in the rest of the liver and kidney.

Dose descriptor:

NOAEL

Effect level:

31.25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Conclusions:

This 90-day oral RDT study was conducted according to OECD guideline 408 and GLP with 3 dose levels of negatively (ZnOAE100(−)) and positively (ZnOAE100(+)) charged ZnO NPs to determine their no observed adverse effect level (NOAEL) and to identify target organs of toxicity.

No deaths were observed in the animals treated with ZnO NPs with either surface charge. In addition, salivation and white faeces were sporadically observed after administration of both test articles. Salivation, fur loss and scarring were observed in the 125 mg/kg and 500 mg/kg groups of negatively and positively charged ZnO NPs, and white faeces were commonly observed in groups treated with 500 mg/kg of test articles. However, these signs are not considered to be treatment related and no further details were given. No statistically significant differences were observed on body weights between the treated rats and their respective control groups in both sexes. Feed and water consumption were significantly increased mainly in high dose animals and at several occasions in males and females of the mid dose groups. However, these changes in feed and water consumption did not show any dose dependency and were observed sporadically and are not regarded to be treatment related.
 
Urinalysis did not reveal significant changes. The changes in haematological and blood biochemical analysis were commonly observed in the 500 mg/kg groups of both sexes of negatively and positively charged ZnO NPs compared with in their respective control groups. For instance, Mean cell volume (MCV), mean cell haemoglobin (MCH), and mean cell haemoglobin concentration (MCHC) significantly decreased after administration of both test articles, including the 2-week recovery period. Total protein and albumin levels also decreased. These significant decreases in haematological and blood biochemical parameters were considered to be related to the administration of negatively and positively charged ZnO NPs.
 
Light yellowish granule-shaped mass in the ventral prostate, reduced right testis, reduced and light brown colour change and adhesion with surrounding tissue in the left kidney, enlarged right kidney, red colour change in the margin of the right kidney, and dark red colour change in the right lateral lobe of the liver were observed in males receiving 500 mg/kg of the negatively charged ZnO NPs. A smaller pituitary gland was observed in females. In the group receiving 31.25 mg/kg of ZnOAE100(−), reduced left seminal vesicle size and light brown colour change in the right kidney were observed in males and females, respectively. In males receiving 125 mg/kg, light yellowish granule-shaped masses were observed in the ventral prostate in three animals, light brown colour change in the liver in two, reduced testis and epididymis size in one, and reduced right seminal vesicle size in one animal. In the females of the 125 mg/kg group, one animal had a light-yellow colour change in the stomach. The males of the 500 mg/kg group had light yellowish granule-shaped masses in the ventral prostate in 13 animals, light-brown colour change in both kidneys in two animals, light brown colour change in both adrenal glands in one animal, and cyst at the margin of the right kidney in one animal. One animal in the female 500 mg/kg group showed a red colour change in the left lobe in the liver and a light-brown colour change in the rest of the liver and kidney.
 
Compared with the control groups, the females in the recovery group receiving 500 mg/kg of ZnOAE100(−) showed an increase in the absolute weight of the submandibular gland (according to the author, data not shown). Compared with the control groups, the group of females receiving 500 mg/kg of ZnOAE100(+) showed a significant increase in the absolute and relative weight of the liver. The males in the 500 mg/kg group showed a significant increase in the relative but not absolute adrenal gland weight (according to the author, data not shown), and the absolute spleen weight decreased and the relative adrenal gland weight increased during the post-treatment recovery period.
 
Histopathological examination showed prominent changes in the stomach, eye, and pancreas compared with those in the negative and vehicle control groups. Squamous cell hyperplasia and vacuolation in non-glandular stomach and intracytoplasmic hyaline droplet, submucosal edema and inflammatory cell infiltration, and mucous cell hyperplasia in glandular stomach were considered to be treatment related because these changes were dose dependent and/or appeared prominently compared with those in the negative and vehicle controls. However, because these changes in the stomach induced tended to return to normal during the recovery period, they were less likely to cause functional disturbances. In addition, acinar cell apoptosis and chronic inflammation was observed in the pancreas in the male and female groups receiving 500 mg/kg of both test articles. These lesions in the pancreas were resolved during the recovery period, but they are considered to be toxicologically significant because they seemed to be severe enough to induce functional abnormalities. Suppurative inflammation in the prostate gland were observed in males receiving 500 mg/kg and retinal atrophy in the eye in both sexes receiving 500 mg/kg of both test articles. In particular, lesions in the eye are considered to be caused by treatment of the test articles similar to that observed in the incidence and severity in recovery groups.

The no observed adverse effect level (NOAEL) for both test articles was identified as 31.25 mg/kg for both sexes, because the adverse effects were observed at all doses greater than 125 mg/kg bw/day.
The results of this oral RDT study in SD rats can generally be regarded as reliable with restrictions, since the study was conducted based on the OECD guideline 408 and according to GLP. The methods and results are described appropriately, and the conclusions are plausible. Therefore, the study is judged as reliable with restrictions [RL=2].

The following restrictions were noted: The applied doses were not analytically verified and homogeneity and stability were not determined. The vehicle of both test substances was not specified. Further, ophthalmological examination and urinalysis were conducted but results not reported. Clinical signs were recorded and some of them reported in the results part but no information was given on the incidence and serverity (e.g. tabular overview of clinical signs). The test substance zinc oxide was modified by changing the surface charge, thus beside zinc oxide additional substances (L-serine and sodium citrate) were administered. A detailed clinical observation and the functional observation battery were not conducted. Historical control data and individual data were not provided.

Executive summary:

This 90-day oral RDT studywas conducted according to OECD guideline 408 and GLP with 3 dose levels of negatively (ZnO^AE100(−))and positively (ZnO^AE100(+)) charged ZnO NPs to determine their no observed adverse effect level (NOAEL) and to identify target organs of toxicity.

Six-week-old Crl:CD(SD) rats (Orient Bio Inc., Gyeonggi-do, Korea) were used for a dose-range finding and the main 90-day toxicity study. The dose level for the main study were selected based on the results of the 14-day DRF study due significant effects at 1 000 mg/kg and 2 000 mg/kg body weight. Dose levels of 31.25, 125 and 500 mg/kg bw/d were chosen for oral administration of both negatively and positively charged ZnO NPs, and two control group animals received distilled water or the vehicle solution. Recovery was observed during the 2 weeks after the end of the administration in the 500 mg/kg bw/d, vehicle control, and negative control groups. Negative control, vehicle control and high-dose groups consisted of 15 rats of each sex, and the low and middle dose groups consisted of ten rats of each sex. The test article was administered into the stomach by oral gavage 7 days/week once daily for 90 days. Test parameters included clinical observation, body weight, feed and water consumption, urinalysis, ophthalmological test, necropsy, organ weight, haematological and biochemical analysis, and histopathological observation based on the recommendations of the OECD guideline.

No deaths were observed in the animals treated with ZnO NPs with either surface charge. In addition, salivation and white faeces were sporadically observed after administration of both test articles. Salivation, fur loss and scarring were observed in the 125 mg/kg and 500 mg/kg groups of negatively and positively charged ZnO NPs, and white faeces were commonly observed in groups treated with 500 mg/kg of test articles. However, these signs are not considered to be treatment related. No statistically significant differences were observed on body weights between the treated rats and their respective control groups in both sexes. Feed and water consumption were significantly increased mainly in high dose animals and at several occasions in males and females of the mid dose groups. However, these changes in feed and water consumption did not show any dose dependency and were observed sporadically and are not regarded to be treatment related.

 

Urinalysis did not reveal significant changes. The changes in haematological and blood biochemical analysis were commonly observed in the 500 mg/kg groups of both sexes of negatively and positively charged ZnO NPs compared with in their respective control groups. Mean cell volume (MCV), mean cell haemoglobin (MCH), andmean cell haemoglobin concentration (MCHC) significantly decreased after administration of both test articles, including the 2-week recovery period. Total protein and Albumin levels also decreased. These significant decreases in haematological and blood biochemical parameters were considered to be related to the administration of negatively and positively charged ZnO NPs.

 

Light yellowish granule-shaped mass in the ventral prostate, reduced right testis, reduced and light brown colour change and adhesion with surrounding tissue in the left kidney, enlarged right kidney, red colour change in the margin of the right kidney, and dark red colour change in the right lateral lobe of the liver were observed in males receiving 500 mg/kg of the negatively charged ZnO NPs. A smaller pituitary gland was observed in females. In the group receiving 31.25 mg/kg of ZnO^AE100(−), reduced left seminal vesicle size and light brown colour change in the right kidney were observed in males and females, respectively. In males receiving 125 mg/kg, light yellowish granule-shaped masses were observed in the ventral prostate in three animals, light brown colour change in the liver in two, reduced testis and epididymis size in one, and reduced right seminal vesicle size in one animal. In the females of the 125 mg/kg group, one animal had a light-yellow colour change in the stomach. The males of the 500 mg/kg group had light yellowish granule-shaped masses in the ventral prostate in 13 animals, light-brown colour change in both kidneys in two animals, light brown colour change in both adrenal glands in one animal, and cyst at the margin of the right kidney in one animal. One animal in the female 500 mg/kg group showed a red colour change in the left lobe in the liver and a light-brown colour change in the rest of the liver and kidney.

 

Compared with the control groups, the females in the recovery group receiving 500 mg/kg of ZnO^AE100(−)showed an increase in the absolute weight of the submandibular gland. Compared with the control groups, the group of females receiving 500 mg/kg of ZnO^AE100(+)study showed a significant increase in the absolute and relative weight of the liver. The males in the 500 mg/kg group showed a significant increase in the relative adrenal gland weight, and the absolute spleen weight decreased during the post-treatment recovery period.

 

Histopathological examination showed prominent changes in the stomach, eye, and pancreas compared with those in the negative and vehicle control groups. Squamous cell hyperplasia and vacuolation in non-glandular stomach and intracytoplasmic hyaline droplet, submucosal edema and inflammatory cell infiltration, and mucous cell hyperplasia in glandular stomach were considered to be treatment related because these changes were dose dependent and/or appeared prominently compared with those in the negative and vehicle controls. However, because these changes in the stomach induced tended to return to normal during the recovery period, they were less likely to cause functional disturbances. In addition, acinar cell apoptosis and chronic inflammation was observed in the pancreas in the male and female groups receiving 500 mg/kg of both test articles. These lesions in the pancreas were resolved during the recovery period, but they are considered to be toxicologically significant because they seemed to be severe enough to induce functional abnormalities. Suppurative inflammation in the prostate gland were observed in males receiving 500 mg/kg and retinal atrophy in the eye in both sexes receiving 500 mg/kg of both test articles. In particular, lesions in the eye are considered to be caused by treatment of the test articles similar to that observed in the incidence and severity in recovery groups.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The methods were not described in detail and not all parameters examined were presented in the results. The clinical signs observed and histopathological findings have not been tabulated by group or sex. Not all parameters of the haematological and clinical biochemical examination have been shown or only for certain groups. The levels of urea in blood were not measured. Grip strength and motor activity were not tested in all animals (n=5/group from the control and high-dose groups. The results of the urinalysis was not shown. Only the organ weights of the heart of the animals in all groups of the main study, and the weights of the kidney, epididymis, submaxillary gland of all groups in the 14-day recovery study were shown in tabular form in the results. The organ weights of the other organs were missing in the results. A histopathological examination of the parathyroid, salivary gland and a section of bone marrow is missing. It was not specified whether the Peyer`s patches were included in the histopathological examination of the small and large intestines. During the histopathological examination of the spinal cord, it was not specified whether the 3 levels of cervical, mid-thoracic and lumbar, were taken into account. The study of the pancreas, stomach, and eyes was performed for all groups, but only the results from the 90-day study were shown in tabular form. The results of the histopathological examination of all organs in the control and high-dose groups were not shown.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998-09-21

Deviations:

yes

Remarks:

No measure of urea in the blood. No histopathological examination of the parathyroid, salivary gland and bone marrow. Mortality was observed once daily instead of twice daily.

GLP compliance:

yes

Remarks:

OECD Principles of Good Laboratory Practice

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ZnO-310 ultrafine zinc oxide; Sumitomo Osaka Cement Co, Ltd, Tokyo, Japan

INFORMATION ON NANOMATERIALS
- Chemical Composition: ZnO
- Particle size: average diameter: 29±3 nm (in deionized water, analysed by Kim, K.M. et al. (2012))*

*References:
- Kim, K.M., Kim, T.H., Kim, H.M. et al.: Colloidal behaviors of ZnO nanoparticles in various aqueous media. Toxicol Environ Health Sci. 2012; 4(2):121–131.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Co, Ltd., Seongnam, Korea
- Age at study initiation: 6 weeks old (5 weeks old at purchase + 7 days of acclimation)
- Weight at study initiation: approx. 185 g (males); approx. 150 g (females) (calculated based on the body weights at study initiation from the graphed changes in body weight in the results)
- Housing: housed in wire cages (maximum of two rats per cage)
- Diet (ad libitum): gamma-ray-irradiated rodent diet; source: Cargill Agri Purina Korea Inc, Seongnam, Korea
- Water (ad libitum): filtered water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 50% ± 20%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-serine buffer (1M Na2CO3, 20 mM HEPES buffer, and L-Serine)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Surface-charge modification was performed with serine, as previously reported (Kim, K.-M. et al. (2012)).* To produce the HEPES-serine buffer, HEPES buffer solution was prepared and pH-adjusted to 6 using 1M Na2CO3. To this, 1 wt/v% L-Serine (Sigma-Aldrich, St Louis, MO, USA) was added. For the high 500 mg/mL dose, the test material was weighed and HEPES-serine buffer added. The middle (250 mg/mL) and low (125 mg/mL) doses were diluted by suspending the modified ZnO NPs in sterile distilled water. Preparation of the test substance for each group for the treatment period was carried out every day.
- Administration volume: 10 mL/kg

VEHICLE
- Justification for use and choice of vehicle: to obtain a positive charge on the ZnO NPs.
- Concentration in vehicle: 500 mg/mL in the high-dose, 250 mg/mL in the mid-dose and 125 mg/mL in the low-dose solution.

*References:
- Kim, K.-M., Kim, T.H., Kim, H.M., et al. Colloidal behaviors of ZnO nanoparticles in various aqueous media. Toxicol Environ Health Sci.; 4(2): 121–131.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of ZnO(SM20(+)) were confirmed using the validation and verification of the concentration of the formulation method outlined in Korea Testing and Research Institute (KTR) study number TBH-1367. The concentration of each preparation was measured at 1 and 45 days and 90 days prior to administration; all the preparations were appropriate within 100 ± 15%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males in main study (three dose groups, negative and vehicle control group); 5 animals for recovery study (high-dose, negative and vehicle control group); 2 animals for organ distribution study (high-dose, negative and vehicle control group)

Control animals:

yes, concurrent vehicle

yes, sham-exposed

Details on study design:

- Dose selection rationale: the dose levels were determined based on the results of the dose-finding studies for 14-day repeated oral-toxicity studies (KTR study number: TBH-1092). The significant adverse effects were observed at dose levels of 2,000 and 1,000 mg/kg; therefore, 500 mg/kg was selected as the high dose for the high-dose group, and then the middle- and low-dose groups were set up by the two-fold intervals as 250 mg/kg and 125 mg/kg, respectively.
- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Rationale for selecting satellite groups: to assess the persistence and reversibility of toxicity in the negative control, vehicle control, and high-dose groups.
- Post-exposure recovery period in satellite groups: 2 weeks
- Rationale for selecting additional animals: a distributional study was also carried out for the systemic distribution of ZnO(SM20(+)) NPs in plasma, tissue, and feces.

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs and mortality): general symptoms, presence of toxic symptoms, and death were observed once a day after administration of the solutions during the testing period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: main studies for all animals, clonic and tonic movements, repetitive behaviour (hyperactive grooming, circling) or abnormal behaviour, aggression, and motor coordination, gait, and posture and handling changes were observed once per week.

BODY WEIGHT: Yes
- Time schedule for examinations: at animal acquisition, grouping, and once a week after the initiation of treatment.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes, calculated as g/rat/day (n=5 animals per group (the 2 animals/cage were counted as one animal in the calculation)).

WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week after the initiation of treatment.
- The water consumption were calculated as g/rat/day (n=5 animals per group (the 2 animals / cage were counted as one animal in the calculation)).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before grouping and during the final week of the 90-day oral-toxicity study.
- Dose groups that were examined: high-dose, vehicle and negative control groups (n=5 animals per group) from the 90-day study.
- Parameters examined: visual appearance of the eye was inspected; funduscopy was performed through the dilated pupil after dripping mydriatic fluid (Ocu-Tropine, Samil Pharmaceutical Co, Ltd, Seoul, Korea) into the eye with aid of a fundus camera

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 90-day experimental period and at the end of the 14-day recovery study.
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia.
- Animals fasted: Yes, for 18 hours.
- How many animals: all animals in the main study and all animals in the recovery study.
- Parameters examined: total leukocyte count, differential leukocyte count, total erythrocyte count, haemoglobin concentration, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte, platelets, blood clotting time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 90-day experimental period.
- Animals fasted: Yes, for 18 hours.
- How many animals: all animals in the main study.
- Parameters examined: total protein, albumin, albuminlb/globulin ratio, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, blood urea nitrogen, total cholesterol, triglycerides, glucose, calcium, inorganic phosphorus, creatine kinase, sodium, potassium, and chloride.

URINALYSIS: Yes (n=5 animals per group, high dose-group, negative and vehicle control group)
- Time schedule for collection of urine: during the last week of the study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: nitrite, protein, glucose, ketone, urobilinogen, and bilirubin levels, specific gravity, pH, leukocyte count, the presence of blood and microscopic examination of the sediment in the 3-hour urine; urinary volume (24-hour collected urine)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90-day experimental period
- Dose groups that were examined (sensory activity): all animals in the main study.
- Dose groups that were examined (grip strength and motor activity): negative and vehicle control groups and high-dose group (n=5 animals per group).
- Battery of functions tested: sensory activity: assessing the grasp response, touch escape, vocalization, pupil reflex, blink reflex and response to toe pinch, tail pinch, and finger approach; grip strength: in the front and hind limbs/feet was determined by using a rat grip strength measurement system (1027 CSX Grip Strength Meter) and grip strength was measured three times and averaged; motor activity: monitoring system (Columbus Instruments) and Truscan 99 data acquisition software (version 2.0).

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross examination of the external body surface, all orifices, cranial cavity, all organs of the chest cavity, and the abdominal cavity was performed.

ORGAN WEIGHT:
The liver, kidney, spleen, adrenal gland, testis, ovaries, brain, pituitary gland, lung, heart, thymus, uterus, prostate, epididymis, submaxillary gland were weighed for their wet weight then the relative organ-weight-to-body-weight ratio was calculated.

HISTOPATHOLOGY: Yes
The liver, kidney, adrenal gland, heart, lung, brain, pituitary gland, seminal vesicle, spleen, testis, ovaries, epididymis, prostate gland, uterus, vagina, tongue, trachea, esophagus, thymus, thyroid gland, stomach, duodenum, urinary bladder, small intestine, large intestine, eyeball, submandibular gland, pancreas, skin, mammary gland, sternum, femur, spinal cord, sciatic nerve, and mesenteric lymph node were removed and fixed in 10% neutral buffered formalin solution; eyes were fixed in Davidson solution; and the testis and epididymis were fixed in Bouin solution. Histopathologic examination of the control and high-dose groups only was undertaken. However, study of the pancreas, stomach, and eyes was performed for all groups.

Other examinations:

DISTRIBUTION of ZnO NPs IN THE PLASMA, ORGANS AND FECES: time schedule: prior to necropsy, a 1 mL blood sample was obtained from the tail vein and some stool samples were collected. Parameters investigated: Zinc content in samples from plasma, tissues (brain, liver, kidney, testis, ovary, spleen, lung, stomach, small intestine, and large intestine) and feces were determined. The distribution of the ZnO(SM20(+)) NPs was analysed on the basis of Zn content in each organ and biological fluids (blood plasma and feces) using inductively coupled plasma atomic absorption spectrometer.

Statistics:

To evaluate the homogeneity of variance, Levene’s tests were performed, and one-way analysis of variance was undertaken to determine significance in terms of the body weight, feed intake, water consumption, organ weight, and haematological and blood biochemical data. If the data had homogeneous variances and significant differences between treatment groups statistically, Scheffé’s test was conducted as the post hoc test. If the data had heterogeneous variance and significant differences were found between groups, Dunnett’s T3 was performed as the post hoc test. All statistical analysis was performed using SPSS software (v 19.0; IBM Corporation, Armonk, NY, USA).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation was observed immediately after administration in both sexes. The salivation was noted firstly in the 500 mg/kg group on day 11 and reported in all NP-treated groups from that time forward.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main study:
- 250 and 500 mg/kg bw/day: MCV and MCH were significantly decreased in males compared to the negative and vehicle control groups (p<0.01).
- 500 mg/kg bw/day: the total leukocyte count (p<0.05) and erythrocyte count (p<0.01) were significantly increased in males compared to both control groups. MCV and MCH levels (p<0.05 and p<0.01) were significantly decreased in females compared to the both control groups.
The MCHC and HGB levels (p<0.01 and p<0.05) in both sexes were significantly decreased compared to both controls. Hct level in males were significantly decreased compared with in the negative control groups (p<0.05).

Recovery study:
- 500 mg/kg bw/day:
the decreased HGB, MCV, MCH, and MCHC levels in males and females were not recovered to negative-control level in the recovery group. The total erythrocyte counts (p<0.05) were significantly increased in both sexes compared to both controls. MCV levels (p<0.01) in both sexes were significantly decreased compared to both controls. There was a significant decrease in MCH levels in males (p<0.01) and females (p<0.05) compared to both controls. MCHC level in males was significantly decreased compared to negative control group. The MCHC level in females was significantly decreased compared to both controls (p<0.05).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 125, 250 and 500 mg/kg: the albumin levels were significantly decreased (P<0.05) in males in the 125 and 250 mg/kg compared to the vehicle control group and in the 500 mg/kg group compared to both controls.
- 250 and 500 mg/kg bw/day: total protein levels were significantly decreased (P<0.05) in males and females compared with the control groups. The albumin levels were significantly decreased (P<0.05) in females compared to the negative and vehicle control group.
- 500 mg/kg bw/day: creatine kinase and chloride levels were significantly (P<0.05) increased in males compared to the vehicle control group. The glucose levels were significantly (P<0.05) decreased in males compared to the negative control group. In females alkaline phosphatase levels were significantly (P<0.05) increased compared to both control groups.

Endocrine findings:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 125, 250 and 500 mg/kg bw/day: acinar cell apoptosis and chronic inflammation of the pancreas in all groups were observed. Various gastric lesions of different grades were observed. In all males and females in all groups, squamous cell hyperplasia, squamous cell vacuolation, and subepithelial inflammatory cell infiltration in non-glandular stomach and submucosal inflammatory cells infiltration, superficial epithelial degeneration/regeneration, intracytoplasmic hyaline droplet, mucous cell hyperplasia, and eosinophilic chief cells in glandular stomach were observed.
- 500 mg/kg bw/day: two cases of retinal atrophy were found in males and females.

recovery study:
- 500 mg/kg bw/day: two cases of retinal atrophy were found in males, also three cases of retinal atrophy were identified in females. In males and females, eye retinal atrophy was of the same grade of lesion as those in main study.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

DISTRIBUTION OF ZnO NPs IN THE PLASMA, ORGANS AND FECES:
- ZnO concentration was increased in the liver, kidney, intestine, plasma, and lung in the study groups compared with in the negative control and vehicle control groups.

Details on results:

CLINICAL SIGNS:
- a wound, fur loss, and red tears were observed once each in each nanoparticle treatment group.
- There were no moribund animals related to the administration of the test article during the experimental period in either sex.

MORTALITY:
- there were no dead animals related to the administration of the test article during the experimental period in either sex.

BODY WEIGHT AND WEIGHT CHANGES:
body-weight changes were not shown to be statistically significant in the study groups compared with in the control groups.

FOOD CONSUMPTION:
main study: 125 and 500 mg/kg bw/day: food consumption was decreased significantly in week 1, 4 and 6 in the 125 mg/kg bw/day group and in week 1 to 6, 8, 9, 11 and 13 in the 500 mg/kg bw/day group in males compared with in the control group. The food consumption was significantly decreased only in week 1 in females in the 125 mg/kg group compared to the negative and vehicle controls (p<0.05).
- no significant changes in food consumption were observed in the 14-day recovery study.

WATER CONSUMPTION:
- main study: 125, 250 and 500 mg/kg bw/day: water consumption was decreased significantly in males in the 500 mg/kg group in week 1, 5, 6, 7, 9, 10 and 11 after administration, in week 5 and 9 - 11 in the 125 mg/kg group and in week 7 in the 250 mg/kg group compared with in the control groups. It was also significantly decreased (p<0.01) in females in week 10 and 11 in the 125 mg/kg group compared with in the negative control group.
- No significant changes in water consumption were observed in the 14-day recovery study.

OPHTALMOLOGICAL FINDINGS
- no test item-related effects observed.

URINALYSIS
- no test item-related effects observed.

BEHAVIOUR (FUNCTIONAL FINDINGS)
- treatment-related abnormal behavior and functionality were not observed when behavior, sensory reflex, grip strength, and motor activity were assessed.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN 7 BODY WEIGHT RATIOS:
- main study: 500 mg/kg bw/day: absolute organ weight of the heart was decreased significantly in males and the weight of submaxillary glands (data not shown) was increased significantly, compared with both controls. The relative weight of heart in males and the absolute and relative weight of heart in females showed no significant differences compared to both controls.
- In terms of relative organ weight, there were no statistical differences between the treated and control groups in either sex.
- Recovery study: 500 mg/kg bw/day: the absolute weights of the submaxillary gland and kidney, and the relative weight of the epididymis were increased significantly in males compared with both controls. No significant differences were observed in the organ weights of the submaxillary gland, kidney and epididymis in females compared to both controls.

GROSS PATHOLOGICAL FINDINGS:
- main study: 500 mg/kg bw/day: malformation of genitalia or atrophy of the seminal vesicle was observed in a single male, and lung discolouration was observed in a female. These were considered not to be related to treatment, as they were low in incidence and there was a lack of a dose relationship compared with the background data kept in the authors facility.

HISTOPATHOLOGICAL FINDINGS:
recovery study: 500 mg/kg bw/day: the lesions observed in the pancreas and stomach disappeared.

DISTRIBUTION OF ZnO NPs IN THE PLASMA, ORGANS AND FECES
- no differences in organ distribution in terms of sex. Nanoparticles were neither detected nor found to be increased in the brain, testis, ovary, spleen and stomach of study-group animals compared with control-group animals.

Dose descriptor:

LOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

In this RDT study, positively charged ZnO nanoparticles at doses of 125, 250 and 500 mg/kg bw/day were repeatedly administered by gavage in SD rats for 90 days. There was no death related to administration of the test article during the experimental period of either sex. There were no significant toxicological changes in the study animals compared with control animals, of either sex, in terms of functional assessment tests, changes in body weight, food and water consumption, urinalysis, ophthalmological tests, necropsy findings, or organ weights. In terms of clinical signs, salivation was observed immediately after administration in both sexes. Haematological analysis revealed that the total erythrocyte and total leukocyt counts were significantly increased in males, and HGB, Hct, MCV, MCH, and MCHC levels were decreased significantly in both sexes in the 500 mg/kg bw/day group compared with controls. Retinal atrophy in the eyes was observed in males and females in the high-dose group of the main study and in recovery group animals. In all treatment groups, various kinds of gastric inflammatory and degenerative lesions with regeneration were observed. Acinar cell apoptosis and chronic inflammation of the pancreas were observed in all groups. The absorption and accumulation of Zn increased with dose increment in liver, kidney, intestine, plasma, and lung, while there was little or no increase in these in the brain, testis, ovary, spleen, and stomach. The ZnO NPs were also dose-dependently excreted into the faeces.
According to the authors, the significant toxic changes were observed to be below 125 mg/kg bw/day, so the no-observed-adverse-effect level was not determined, but the lowest-observed-adverse-effect level was considered to be 125 mg/kg bw/day in both sexes.

The results of this oral RDT study in SD rats can generally be regarded as reliable with restrictions, since the study was conducted essentially equivalent to OECD guideline 408 (1998) and under GLP. The methods and results are described appropriately, and the conclusions are plausible.

However, the methods were not described in detail and not all parameters examined were presented in the results. Furthermore, some parameters are missing from the investigations carried out.
The authors reported that clinical signs were observed. Salivation was observed immediately after administration in both sexes. The salivation was noted firstly in the 500 mg/kg bw/day group on day 11 and reported in all NP-treated groups from that time forward. A wound, fur loss, and red tears were observed once each in each NP treatment group. However, the clinical signs observed have not been tabulated by group or sex, which means that it is not possible to determine how the symptoms differ between the sexes and whether these were dose-related.
A haematological examination was performed, but only those parameters were shown in the results that showed significant changes. However, since not all parameters were shown in the results for the animals in the main study and recovery study, a comparison of the haematological data between the two studies is not possible. It is therefore unclear whether the haematological changes observed in the 90-day study were still present or whether they have subsided again in the recovery study.
A clinical biochemical examination was performed, but only those parameters were shown in the results that showed significant changes. However, since only some parameters were shown for the animals in the main study, but none were shown for the animals in the recovery study, a comparison of the clinical-biochemical data between the two studies is not possible. In addition, the urea in the blood was not measured. Furthermore, it was not stated in the publication whether a clinical chemical examination of the blood was also carried out on the animals in the 14-day recovery study.
Grip strength and motor activity were not tested in all animals (n=5/group from the negative, vehicle control and high-dose groups) at the end of the experimental period. However, investigations on grip strength and motor activity should be made on all animals according to OECD 408, and investigations in only half of the animals are not representative of the entire group.
A urinalysis was also carried out, but the results of the parameters examined were not shown in the publication.
The liver, spleen, adrenal gland, testis, ovaries, brain, pituitary gland, lung, thymus, uterus, prostate and epididymis and submaxillary glandwere weighed, but not all organ weights were shown in the results for the animals in the main study or recovery study. Therefore, a comparison of the organ weights between the two studies is not possible.
Furthermore, a histopathological examination of the parathyroid, salivary gland and a section of bone marrow is missing. It was not specified whether the Peyer`s patches were included in the histopathological examination of the small and large intestines. During the histopathological examination of the spinal cord, it was not specified whether the 3 levels of cervical, mid-thoracic and lumbar, were taken into account. In addition, the histopathological results were incomplete. The study of the pancreas, stomach, and eyes was performed for all groups, but only the results of all groups from the 90-day study were shown in tabular form. In addition, the results of the histopathological examination of all organs in the control and high-dose groups were not shown in the publication. Due to these missing results, it is unclear whether and which histopathological changes in the animals from the 90-day study recovered in the 14-day recovery study. In addition, since the results of the other organs from the high-dose groups are missing in the main study, it is unclear whether there were changes in the other organs or not.
In addition, observations of the mortality of all animals were only carried out once a day after administration of the solutions during the test period and not at least twice a day as required in OECD guideline 408.
According to OECD 408, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (NOAEL) at the lowest dose level. However, a NOAEL could not be determined in the present study, since treatment-related effects were observed in all three dose groups, especially with regard to the histopathological findings. Therefore, the selection of the dose levels was unsuitable according to OECD 408.

In the present study, haematological analysis revealed that the total erythrocyte and total leukocyt counts were significantly increased in males, and HGB, Hct, MCV, MCH, and MCHC levels were decreased significantly in both sexes in the 500 mg/kg bw/day group compared with controls.
The total leukocyte count, erythrocyte count in males, the MCH and MCV in females, and the HGB, Hct, MCHC levels in both sexes in the main study, as well as the HGB and MCHC levels in the recovery study (both sexes) in the high dose groups were statistically significant compared to the controls, but they were in the normal range compared to the control groups and/or compared to the historical control data of Crj:CD (SD) rats (Lee, J.M. et al. (2012)).*
In the 90-day study, the changes observed in total leukocytes, total erythrocyte, MCHC levels in males, and HGB, Hct, MCV and MCH in both sexes were dose-dependent. These haematological effects are nevertheless physiologically relevant, since they were also observed in other studies in which zinc was administered orally. However, it is unclear whether the blood formation in the bone marrow was influenced by the ZnO(SM20(+)), as no histopathological examination of the bone marrow was carried out. Furthermore, various kinds of gastric inflammatory and degenerative lesions with regeneration were observed in all treatment groups, as well as acinar cell apoptosis and chronic inflammation of the pancreas were observed in all groups. Even when these histopathological findings in the stomach and pancreas disappeared in the recovery study and therefore do not represent any adverse effects, they are nevertheless relevant for the toxicological assessment. Other studies, in which zinc was also administered orally, also showed effects in the stomach and pancreas. Furthermore, acinar cell apoptosis was observed in the pancreas which was also shown in a microscopic picture, but the specified microscopic magnification in the picture is too low. A larger magnification could be selected for the illustration in the publication in order to be able to show the apoptosis in the cells.

Therefore, the study is judged as reliable with restrictions because it can be considered as a guideline study without detailed documentation.

*References:
- Lee, J.M., Lee, M.A., Do, H.N., Song, Y.I., Bae, R.J., Lee, H.Y., Park, S.H., Kang, J.S., Kang, J.K.: Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats. Lab Anim Res. 2012 Jun; 28(2):115-21.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The methods were not described in detail and not all parameters examined were presented in the results. The clinical signs observed and histopathological findings have not been tabulated by group or sex. Not all parameters of the haematological and clinical biochemical examination have been shown or only for certain groups. The levels of urea in blood were not measured. The histopathological examination of the parathyroid, salivary gland and a section of bone marrow was missing. It was not specified whether the Peyer`s patches were included in the histopathological examination of the small and large intestines. During the histopathological examination of the spinal cord, it was not specified whether the 3 levels of cervical, mid-thoracic and lumbar, were taken into account. The results of food and water consumption were not shown. The results of the urinalysis were not shown. Grip strength and motor activity were not tested in all animals (n=5/group from both control groups and high-dose groups). Not all organ weights were shown in the results for the animals in the main and recovery study.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998-09-21

Deviations:

yes

Remarks:

No measurement of blood urea levels. No histopathological examination of the parathyroid, salivary gland and bone marrow. Mortality was observed once daily instead of twice daily.

GLP compliance:

yes

Remarks:

guidelines of the OECD of Good Laboratory Practices

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: ZnO-310 ultrafine zinc oxide; Sumitomo Osaka Cement Co, Ltd., Tokyo, Japan

INFORMATION ON NANOMATERIALS
- Chemical Composition: ZnO
- Particle size: average diameter: 29±3 nm (in deionized water, analysed by Kim, K.M. et al. (2012))*

*Reference:
- Kim, K.M., Kim, T.H., Kim, H.M. et al.: Colloidal behaviors of ZnO nanoparticles in various aqueous
media. Toxicol Environ Health Sci. 2012; 4(2):121–131.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Co, Ltd., Seongnam, Korea
- Age at study initiation: 6 weeks old (5 weeks old at purchase + 7 days of acclimation)
- Weight at study initiation: approx. 185 g (males); approx. 160 g (females) (calculated based on the body weights at study initiation from the graphed changes in body weight in the results)
- Housing: housed in wire cages (maximum of two rats per cage)
- Diet (ad libitum): gamma-ray-irradiated rodent diet; source: Cargill Agri Purina Korea Inc, Seongnam, Korea
- Water (ad libitum): filtered water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES)-citrate buffer (1M Na2CO3, 20 mM HEPES buffer and sodium citrate)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The surface charge modification was performed by using sodium citrate to add topical negative charges to the ZnO NPs, as previously reported (Kim, K.-M. et al. (2012)).* The HEPES buffer solution was first adjusted to pH 7 using 1M Na2CO3, and then sodium citrate was added to the HEPES buffer to produce HEPES-citrate buffer (2% citrate). Next, the ZnO NPs were suspended in the HEPES-citrate buffer for chemical modification, as described previously, weighed, and resuspended in HEPES-citrate buffer solution to yield a high-dose (500 mg/mL) NP solution. The mid-dose (250 mg/mL) and low-dose (125 mg/mL) NP solutions were diluted by suspending the modified ZnO NPs in sterile distilled water instead of HEPES-citrate buffer. Preparation of freshly modified ZnO NPs for use in each experimental group was done daily over the course of the 90-day study.
- Administration volume: 10 mL/kg

VEHICLE:
- Justification for use and choice of vehicle: to add topical negative charges to the ZnO NPs.
- Concentration in vehicle: 500 mg/mL in the high-dose, 250 mg/mL in the mid-dose and 125 mg/mL in the low-dose solution.

*References:
- Kim, K.-M., Kim, T.H., Kim, H.M., et al.: Colloidal behaviors of ZnO nanoparticles in various aqueous media. Toxicol Environ Health Sci.; 4(2): 121–131.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the resultant ZnO(SM20(−)) NPs were confirmed using method validation and verification of the formulation concentration according to protocols established by the Korea Testing and Research Institute (KTR), study number TBH-1026. The concentration of each preparation was measured on days 1, 45 and 90, just prior to administration to the rats. All preparations were confirmed within 100%±15%.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals in main study (three dose groups, negative and vehicle control group); 5 animals for recovery study (high-dose, negative and vehicle control group); 2 animals for organ distribution study (highdose, negative and vehicle control group)

Control animals:

yes, concurrent vehicle

yes, sham-exposed

Details on study design:

- Dose selection rationale: the dose levels in the 90-day oral toxicity study were determined based on the results of a previously conducted dose-finding, 14-day repeated oral toxicity study (KTR, study number TBH-1091). Significant adverse effects were observed at dose levels of 500 and 1,000 mg/kg in the dose-finding study; therefore, 500 mg/kg was selected for use in the high-dose group, and the mid- and low-dose groups were based on two-fold intervals of NP dilution.
- Fasting period before blood sampling for clinical biochemistry: 18 hours
- Rationale for selecting satellite groups: to assess the persistence and reversibility of toxicity by a negative control recovery group, vehicle control recovery and a high-dose NP recovery group.
- Post-exposure recovery period in satellite groups: 14 days
- Rationale for selecting additional animals: toxicokinetic and distribution studies were also conducted to determine the systemic distribution of the ZnO NPs.

Positive control:

not specified

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: (clinical signs and mortality): All animals in the primary oral toxicity study were observed once per day after the daily ZnO(SM20(-)) NP administration for general symptoms, the presence of any adverse/toxic symptoms, and/or the occurrence of death.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all animals were observed once per week for chronic and tonic movements, repetitive behavior (excessive grooming, hyperactivity, or repetitive circling), abnormal behavior, aggression, motor coordination or lack thereof, gait, posture, and handling changes in response to NP administration.

BODY WEIGHT: Yes
- Time schedule for examinations: at animal acquisition, grouping, and once per week after the initiation of treatment.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, calculated as g/rat/day (n=5 animals per group (the 2 animals/cage were counted as one animal in the calculation)).

WATER CONSUMPTION: Yes (n=5 animals per group)
- Time schedule for examinations: once per week after initiation of treatment.
- water consumption was calculated as g/rat/day (n=5 animals per group (the 2 animals/cage were counted as one animal in the calculation)).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before grouping and during the final week of the 90-day oral toxicity study.
- Dose groups that were examined: vehicle control, negative control and high-dose groups (n=5 animals per group).
- Parameters examined: visual appearance of the eye was inspected; funduscopy was performed through the dilated pupil after dripping mydriatic fluid (Ocu-Tropine, Samil Pharmaceutical Co, Ltd, Seoul, Korea) into the eye with aid of a fundus camera.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the 90-day experimental period and at the end of the 14-day recovery study.
- Anaesthetic used for blood collection: Yes, anesthetized with inhaled isoflurane.
- Animals fasted: Yes, for 18 hours.
- How many animals: all animals in the main study and all animals in the recovery study.
- Parameters examined: total leukocyte count, differential leukocyte count, total erythrocyte count, haemoglobin concentration, haematocrit, mean cell volume, mean cell haemoglobin (average mass of haemoglobin per red blood), mean cell haemoglobin concentration, total reticulocyte count, total eosinophil count, and total platelet count, blood clotting time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the 90-day experimental period and at the end of the 14-day recovery study.
- Animals fasted: Yes, for 18 hours.
- How many animals: all animals in the main study and all animals in the recovery study.
- Parameters examined: total protein, albumin, albumin/globulin ratio, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, blood urea nitrogen, total cholesterol, triglycerides, glucose, calcium, inorganic phosphorus, and creatine kinase, sodium, potassium, and chloride.

URINALYSIS: Yes (n=5 animals per group, high dose-group, negative and vehicle control group)
- Time schedule for collection of urine: during the last week of the 90 day study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: nitrite, protein, glucose, ketone, urobilinogen, and bilirubin levels, specific gravity, pH, leukocyte count, the presence of blood and microscopic examination of the sediment in the 3-hour urine; Urinary volume in the 24-hour urine.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90-day experimental period.
- Dose groups that were examined (sensory activity): all animals in the primary oral toxicity study.
- Dose groups that were examined (grip strength and motor activity): negative, vehicle control groups and high-dose group (n=5 animals per group).
- Battery of functions tested: sensory activity: assessing the grasp response, touch escape, vocalization, pupil reflex, blink reflex and response to toe pinch, tail pinch, and finger approach; grip strength: in the front and hind limbs/feet was determined by using a rat grip strength measurement system (1027 CSX Grip Strength Meter) and grip strength was measured three times and averaged; motor activity: monitoring system and Truscan 99 data acquisition software.

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross examination was performed of the external body surface of each rat, all orifices, the cranial cavity, all organs of the chest cavity, and the abdominal cavity.

ORGAN WEIGHT:
The liver, kidney, spleen, adrenal glands, testes, ovaries, brain, pituitary gland, lung, heart, thymus, uterus, prostate gland, epididymis, and submaxillary gland were removed, subjected to necroscopic analysis, and weighed to determine the wet weight of each organ. The relative organ-weight-to-bodyweight ratio was then calculated for each organ.

HISTOPATHOLOGY: Yes
Next, the liver, kidney, adrenal gland, heart, lung, brain, pituitary gland, seminal vesicle, spleen, testes, ovaries, epididymis, prostate gland, uterus, vagina, tongue, trachea, esophagus, thymus, thyroid gland, stomach, duodenum, urinary bladder, small intestine, large intestine, eyeball, submandibular gland, pancreas, skin, mammary gland, sternum, femur, spinal cord, sciatic nerve, and mesenteric lymph node were removed and fixed in 10% neutral buffered formalin solution. The eyeball was fixed in Davidson solution, and the testes and epididymis were fixed in Bouin solution. Organs were stained with haematoxylin and eosin (H&E) or Alcian blue as appropriate and subjected to histopathological analysis. The analysis was only performed for the vehicle and negative control and high-dose groups, with the exception of the pancreas, stomach, and eyes, which were included in all groups.

Other examinations:

DISTRIBUTION OF ZnO NPs IN THE PLASMA, ORGANS AND FECES: time schedule: prior to necropsy, blood samples were obtained from the tail vein and some stool samples were collected. Parameter investigated: determination of zinc content in samples from plasma, tissues (brain, liver, kidney, testis, ovary, spleen, lung, stomach, small intestine, and large intestine) and feces. Distribution of the ZnO(SM20(-)) NPs was analysed on the basis of Zn content in each organ biological fluids (blood plasma and feces) by ICP-AAS spectrometer.

Statistics:

Levene’s test was performed to evaluate the homogeneity of variance. A one-way ANOVA (analysis of variance) for significance was performed to evaluate the bodyweight, food intake, water consumption, organ weight, and haematological and blood biochemical data. In cases where the data showed homogeneous variances and statistically significant differences between treatment and control groups, Scheffe’s post hoc test was conducted. In cases where the data showed heterogeneous variances and statistically significant differences between groups, Dunnett’s T3 post hoc test was conducted. All statistical analyses were performed by using freely available SPSS (IBM Corporation, Armonk, NY, USA) software, version 19.0.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 125, 250 and 500 mg/kg bw/day: excessive salivation was observed in the 125 mg/kg bw/day group from days 33 to 63, in the 250 mg/kg group from day 25 up until the end of treatment, and in the 500 mg/kg bw/day group from day 14 up until the end of treatment. Salivation was more frequent in the 500 mg/kg bw/day group relative to the other groups.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- main study: 125, 250 and 500 mg/kg bw/day: statistically significant increases (P<0.05) were observed in the 125 mg/kg bw/day group (both males and females) at weeks 6, 9, 12, and 13 compared with the control groups; in the 250 mg/kg bw/day group at weeks 5, 8, 9, 10, 11, 12, and 13 (P<0.05 or P<0.01); and in the 500 mg/kg bw/day group at weeks 3, 4, 5, 6, 8, 9, and 13 (P<0.05 or P<0.01). When stratified by sex, food consumption significantly increased (P<0.05) in the female 250 mg/kg bw/day group at week 1 compared with the control groups, in the female 500 mg/kg bw/day group (P<0.05 or P<0.01) from weeks 1 to 13.
- recovery study: 500 mg/kg bw/day: statistically significant increases were observed during both weeks of the post-treatment recovery period (P<0.05 or P<0.01). Food consumption significantly increased in the females at week 2.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

main study:
- 500 mg/kg bw/day: water consumption significantly increased in males in all groups at various time points. This was most obvious in the 500 mg/kg bw/day group compared with the controls and occurred at weeks 1, 2, 3, 4, and 12. In females, water consumption was significantly increased (P<0.05 or P<0.01) in the 500 mg/kg bw/day group at weeks 1, 2, 3, 4, and 5.

recovery study:
- 500 mg/kg bw/day: water consumption significantly increased in males during week 2 (P<0.05 or P<0.01). In females, water consumption was significantly increased (P<0.05 or P<0.01) in 500 mg/kg bw/day recovery group at week 2.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 500 mg/kg bw/day; one case of opacity of the eye in the male was observed.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 250 and 500 mg/kg bw/day: concentration of HGB was signif. decreased in males compared with vehicle control group. MCH were significantly decreased (P<0.05) in males compared with both controls.
- 500 mg/kg bw/day: MCV levels were significantly decreased (P<0.05) in the males compared with the vehicle controls. The MCHC levels were significantly decreased in both sexes compared to both controls (p<0.05). Total erythrocyte count in females was significantly increased in females compared to both controls (p<0.05). MCV and MCH levels were significantly decreased (p<0.01) in the females compared to both controls. HGB level was significantly decreased (p<0.05) in females compared to vehicle controls.
recovery study:
- 500 mg/kg bw/day: erythrocyte counts were significantly increased (p<0.05) in the males compared to vehicle controls. Total erythrocyte counts were significantly increased (p<0.05) in females compared to both controls. MCH was significantly decreased in females compared to both controls (p<0.05). MCHC was significantly decreased in females compared to vehicle controls (p<0.05) and HCT level in females was significantly increased compared to negative controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 250 and 500 mg/kg bw/day: total serum protein level was significantly decreased in males compared to negative controls (p<0.01) and vehicle controls (p<0.05). total serum protein level in females was also significantly decreased (p<0.01) in the 500 mg/kg bw/day compared to both controls and in the 250 mg/kg bw/day compared negative controls.
- 500 mg/kg bw/day: creatine kinase levels were significantly increased (p<0.05) in males compared to both controls. The albumin level in females was significantly decreased compared to negative controls (p<0.01) and vehicle controls (p<0.05).

Endocrine findings:

not specified

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

main study:
- 500 mg/kg bw/day: one case of a small testis with seminal vesicle atrophy in a male. One case of a brownish change on the left lateral lobe of the liver was observed in a female.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

main study:
125, 250 and 500 mg/kg bw/day: various kinds of gastric lesions in varying grades were observed. Epithelial vacuolation was observed in the forestomach and basal cell hyperplasia, epithelial hyperplasia, epithelial vacuolation, hyperkeratosis, and submucosal inflammatory cell infiltration were observed in the limiting ridge. In the glandular stomach, erosive lesions, infiltrating epithelial globule leukocytes, submucosal edema, and inflammation, chief cell-like cells with eosinophilic cytoplasmic granules, and a reduced number of parietal cells were all observed (grade: minimal-to-severe).
- 250 and 500 mg/kg bw/day: minimal-to-severe grade retinal atrophy was observed in males in the mid- and high-dose group and in females in the high-dose group.
- 500 mg/kg bw/day: acinar cell apoptosis, ductular hyperplasia, periductular lymphoid cell infiltration, and regenerative acinar cells were all observed in the pancreas of the males and females.

recovery study: the grade and incidence of lesions observed in the main study was reduced in the recovery group (data not shown).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

DISTRIBUTION OF ZnO NPs IN THE PLASMA, ORGANS AND FECES:
Zn concentrations dose-dependently increased in the liver, kidney, intestine, and plasma of the experimental compared with the control groups. The ZnO NPs were also dose-dependently excreted into the feces

Details on results:

CLINICAL SIGNS:
- Other adverse symptoms included piloerection, fur loss, soft stools, diarrhoea, the appearance of wound on the skin and stain around the mouth, soiled fur, and opacity of the eye.
- However, no dose-response effects of the ZnO(SM20(−)) NPs were seen for any of these symptoms.

MORTALITY
- None of the animals died in any of the study groups during the course of the investigation.

BODY WEIGHT AND WEIGHT CHANGES
- bodyweight changes in the experimental and control groups were not significantly different for either sex.

WATER CONSUMPTION:
- main study: 125 and 250 mg/kg bw/day: water consumption significantly increased in males in all groups at various time points. It was seen in the 250 mg/kg bw/day group at weeks 3 and 4 (P<0.05), and in the 125 mg/kg bw/day group at week 4 (P<0.05). In females, water consumption was significantly increased (P<0.05 or P<0.01) in the 500 mg/kg bw/day group at weeks 1, 2, 3, 4, and 5.

HAEMATOLOGICAL FINDINGS:
- main study: 250 mg/kg bw/day: eosinophil counts (EOS) were significantly increased (P<0.05) in the males compared to negative controls. HCT level were significantly decreased in males compared to vehicle controls (p<0.05).
- None of the other investigated haematological parameters (leukocyte, platelet or reticulocyte counts) differed significantly between the groups.

CLINICAL BIOCHEMISTRY FINDINGS:
- main study: 250 mg/kg bw/day: albumin level in males was significantly decreased compared to the negative controls (p<0.01) and vehicle controls (p<0.05).
- None of the other investigated biochemical parameters (albumin/globulin ratio, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, creatinine, blood urea nitrogen, total cholesterol, triglycerides, glucose, calcium, inorganic phosphorus, sodium, potassium, and chloride) differed significantly between the groups.

URINALYSIS FINDINGS:
- no abnormal urinary changes were observed in any of the treated animals compared with the controls.

BEHAVIOUR (FUNCTIONAL FINDINGS):
- abnormal behavior and functionality were not observed in any of the parameters for behavioral assessment or sensory reflex evaluation. Grip strength and motor activity were not treatment-related.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN 7 BODY WEIGHT RATIOS:
- main study: 500 mg/kg bw/day: relative organ weight of the adrenal gland was significantly increased (p<0.05) in males compared to the vehicle control group.
- recovery study: 500 mg/kg bw/day: the absolute weight of the submaxillary gland was significantly increased (p<0.05) in the males compared with the vehicle controls. Relative uterus weight was significantly increased (p<0.05) in females compared to vehicle control group.
- the absolute and relative organ weights in the 90-day oral toxicity study were statistically similar between the experimental groups and the control groups - for the absolute adrenal gland weight in the males, and the absolute ovary and uterus weights in the females.
- Nonetheless, these organ weight differences showed no dose dependency or correlation with the histopathologic findings described in the histopathological examination section.

GROSS PATHOLOGICAL FINDINGS
- 250 mg/kg bw/day: one case of a mass in the thymus in a male was observed.
- control group: one case of black spot was observed on the right adrenal gland in a female.
- Necropsy findings showed no dose dependency of any of these findings.

HISTOPATHOLOGICAL FINDINGS:
- main study: most of the organs appeared normal in the experimental groups.

DISTRIBUTION OF ZnO NPs IN THE PLASMA, ORGANS AND FECES:
- no clear differences were observed between the data for the male and female rats. Little or no increase in the Zn concentration in the brain, testis, ovary, spleen, stomach, or lung, with the exception of the stomach in the female 500 mg/kg group.

Dose descriptor:

LOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

In this RDT study, negatively charged ZnO NPs (ZnOSM20(-)) at doses of 125, 250 and 500 mg/kg bw/day were repeatedly administered by gavage in SD rats for 90 days. None of the animals died, but number of adverse symptoms were associated with the NPs, including salivation in all of the test animals. Evaluation of sensory responses, motor activity, weight changes, and urinalysis were unremarkable. At some time points, food intake, and water consumption were increased in the experimental groups, but this phenomenon was not thought to be treatment-related due to the lack of dose dependency. No effects on body weight gains were observed. Haematological and blood biochemical analyses revealed small but significant decreases in the amount of HGB, MCV, MCH and MCHC in the male 250 and/or 500 mg/kg groups and in the female 500 mg/kg bw/day group. In addition, total erythrocytes in females in the 500 mg/kg bw/day group were significantly increased. Moreover, total serum protein and albumin levels were significantly decreased in the 250 and/or 500 mg/kg bw/day groups for both sexes.
Apoptosis of pancreatic acinar cells, infiltration of periductular lymphoid cells, ductular epithelial hyperplasia and increased numbers of regenerated acinar cells were observed in high dose males and females. Retinal atrophy of the eye was observed in the 250 and 500 mg/kg male groups and the 500 mg/kg female group, and various histopathological lesions were also observed in the stomach of the ZnO NP-treated rats. In the recovery group, the pancreas and stomach lesions resolved, but the retinal atrophy did not. According to the authors, these results indicate that the target organs of the ZnO NPs are the pancreas, stomach, and eye.

Toxicokinetic data showed similar dose- and time-dependent increases in the accumulation and absorption of Zn in the liver, kidney, large intestine, and small intestine of both male and female rats.

According to the authors, a NOAEL of the ZnO(SM20(-)) nanoparticles was not determined based on the results of this study, and the lowest dose level of 125 mg/kg in both sexes was considered to be a LOAEL.

The results of this oral RDT study in SD rats can generally be regarded as reliable with restrictions, since the study was conducted based on the OECD guideline 408 and according to GLP. The methods and results are described appropriately, and the conclusions are plausible.

However, the methods were not described in detail and not all parameters examined were presented in the results. Furthermore, some parameters are missing from the investigations carried out.
The authors reported that clinical signs were observed. Salivation was observed in all treatment groups and other adverse symptoms, but the clinical signs observed have not been tabulated by group or sex. Therefore, it is not possible to determine how the symptoms differ between the sexes and whether these were dose-related.

A haematological examination was performed, but only those parameters were shown that showed significant changes. However, since not all parameters were shown in the results for the animals in the main study and recovery study, a comparison of the haematological data between the two studies is not possible. It is therefore unclear whether the haematological changes observed in the 90-day study were still present or whether they have subsided again in the 14-day study.
A clinical biochemical examination was performed, but only those parameters were shown in the results that showed significant changes. However, since only some biochemical parameters were shown for the animals in the main study, but none were shown for the animals in the recovery study, a comparison of the clinical-biochemical data between the two studies is not possible. Furthermore, it was not stated in the publication whether a clinical chemical examination of the blood was also carried out on the animals in the 14-day recovery study. It is therefore unclear whether the clinical biochemistry changes observed in the 90-day study were still present or whether they have subsided again in the 14-day study.
The histopathological examination of the parathyroid, salivary gland and a section of bone marrow was missing. Furthermore, it was not specified whether the Peyer`s patches were included in the histopathological examination of the small and large intestines. During the histopathological examination of the spinal cord, it was not specified whether the 3 levels of cervical, mid-thoracic and lumbar, were taken into account. The histopathological findings have not been tabulated by group or sex. Therefore, it is not possible to determine how the findings differ between the sexes and whether these were dose-related.
The results of food and water consumption were also not shown in the publication.
A urinalysis was also carried out, but the results of the parameters examined were not shown in the publication.
The liver, spleen, adrenal gland, testis, ovaries, brain, pituitary gland, lung, thymus, uterus, prostate, epididymis and submaxillary gland were weighed, but not all organ weights were shown in the results for the animals in the main study or recovery study. Therefore, a comparison of the organ weights between the two studies is not possible.
Grip strength and motor activity were not tested in all animals (n=5/group from the negative, vehicle control and high-dose groups) at the end of the experimental period. However, investigations on grip strength and motor activity should be made on all animals according to OECD 408, and investigations in only half of the animals are not representative of the entire group.
In addition, observations of the mortality of all animals were only carried out once a day after administration of the solutions during the test period and not at least twice a day as required in OECD guideline 408.
According to OECD 408, a descending sequence of dose levels should be selected with a view to demonstrating any dosage related response and a no-observed-adverse-effect level (NOAEL) at the lowest dose level. However, a NOAEL could not be determined in the present study, since treatmentrelated effects were observed in all three dose groups, especially with regard to the histopathological findings. Therefore, the selection of the dose levels was unsuitable according to OECD 408.

In the present study, haematological and blood biochemical analyses revealed significant decreases in the amount of HGB, Hct, MCV, MCH and MCHC in the male 250 and/or 500 mg/kg bw/day groups and female 500 mg/kg bw/day group. They also said that total serum protein and albumin levels were significantly decreased in the 250 and/or 500 mg/kg bw/day groups for both sexes.

The observed significant changes of HCT (females), MCH (males), HGB, MCV and MCHC (both sexes) in the mid- and/or high-dose groups in the main study, as well as the significant changes of total erythrocyte count (both sexes), HCT, MCH and MCHC (females) were statistically significant compared to the control groups, but they were in the normal range compared to the control groups and/or compared to the historical control data of Crj:CD (SD) rats (Lee, J.M. et al. (2012)).*
In the 90-day study, the changes observed in HGB levels in males, and total erythrocyte, MCV and MCH levels in females were dose-dependent. These haematological effects are nevertheless physiologically relevant, since they were also observed in other studies in which zinc was administered orally. However, it is unclear whether the blood formation in the bone marrow was influenced by the ZnO(SM20(-)), as no histopathological examination of the bone marrow was carried out.
According to the authors, there were lesions in the pancreas and stomach, and retinal atrophy was observed in the 250 and 500 mg/kg bw/day group. Since the results of the histopathological examination are not shown in tabular form, it is unclear how many animals in each group showed abnormalities, and whether their incidence or severity were dose-dependent. Furthermore, one case of opacity of the eye in the male high-dose group was observed during the ophthalmic examination. However, it was not indicated whether this finding was made prior to grouping or in the final week of the 90-day study. Furthermore, even when the grade and incidence of these lesions was reduced in the recovery group and therefore do not represent any adverse effects, they are nevertheless relevant for the toxicological assessment. Other studies, in which zinc was also administered orally, also showed effects in the stomach and
pancreas.

Therefore, the study is judged as reliable with restrictions because it can be considered as a guideline study without detailed documentation.

*Reference:
- Lee, J.M., Lee, M.A., Do, H.N., Song, Y.I., Bae, R.J., Lee, H.Y., Park, S.H., Kang, J.S., Kang, J.K. Historical control data from 13-week repeated toxicity studies in Crj:CD (SD) rats. Lab Anim Res. 2012; 28(2): 115-21.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 - .. September 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not performed under GLP conditions. Only male rats were used.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

This study is a dose-range finding study

Deviations:

yes

Remarks:

only male rats were used

Principles of method if other than guideline:

No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.77 - <= 1.99 µm

Geometric standard deviation (GSD):

2.08

Remarks on MMAD:

MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.

Dose / conc.:

10.9 mg/m³ air

Remarks:

Test Group 1
(T 0420, 12 mg/m³)

Dose / conc.:

20.7 mg/m³ air

Remarks:

Test Group 2
(T 0420, 24 mg/m³)

Dose / conc.:

10.8 mg/m³ air

Remarks:

Test Group 3
(T 0421, 12 mg/m³)

Dose / conc.:

21.3 mg/m³ air

Remarks:

Test Group 4
(T 0421, 24 mg/m³)

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

No. of animals per sex per dose:

5 animals were used per group (total of 5 groups and total of 25 animals).

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: /

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly (Monday and Friday) thereafter until one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights of last weighing. Those of the weekends will be calculated as the difference of Monday to the previous Friday.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
Food consumption was determined weekly, from Monday to Friday and from Friday to Monday. Food consumption was calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: all
- Number of animals: 25

LUNG BURDEN: No

OTHER: /

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

No information provided

Clinical signs:

no effects observed

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.

Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively.

Food efficiency:

not specified

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

/

Ophthalmological findings:

not specified

Description (incidence and severity):

/

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.

Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).

In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

/

Endocrine findings:

not specified

Description (incidence and severity):

/

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

not specified

Description (incidence and severity):

/

Immunological findings:

not specified

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.

Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.

The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.

In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

Details on results:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations,.

The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.

During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).

Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).

BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities

In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.

Regarding pathology, the lungs and nasal cavity were the target organs.

In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Effect level:

mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Remarks on result:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Critical effects observed:

not specified

Concentration measurements in the exposure system

Study means and standard deviations of test substance concentrations:

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness dust generation (%)
		Mean	SD
1	12	10.94	0.41	18	59.9 %
2	24	20.67	0.89	28	74.7 %
3	12	10.76	0.37	19	56.7 %
4	24	21.28	1.02	42	50.3 %

Results of the particle size analyses

All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.

Cascade impactor measurements:

T 420			T 0421
	MMAD / GSD	% < 3 µm MMAD		MMAD / GSD	% < 3 µm MMAD
Test group 1 Measurement 1	1.90 µm / 2.03	74.2 %	Test group 3 Measurement 1	1.99 µm / 1.99	72.4 %
Test group 1 Measurement 2	1.79 µm / 2.04	76.5 %	Test group 3 Measurement 2	1.92 µm / 2.17	71.7 %
Test group 2 Measurement 1	1.85 µm / 2.02	75.3 %	Test group 4 Measurement 1	1.77 µm / 1.97	78.2 %
Test group 2 Measurement 2	1.77 µm / 2.17	75.2 %	Test group 4 Measurement 2	1.79 µm / 2.26	73.5 %

In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.

Particle size distribution measured by APS:

	Measurement date	MMAD / GSD		Measurement date	MMAD / GSD
Test group 1	26 Jun 20	2.47 µm/2.24	Test group 3	26 Jun 20	3.23 µm/1.87
		2.39 µm/2.22			3.38 µm/1.98
		2.47 µm/2.34			3.33 µm/1.91
	02 Jul 20	2.28 µm/2.37		02 Jul 20	2.93 µm/1.97
		2.13 µm/2.25			3.11 µm/2.21
		2.10 µm/2.23			3.46 µm/2.39
Test group 2	25 Jun 20	1.94 µm/2.66	Test group 4	25 Jun 20	2.41 µm/2.16
		1.82 µm/2.54			2.40 µm/2.18
		1.82 µm/2.52			2.29 µm/2.09
	03 Jul 20	2.41 µm/2.46		03 Jul 20	2.76 µm/2.25
		2.50 µm/2.37			2.67 µm/2.22
		2.50 µm/2.55			2.59 µm/2.17

 

BAL RESULTS

Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Cells	7.4*	8.9**	4.8**	8.6**
Eosinophils	4.1	16.0	6.7	11.0
Lymphocytes	4.9*	7.4**	4.0*	7.5**
Macrophages	0.6	1.1	1.1	0.7
Neutrophils	620.8*	722.3**	333.3**	734.0**
Monocytes	105.2*	91.2**	48.0**	71.4**
Epithelial cells	+	+	+	+

One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

+ increase could not be calculated because of zero activity in controls

Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Protein	14.4**	18.6**	5.4**	17.5**
GGT	4.6**	4.1**	2.9**	4.3**
LDH	11.0**	11.2**	4.6**	11.7**
ALP	16.6**	23.5**	7.0**	27.4**
NAG	2.7*	2.7*	1.7**	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

 

Histopathology

Treatment-related findings were observed in organs listed in the tables below with incidences and grading:

Incidence and grading of histological findings in larynx:

Larynx (Level I)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Epithelial alteration	1	0	4	1	2
·         Grade 1	1		4	1	2

The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).

Incidence and grading of histological findings in lungs

Lungs	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Infiltration, granulocytic	0	5	5	5	5
·         Grade 1		4		3	2
·         Grade 2		1	5	2	3
Histiocytosis, alveolar	0	5	5	5	5
·         Grade 1				1
·         Grade 2		4		2
·         Grade 3		1	5	2	3
·         Grade 4					2

In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.

Incidence and grading of histological findings in the nasal cavity:

Nasal cavity (Level IV)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Degeneration/regeneration, olfactory epithelium	0	5	5	5	5
·         Grade 1		3
·         Grade 2		2		3
·         Grade 3			5	2	1
·         Grade 4					4

Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.

Incidence and grading of histological findings in the tracheobronchial lymph nodes:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Histiocytosis (m)focal	0	3	3	4	5
·         Grade 1				2	1
·         Grade 2		2	3	2	4
·         Grade 3		1

Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Pathology

Weight parameters

Absolute weights

When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of absolute lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	113%	135%**	114%**	141%**

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of relative lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	117%	138%**	119%**	154%**

*p <= 0.05; **p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross lesions

In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.

Incidence of gross lesions observed during necropsy:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Enlarged	0	2	1	0	2

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:

Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.

Executive summary:

Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.

The following is a summary of the most relevant results:

Test group 4 (T0421, 24 mg/m³)

• Impaired body weight development, reduced food consumption


• Increased absolute and relative neutrophil cell counts in blood


• Decrease relative lymphocyte counts in blood


• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (141%/154%)


• Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals


• Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 3 (T0421 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (114%/119%)


• Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 2 (T0420, 24 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (135%/138%)


• Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

 

Test group 1 (T0420, 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 - .. September 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not performed under GLP condtions. Only male rats were used.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

This study is a dose-range finding study

Deviations:

yes

Remarks:

only male rats were used

Principles of method if other than guideline:

No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 93.8% for T0421.
- Test substance No.: 20/0051-1 for T0421.
- Batch identification: T0421.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: Aug 2020 for T0421.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.77 - <= 1.99 µm

Geometric standard deviation (GSD):

2.08

Remarks on MMAD:

MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.

Dose / conc.:

10.9 mg/m³ air

Remarks:

Test Group 1
(T 0420, 12 mg/m³)

Dose / conc.:

20.7 mg/m³ air

Remarks:

Test Group 2
(T 0420, 24 mg/m³)

Dose / conc.:

10.8 mg/m³ air

Remarks:

Test Group 3
(T 0421, 12 mg/m³)

Dose / conc.:

21.3 mg/m³ air

Remarks:

Test Group 4
(T 0421, 24 mg/m³)

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

No. of animals per sex per dose:

5 animals were used per group (total of 5 groups and total of 25 animals).

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: /

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly (Monday and Friday) thereafter until one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights of last weighing. Those of the weekends will be calculated as the difference of Monday to the previous Friday.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
Food consumption was determined weekly, from Monday to Friday and from Friday to Monday. Food consumption was calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: all
- Number of animals: 25

LUNG BURDEN: No

OTHER: /

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

No information provided

Clinical signs:

no effects observed

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.

Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively.

Food efficiency:

not specified

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

/

Ophthalmological findings:

not specified

Description (incidence and severity):

/

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.

Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).

In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

/

Endocrine findings:

not specified

Description (incidence and severity):

/

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

not specified

Description (incidence and severity):

/

Immunological findings:

not specified

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.

Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.

The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.

In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

Details on results:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations.

The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.

During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).

Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).

BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities

In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.

Regarding pathology, the lungs and nasal cavity were the target organs.

In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Effect level:

mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Remarks on result:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Critical effects observed:

not specified

Concentration measurements in the exposure system

Study means and standard deviations of test substance concentrations:

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness dust generation (%)
		Mean	SD
1	12	10.94	0.41	18	59.9 %
2	24	20.67	0.89	28	74.7 %
3	12	10.76	0.37	19	56.7 %
4	24	21.28	1.02	42	50.3 %

Results of the particle size analyses

All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.

Cascade impactor measurements:

T 420			T 0421
	MMAD / GSD	% < 3 µm MMAD		MMAD / GSD	% < 3 µm MMAD
Test group 1 Measurement 1	1.90 µm / 2.03	74.2 %	Test group 3 Measurement 1	1.99 µm / 1.99	72.4 %
Test group 1 Measurement 2	1.79 µm / 2.04	76.5 %	Test group 3 Measurement 2	1.92 µm / 2.17	71.7 %
Test group 2 Measurement 1	1.85 µm / 2.02	75.3 %	Test group 4 Measurement 1	1.77 µm / 1.97	78.2 %
Test group 2 Measurement 2	1.77 µm / 2.17	75.2 %	Test group 4 Measurement 2	1.79 µm / 2.26	73.5 %

In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.

Particle size distribution measured by APS:

	Measurement date	MMAD / GSD		Measurement date	MMAD / GSD
Test group 1	26 Jun 20	2.47 µm/2.24	Test group 3	26 Jun 20	3.23 µm/1.87
		2.39 µm/2.22			3.38 µm/1.98
		2.47 µm/2.34			3.33 µm/1.91
	02 Jul 20	2.28 µm/2.37		02 Jul 20	2.93 µm/1.97
		2.13 µm/2.25			3.11 µm/2.21
		2.10 µm/2.23			3.46 µm/2.39
Test group 2	25 Jun 20	1.94 µm/2.66	Test group 4	25 Jun 20	2.41 µm/2.16
		1.82 µm/2.54			2.40 µm/2.18
		1.82 µm/2.52			2.29 µm/2.09
	03 Jul 20	2.41 µm/2.46		03 Jul 20	2.76 µm/2.25
		2.50 µm/2.37			2.67 µm/2.22
		2.50 µm/2.55			2.59 µm/2.17

 

BAL RESULTS

Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Cells	7.4*	8.9**	4.8**	8.6**
Eosinophils	4.1	16.0	6.7	11.0
Lymphocytes	4.9*	7.4**	4.0*	7.5**
Macrophages	0.6	1.1	1.1	0.7
Neutrophils	620.8*	722.3**	333.3**	734.0**
Monocytes	105.2*	91.2**	48.0**	71.4**
Epithelial cells	+	+	+	+

One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

+ increase could not be calculated because of zero activity in controls

Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Protein	14.4**	18.6**	5.4**	17.5**
GGT	4.6**	4.1**	2.9**	4.3**
LDH	11.0**	11.2**	4.6**	11.7**
ALP	16.6**	23.5**	7.0**	27.4**
NAG	2.7*	2.7*	1.7**	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

 

Histopathology

Treatment-related findings were observed in organs listed in the tables below with incidences and grading:

Incidence and grading of histological findings in larynx:

Larynx (Level I)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Epithelial alteration	1	0	4	1	2
·         Grade 1	1		4	1	2

The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).

Incidence and grading of histological findings in lungs

Lungs	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Infiltration, granulocytic	0	5	5	5	5
·         Grade 1		4		3	2
·         Grade 2		1	5	2	3
Histiocytosis, alveolar	0	5	5	5	5
·         Grade 1				1
·         Grade 2		4		2
·         Grade 3		1	5	2	3
·         Grade 4					2

In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.

Incidence and grading of histological findings in the nasal cavity:

Nasal cavity (Level IV)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Degeneration/regeneration, olfactory epithelium	0	5	5	5	5
·         Grade 1		3
·         Grade 2		2		3
·         Grade 3			5	2	1
·         Grade 4					4

Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.

Incidence and grading of histological findings in the tracheobronchial lymph nodes:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Histiocytosis (m)focal	0	3	3	4	5
·         Grade 1				2	1
·         Grade 2		2	3	2	4
·         Grade 3		1

Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Pathology

Weight parameters

Absolute weights

When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of absolute lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	113%	135%**	114%**	141%**

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of relative lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	117%	138%**	119%**	154%**

*p <= 0.05; **p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross lesions

In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.

Incidence of gross lesions observed during necropsy:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Enlarged	0	2	1	0	2

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:

Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.

Executive summary:

Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.

The following is a summary of the most relevant results:

Test group 4 (T0421, 24 mg/m³)

• Impaired body weight development, reduced food consumption


• Increased absolute and relative neutrophil cell counts in blood


• Decrease relative lymphocyte counts in blood


• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (141%/154%)


• Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals


• Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 3 (T0421 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (114%/119%)


• Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

 

Test group 2 (T0420, 24 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (135%/138%)


• Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 1 (T0420, 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2020 - ...June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study without restrictions performed under GLP conditions.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (uncoated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.52 - <= 2.01 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.52-2.01 μm (geometric standard deviation = 4.04--2.28)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

0.52 mg/m³ air (analytical)

Remarks:

SD: 0.10 mg/m3; target concentration: 0.5 mg/m³: Test Group 1 (Parental animals F0)

Dose / conc.:

2 mg/m³ air (analytical)

Remarks:

SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 2 (Parental animals F0)

Dose / conc.:

9.97 mg/m³ air (analytical)

Remarks:

SD: 1.23 mg/m3, target concentration: 10 mg/m³: Test Group 3 (Parental animals F0); Test Group 13 (male animals for particle detection); Test Group 23 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ±20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the
control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g

Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

Food efficiency:

not examined

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

/

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.

Immunological findings:

not examined

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
--> These effects were observed as treatment-related, adverse effects
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 9 males and 10 females

Test group 23 (Recovery group R1, 10 mg/m³)
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4
female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal
Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 1 female

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
Test item 1 (Zinc oxide T0420)
The following treatment-related, adverse effects were observed:
Test group 3 (10 mg/m³):
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ -Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:
During pre-exposure period, none of the male and female rats showed any clinical signs and findings different from normal.
Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.

BODY WEIGHT AND WEIGHT GAIN
Body weight change:
The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g
Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

HAEMATOLOGICAL FINDINGS:
In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.

CLINICAL CHEMISTRY:
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)

GROSS PATHOLOGY
Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.


HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: no findings observed

Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in the bronchoalveolar lavage (BAL) of males and females of
test group 3 (10 mg/m3 Zinc oxide T0420) total cell counts, as well as absolute and relative
lymphocyte, neutrophil cell (PMN) and monocyte counts as well as absolute eosinophil cell
counts (not significantly) were increased. Relative macrophage counts were significantly
decreased. These alterations were regarded as treatment related and adverse.
In the BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) absolute and relative lymphocyte, neutrophil cell and monocyte counts were already significantly increased whereas
relative macrophage counts were significantly decreased. In males of test group 1 (0.5 mg/m3
Zinc oxide T0420) absolute and relative lymphocyte counts were significantly increased. In
females of test group 2 absolute and relative monocyte counts as well as relative neutrophil
counts were significantly increased whereas relative macrophage counts were significantly
decreased. However, in the BAL of both sexes of test group 2 as well as in BAL of males of
test group 1 total cell counts were not altered, and the differential cell counts were only
marginally changed (below 10fold). Therefore, the cell count changes in BAL of both sexes in
test group 2 and in BAL of males of test group 1 were regarded as treatment related but non adverse.

Recovery animals:
After the 8-week recovery period, no significant changes in BAL cytology were observed in
BAL of both sexes of test group 23 (10 mg/m3 Zinc oxide T0420).
Proteins/enzymes:
Parental animals:
After the administration period, in BAL of males and females of test group 3 (10 mg/m3 Zinc
oxide T0420) total protein levels as well as lactate dehydrogenase (LDH) and alkaline
phosphatase (ALP) activity were moderately, significantly increased whereas β-N-Acetyl
glucosaminidathe zinc content in lungs, liver, heart and brain of parse (NAG) in males of this test group and γ-Glutamyl-transferase (GGT) activity
in both sexes were marginally but also significantly increased. These alterations were regarded
as treatment related and adverse.
Additionally, in BAL of females of test group 3 (10 mg/m3 Zinc oxide T0420) NAG activity was
significantly increased, and in BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) LDH,
ALP and GGT activities and in females of this test group ALP and GGT activities were
significantly increased. However, the changes were marginally (below 2fold). Therefore, these
alterations were regarded as treatment related but non-adverse.
Recovery animals:
After the 8-week recovery period, in BAL of males and females of test group 23 (10 mg/m3
Zinc oxide T0420) no protein level and enzyme activity changes were observed.


OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. In all other examined organs, the zinc level was comparable with the control.

- Electron microscopy:
Electron microscope analysis of particulate matter in organs and tissues: (see section overall remarks/attachments)

Dose descriptor:

NOAEC

Remarks:

local toxicity

Effect level:

0.52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: At the target mid concentration of 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

0.52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: At the target low concentration of 0.5 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one female animal

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

9.97 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology

Critical effects observed:

yes

Lowest effective dose / conc.:

2 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.

To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 

Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.

The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).

All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.

Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).

To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2020 - ...June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study without restrictions performed under GLP conditions.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (coated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 93.8% for T0421.
- Test substance No.: 20/0051-1 for T0421.
- Batch identification: T0421.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: Aug 2020 for T0421.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.6 - <= 2.53 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.60-2.53 μm (geometric standard deviation = 3.08-2.23)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

0.52 mg/m³ air (analytical)

Remarks:

SD: 0.16 mg/m3; target concentration: 0.5 mg/m³: Test Group 4 (Parental animals F0)

Dose / conc.:

2.01 mg/m³ air (analytical)

Remarks:

SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 5 (Parental animals F0)

Dose / conc.:

10.07 mg/m³ air (analytical)

Remarks:

SD: 0.96 mg/m3, target concentration: 10 mg/m³: Test Group 6 (Parental animals F0); Test Group 16 (male animals for particle detection); Test Group 26 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the
control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period
on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these
groups. The findings in the eye was considered incidental due to missing concentration response
relationship.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.


The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

Food efficiency:

not examined

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

/

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

Immunological findings:

not examined

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%), Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³): Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³):
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 10 males and 5 females
--> These effects were observed as treatment-related, adverse effects
Test group 26 (Recovery group R1, 10 mg/m³)
No treatment-related adverse findings

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity,
level IV, exemplarily) in 6 males and 10 females

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female
Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase
(ALP) activities in BAL of both sexes
• Increased β-Glutamyl-transferase (GGT) activity in BAL of males

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these groups. The findings in the eye was considered incidental due to missing concentration response relationship.

BODY WEIGHT AND WEIGHT GAIN
In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.


The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

HAEMATOLOGICAL FINDINGS:
At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.


CLINICAL CHEMISTRY:
Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.


NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³): Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
--> These effects were observed as treatment-related, adverse effects


GROSS PATHOLOGY

Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³):
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 10 males and 5 females
--> These effects were observed as treatment-related, adverse effects
Test group 26 (Recovery group R1, 10 mg/m³)
No treatment-related adverse findings

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: One female of test group 26 (test item 2, 10 mg/m³) revealed also a minimal epithelial alteration at the base of the epiglottis. These findings were considered to be treatment-related.


Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in BAL of males and females of test group 6 (10 mg/m3 Zinc oxide T0421) total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts were significantly increased. Additionally, absolute eosinophil cell counts were increased (not significantly) in BAL of males whereas relative macrophage counts were significantly decreased in BAL of males and females of test group 6. These alterations were regarded as treatment related and adverse.
In BAL of males in test group 5 (2 mg/m3 Zinc oxide T0421) absolute and relative neutrophil cell and monocyte counts as well as relative lymphocyte counts were already significantly increased. In BAL of both sexes of test group 5 relative macrophage counts were significantly decreased, and in females of this test group relative neutrophil cell counts were already significantly increased. However, in both sexes total cell counts in BAL were not significantly altered, and the differential cell counts were only marginally changed (below 10fold).
Therefore, these alterations in BAL cytology were regarded as treatment related but nonadverse.
Recovery animals:
After the 8-week administration period, in BAL of females of test group 26 (10 mg/m3 Zinc oxide T0421) absolute epithelial cells were marginally but significantly increased. This isolated change was regarded as incidental and not treatment related.

Proteins/enzymes:
Parental animals:
After the administration period, in BAL of males and females of test group 6 (10 mg/m3 Zinc oxide T0421) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas γ-Glutamyltransferase (GGT) activity in males of this test group was marginally but also significantly increased. These alterations were regarded as treatment related and adverse.
Additionally, in BAL of both sexes of test group 6 (10 mg/m3 Zinc oxide T0421) β-
-N-Acetyl glucosaminidase (NAG) activity as well as in females of this test group GGT activity were significantly increased. In BAL of males of test group 5 (2 mg/m3 Zinc oxide T0421) LDH, ALP and GGT activities and in females ALP and GGT activities were significantly increased.
However, the changes were marginally (below 2fold). Therefore, these alterations were regarded as treatment related but non-adverse.
Recovery animals:
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total protein levels in BAL were still significantly increased. However, this was the only altered parameter in BAL among these individuals and the increase was only marginal (below 2fold).
Therefore, this isolated change was regarded as incidental and not treatment related.


OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. Conversely, statistically lower zinc content was observed in liver and heart of female animals exposed to 10 mg/m³ coated ZnO. In all other examined organs, the zinc level was comparable with the control.

- Electron microscopy:
Electron microscope analysis of particulate matter in organs and tissues: (see section overall remarks/attachments)

Dose descriptor:

NOAEC

Remarks:

local toxicity

Effect level:

0.52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: histological findings in the nasal cavity of one male and one female rat. at the target mid concentration of 2 mg/m³

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

10.07 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry ; increased neutrophils and lymphocytes in blood at the target conc of 10 mg/m3

Critical effects observed:

yes

Lowest effective dose / conc.:

2.01 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.

To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 

Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.

The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).

All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.

Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).

To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Reference 5

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 November 2020 - ...June 2022

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (uncoated/coated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

micro sized Zinc Oxide
Purity: 99.8%
Name of reference substance 1: Zinc oxide T0242
Reference substance No.: 20/0201-1
Batch identification: 56589
Appearance - physical state / color: Solid / white
Storage conditions: Room temperature
BET: 4.48 m2/g

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.67 - <= 1.48 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.67- 1.48 μm (geometric standard deviation = 3.71-2.40)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

9.68 mg/m³ air (analytical)

Remarks:

SD: 1.47 mg/m3, target concentration: 10 mg/m³: Test Group 7 (Parental animals F0); Test Group 17 (male animals for particle detection); Test Group 27 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for main groups 0 (air control) versus 7 (micro ZnO) as well as 0 versus 8 (Zn sulphate), and recovery groups 20 (air control) versus 23 (T0420), 20 versus 26 (T0421), 20 versus 27 and 20 versus 28
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> Student's t-test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+)
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one-sided-)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
-Weight of the anesthetized animals and absolute and relative organ weights
--> WILCOXON test (two-sided)

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, reference item 1 (test groups 7, 17 and 27):
One male animal of test group 7 (No. 126) showed a mass that was palpable through skin on
study days 66 - 149. No clinical signs of toxicity were noted in any other animals of these
groups.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes of body weight were determined in male
animals:
- Test group 7: day 25: 350.5g (p< 0.05), whereas the control group was 363.4g
- Test group 7: day 32: 362.2g (p< 0.05), whereas the control group was 379.4g
- Test group 7: day 39: 373.5g (p< 0.05), whereas the control group was 390.9g
- Test group 7: day 46: 377.4g (p< 0.05), whereas the control group was 394.9g
- Test group 7: day 60: 398.5g (p< 0.05), whereas the control group was 416.7g
- Test group 7: day 67: 405.2g (p< 0.05), whereas the control group was 424.6g
- Test group 7: day 94: 423.8g (p< 0.01), whereas the control group was 453.5g
- Test group 27: day 32: 361.7g (p< 0.05), whereas the control group was 375.0g
- Test group 27: day 39: 375.9g (p< 0.05), whereas the control group was 390.9g

The following statistically significant body weight changes were determined in male animals:
- Test group 7: day 25-> 32: 11.7 (p< 0.01), whereas the control group was 16.1g
- Test group 27: day 18-> 25: 12.7 (p< 0.01), whereas the control group was 16.9g
- Test group 27: day 74-> 81: 5.4 (p< 0.01), whereas the control group was 9.8g
-> Retarded body weight development in male animals as treatment-related, adverse effects

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

/

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
decreased relative basophil counts in males of test group 7 (10 mg/m3 Zinc oxide T0242); decreased absolute and relative monocyte counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); increased red blood cell (RBC) counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in females of test group 7 (10 mg/m3 Zinc oxide T0242);

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased total bilirubin and sodium values in females of test group 7 (10 mg/m3 Zinc oxide T0242)(males, inorganic phosphate 1.48-1.85 mmol/L; females, albumin 35.27-40.13 g/L; total bilirubin 1.25-2.20 μmol/L; sodium 140.7-143.0 mmol/L).

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
Reference item 1 (Test group 7):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

Immunological findings:

not examined

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%),
Reference item 1- Test group 7 (10mg/m3) (Zinc oxide T0242) : Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 7 males and 10 females
--> These effects were observed as treatment-related, adverse effects
Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female
animals

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all
female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 4 females

Test group 27 (Recovery group R1, 10 mg/m³)
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in
2 males and 3 females

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte
counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β--N-Acetyl glucosaminidase (NAG) activity in BAL of males

Test group 27 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 1 (test groups 7, 17 and 27): One male animal of test group 7 (No. 126) showed a mass that was palpable through skin on study days 66 - 149. No clinical signs of toxicity were noted in any other animals of these groups.


BODY WEIGHT AND WEIGHT GAIN
The following statistically significant changes of body weight were determined in male
animals:
- Test group 7: day 25: 350.5g (p< 0.05), whereas the control group was 363.4g
- Test group 7: day 32: 362.2g (p< 0.05), whereas the control group was 379.4g
- Test group 7: day 39: 373.5g (p< 0.05), whereas the control group was 390.9g
- Test group 7: day 46: 377.4g (p< 0.05), whereas the control group was 394.9g
- Test group 7: day 60: 398.5g (p< 0.05), whereas the control group was 416.7g
- Test group 7: day 67: 405.2g (p< 0.05), whereas the control group was 424.6g
- Test group 7: day 94: 423.8g (p< 0.01), whereas the control group was 453.5g
- Test group 27: day 32: 361.7g (p< 0.05), whereas the control group was 375.0g
- Test group 27: day 39: 375.9g (p< 0.05), whereas the control group was 390.9g

The following statistically significant body weight changes were determined in male animals:
- Test group 7: day 25-> 32: 11.7 (p< 0.01), whereas the control group was 16.1g
-> Retarded body weight development in male animals as treatment-related, adverse effects

FOOD CONSUMPTION
No effect observed

HAEMATOLOGICAL FINDINGS:
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
decreased relative basophil counts in males of test group 7 (10 mg/m3 Zinc oxide T0242); decreased absolute and relative monocyte counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); increased red blood cell (RBC) counts in females of test group 7 (10 mg/m3 Zinc oxide T0242); decreased mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) in females of test group 7 (10 mg/m3 Zinc oxide T0242);

CLINICAL CHEMISTRY:
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased total bilirubin and sodium values in females of test group 7 (10 mg/m3 Zinc oxide T0242)(males, inorganic phosphate 1.48-1.85 mmol/L; females, albumin 35.27-40.13 g/L; total bilirubin 1.25-2.20 μmol/L; sodium 140.7-143.0 mmol/L).

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
Reference item 1 (Test group 7):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%),
Reference item 1- Test group 7 (10mg/m3) (Zinc oxide T0242) : Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
--> These effects were observed as treatment-related, adverse effects

GROSS PATHOLOGY

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 7 males and 10 females
--> These effects were observed as treatment-related, adverse effects
Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female Animals

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
No findings

Recovery animals:
No findings


Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

In males and females of the two reference items, similar findings were observed as described for test group 3 and 6 (test item 1 and 2, 2 mg/m³). Only the severity was slightly higher when compared with the other test items, especially in test group 7 animals.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

The males and females exposed to the reference items showed similar findings as the animals exposed to the test substances.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
Females of test group 7 (reference item 1, 10 mg/m³) and males and females of test group 8 (reference item 2, 22 mg/m³) revealed the same findings in the nasal cavity.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in BAL of males and females of test group 7 (10 mg/m3 Zinc oxide T0422) total cell counts as well as absolute and relative neutrophil cell and monocyte counts and absolute lymphocyte counts were significantly increased whereas relative macrophage counts were significantly decreased. Additionally, in males of this test group absolute macrophage and eosinophil counts (not significantly) were increased. These alterations were regarded as treatment related and adverse.

Recovery animals:
After the 8-week recovery period, no changes were observed in BAL cytology of males and females of test group 27 (10 mg/m3 Zinc oxide T0422).

Proteins/enzymes:

Parental animals:
After the administration period, in BAL of males and females of test group 7 (10 mg/m3 Zinc oxide T0422) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas β -N-Acetyl glucosaminidase (NAG) activity in males and γ-Glutamyl-transferase (GGT) activity in both sexes were marginally but also significantly increased. These alterations were regarded as treatment related and adverse.
Additionally, in females of test group 7 (10 mg/m3 Zinc oxide T0422) NAG activity was also significantly increased, but the change was below 2fold and therefore it was regarded as maybe treatment related but non-adverse.


Recovery animals:
After the 8-week recovery period, in males of test group 27 (10 mg/m3 Zinc oxide T0422) total protein levels and NAG activity were marginally but significantly increased. However, the small increases (below 2fold) of both parameters and no changed BAL cytology counts among these individuals indicated that the total protein and NAG changes were rather incidental than treatment related.

OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. In all other examined organs, the zinc level was comparable with the control.

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

9.68 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested target concentration of 10 mg/m³

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

9.68 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry ; increased neutrophils and lymphocytes in blood at the target conc of 10 mg/m3

Critical effects observed:

yes

Lowest effective dose / conc.:

9.68 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.

To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 

Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.

The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).

All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.

Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).

To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Reference 6

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 November 2020 - ...June 2022

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

The study presented herein is a guideline study with a major deficiency under GLP conditions. Only one concentration level was tested.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (uncoated/coated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name of substance: Zinc sulfate monohydrate
Reference substance No.: 20/0420-1
Batch identification: 201126
Content: Zinc: 36.2 weight-%
Storage stability: Dec 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility
Storage conditions: Room temperature
Appearance - physical state / color solid / white
By Products Chlorides: 0.32 weight-%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.86 - <= 2.74 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 1.86- 2.74 μm (geometric standard deviation = 1.99-2.01)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

21.92 mg/m³ air (analytical)

Remarks:

SD: 1.30 mg/m3, target concentration: 22 mg/m³: Test Group 8 (Parental animals F0); Test Group 18 (male animals for particle detection); Test Group 28 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for main groups 0 (air control) versus 7 (micro ZnO) as well as 0 versus 8 (Zn sulphate), and recovery groups 20 (air control) versus 23 (T0420), 20 versus 26 (T0421), 20 versus 27 and 20 versus 28
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> Student's t-test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+)
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one-sided-)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: WILCOXON-test (two-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians
-Weight of the anesthetized animals and absolute and relative organ weights
--> WILCOXON test (two-sided)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 2 (test groups 8, 18 and 28):
Eleven of the thirteen male animals of test group 8 showed salivation during exposure period on/or study days 4 - 89. In seven male animals of this group respiration sound was noted in addition. Two male animals (Nos: 136 + 138) of this group sparse fur (study day 79 – 84) were noted in addition. Salivation was also noted in three of the three male animals in test group 18.
Salivation was also noted in four of the five male animals in test group 28 during exposure period on/or study days 4 - 89. Respiration sound was observed in three of the five male rats of this group.
--> During exposure period, salivation and respiration sounds were detected in several male and female animals.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes of body weight were determined in male
animals:
- Test group 8: day 18: 329.9g (p< 0.05), whereas the control group was 345.9g
- Test group 8: day 25: 342.5g (p< 0.05), whereas the control group was 363.4g
- Test group 8: day 32: 354.2g (p< 0.01), whereas the control group was 379.4g
- Test group 8: day 39: 364.7g (p< 0.01), whereas the control group was 390.9g
- Test group 8: day 46: 370.4g (p< 0.05), whereas the control group was 394.9g
- Test group 8: day 53: 381.2g (p< 0.05), whereas the control group was 407.9g
- Test group 8: day 60: 387.4g (p< 0.01), whereas the control group was 416.7g
- Test group 8: day 67: 393.1g (p< 0.01), whereas the control group was 424.6g
- Test group 8: day 74: 397.1g (p< 0.01), whereas the control group was 431.2g
- Test group 8: day 81: 406.7g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 88: 411.8g (p< 0.01), whereas the control group was 442.2g
- Test group 8: day 92: 411.7g (p< 0.01), whereas the control group was 447.0g
- Test group 8: day 102: 383.6g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 109: 395.7g (p< 0.01), whereas the control group was 444.5g
- Test group 8: day 116: 399.7g (p< 0.01), whereas the control group was 449.3g
- Test group 8: day 123: 402.7g (p< 0.01), whereas the control group was 452.1g
- Test group 8: day 130: 404.4g (p< 0.01), whereas the control group was 459.9g
- Test group 8: day 137: 410.5g (p< 0.01), whereas the control group was 459.4g
- Test group 8: day 144: 410.6g (p< 0.01), whereas the control group was 463.8g
- Test group 8: day 146: 410.8g (p< 0.01), whereas the control group was 466.6g
- Test group 28: day 18: 327.3g (p< 0.05), whereas the control group was 344.0g
- Test group 28: day 25: 339.2g (p< 0.01), whereas the control group was 360.9g
- Test group 28: day 32: 348.7g (p< 0.01), whereas the control group was 375.0g
- Test group 28: day 39: 361.1g (p< 0.01), whereas the control group was 390.9g
- Test group 28: day 46: 371.5g (p< 0.01), whereas the control group was 399.8g
- Test group 28: day 53: 381.5g (p< 0.01), whereas the control group was 411.9g
- Test group 28: day 60: 386.6g (p< 0.01), whereas the control group was 414.4g
- Test group 28: day 67: 392.7g (p< 0.01), whereas the control group was 425.4g
- Test group 28: day 74: 401.5g (p< 0.01), whereas the control group was 429.7g
- Test group 28: day 81: 410.9g (p< 0.01), whereas the control group was 439.5g
- Test group 28: day 88: 417.2g (p< 0.01), whereas the control group was 446.1g
- Test group 28: day 92: 417.9g (p< 0.01), whereas the control group was 449.2g
- Test group 28: day 102: 423.0g (p< 0.05), whereas the control group was 450.4g
- Test group 28: day 109: 434.0g (p< 0.05), whereas the control group was 457.6g
The following statistically significant changes of body weight were determined in female
animals:
- Test group 8: day 93: 238.9g (p< 0.05), whereas the control group was 251.8g
- Test group 28: day 18: 198.4g (p< 0.01), whereas the control group was 213.1g
- Test group 28: day 25: 209.8g (p< 0.05), whereas the control group was 220.9g
- Test group 28: day 32: 212.5g (p< 0.01), whereas the control group was 228.2g
- Test group 28: day 60: 231.2g (p< 0.05), whereas the control group was 245.6g
- Test group 28: day 116: 251.6g (p< 0.05), whereas the control group was 264.2g

The following statistically significant body weight changes were determined in male animals:
- Test group 8: day 0-> 4: 4.7 (p< 0.01), whereas the control group was 10.4g
- Test group 8: day 11-> 18: 17.1 (p< 0.05), whereas the control group was 22.0g
- Test group 8: day 18-> 25: 12.6 (p< 0.01), whereas the control group was 17.5g
- Test group 8: day 25-> 32: 11.7 (p< 0.05), whereas the control group was 16.1g
- Test group 8: day 74-> 81: 9.6 (p< 0.01), whereas the control group was 5.7g
- Test group 8: day 88-> 92: -0.1 (p< 0.01), whereas the control group was 4.8g
- Test group 8: day 102-> 109: 12.1 (p< 0.05), whereas the control group was 7.6g

- Test group 18: day 74-> 81: 7.3 (p< 0.05), whereas the control group was 0.9g

The following statistically significant body weight changes were determined in female animals:
- Test group 28: day 0-> 4: 5.3 (p< 0.01), whereas the control group was 10.3g
- Test group 28: day 11-> 18: 7.3 (p< 0.01), whereas the control group was 17.7g

-> Retarded body weight development in male/female animals as treatment-related, adverse effects

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in male animals:
•Test group 8: day 0 - 4: +21.3 g (p≤ 0.01), whereas the control group was +24.4 g
• Test group 8: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The following statistically significant changes of mean food consumption were determined in female animals:
Test group 8: day 4 - 11: +16.5 g (p≤ 0.01), whereas the control group was +17.6 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse. The finding in test group 8 was considered not biologically relevant due to its transient nature.

Food efficiency:

not examined

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

/

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Increased hemoglobin values in males of test group 8 (22 mg/m3 Zinc sulfate)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of administration in males of test group 8 (22 mg/m3 Zinc sulfate) total bilirubin values were significantly increased, but this was the only relevantly changed clinical chemistry parameter among these individuals. This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test groups 8 (22 mg/m3 Zinc sulfate)
After the 8-week recovery period, in males of test group 28 (10 mg/m3 Zinc oxide 22 mg/m3 Zinc sulfate) creatinine values were significantly lower compared to controls. However, total bilirubin values were within historical control ranges
(males, total bilirubin 1.34-2.07 μmol/L) whereas creatinine values were marginally below this range (males; creatinine 31.8-37.0 μmol/L). Therefore, total bilirubin increase was regarded as incidental and not treatment related whereas creatinine decrease was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.

Overall motor activity (summation of all intervals):
there were no statistically significant deviations from the control group 0.

Immunological findings:

not examined

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%),
Reference item 2- Test group 8 (22mg/m3) (Zinc sulfate) :
Increase of absolute/relative lung weights in males (125%/138%) and females
(114%/119%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
•Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 5 males and 8 females--> These effects were observed as treatment-related, adverse effects
Test group 28 (Recovery group , Zinc sulfate monohydrate)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³):
Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the
epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9
females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium (nasal
cavity, level IV, exemplarily) in all males and all females

Test group 28 (Recovery group (Zinc sulfate monohydrate):
Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis
in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and
3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female
animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1
male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in
4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:
Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes

Test group 28 (Recovery group Zinc sulfate monohydrate)
• No treatment-related adverse findings in lavage

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20): There were no clinical signs and findings different from normal.
-Exposure period, reference item 2 (test groups 8, 18 and 28):
Eleven of the thirteen male animals of test group 8 showed salivation during exposure period on/or study days 4 - 89. In seven male animals of this group respiration sound was noted in addition. Two male animals (Nos: 136 + 138) of this group sparse fur (study day 79 – 84) were noted in addition. Salivation was also noted in three of the three male animals in test group 18.
Salivation was also noted in four of the five male animals in test group 28 during exposure period on/or study days 4 - 89. Respiration sound was observed in three of the five male rats of this group.
--> During exposure period, salivation and respiration sounds were detected in several male and female animals.


BODY WEIGHT AND WEIGHT GAIN
The following statistically significant changes of body weight were determined in male
animals:
- Test group 8: day 18: 329.9g (p< 0.05), whereas the control group was 345.9g
- Test group 8: day 25: 342.5g (p< 0.05), whereas the control group was 363.4g
- Test group 8: day 32: 354.2g (p< 0.01), whereas the control group was 379.4g
- Test group 8: day 39: 364.7g (p< 0.01), whereas the control group was 390.9g
- Test group 8: day 46: 370.4g (p< 0.05), whereas the control group was 394.9g
- Test group 8: day 53: 381.2g (p< 0.05), whereas the control group was 407.9g
- Test group 8: day 60: 387.4g (p< 0.01), whereas the control group was 416.7g
- Test group 8: day 67: 393.1g (p< 0.01), whereas the control group was 424.6g
- Test group 8: day 74: 397.1g (p< 0.01), whereas the control group was 431.2g
- Test group 8: day 81: 406.7g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 88: 411.8g (p< 0.01), whereas the control group was 442.2g
- Test group 8: day 92: 411.7g (p< 0.01), whereas the control group was 447.0g
- Test group 8: day 102: 383.6g (p< 0.01), whereas the control group was 436.9g
- Test group 8: day 109: 395.7g (p< 0.01), whereas the control group was 444.5g
- Test group 8: day 116: 399.7g (p< 0.01), whereas the control group was 449.3g
- Test group 8: day 123: 402.7g (p< 0.01), whereas the control group was 452.1g
- Test group 8: day 130: 404.4g (p< 0.01), whereas the control group was 459.9g
- Test group 8: day 137: 410.5g (p< 0.01), whereas the control group was 459.4g
- Test group 8: day 144: 410.6g (p< 0.01), whereas the control group was 463.8g
- Test group 8: day 146: 410.8g (p< 0.01), whereas the control group was 466.6g
- Test group 28: day 18: 327.3g (p< 0.05), whereas the control group was 344.0g
- Test group 28: day 25: 339.2g (p< 0.01), whereas the control group was 360.9g
- Test group 28: day 32: 348.7g (p< 0.01), whereas the control group was 375.0g
- Test group 28: day 39: 361.1g (p< 0.01), whereas the control group was 390.9g
- Test group 28: day 46: 371.5g (p< 0.01), whereas the control group was 399.8g
- Test group 28: day 53: 381.5g (p< 0.01), whereas the control group was 411.9g
- Test group 28: day 60: 386.6g (p< 0.01), whereas the control group was 414.4g
- Test group 28: day 67: 392.7g (p< 0.01), whereas the control group was 425.4g
- Test group 28: day 74: 401.5g (p< 0.01), whereas the control group was 429.7g
- Test group 28: day 81: 410.9g (p< 0.01), whereas the control group was 439.5g
- Test group 28: day 88: 417.2g (p< 0.01), whereas the control group was 446.1g
- Test group 28: day 92: 417.9g (p< 0.01), whereas the control group was 449.2g
- Test group 28: day 102: 423.0g (p< 0.05), whereas the control group was 450.4g
- Test group 28: day 109: 434.0g (p< 0.05), whereas the control group was 457.6g
The following statistically significant changes of body weight were determined in female
animals:
- Test group 8: day 93: 238.9g (p< 0.05), whereas the control group was 251.8g
- Test group 28: day 18: 198.4g (p< 0.01), whereas the control group was 213.1g
- Test group 28: day 25: 209.8g (p< 0.05), whereas the control group was 220.9g
- Test group 28: day 32: 212.5g (p< 0.01), whereas the control group was 228.2g
- Test group 28: day 60: 231.2g (p< 0.05), whereas the control group was 245.6g
- Test group 28: day 116: 251.6g (p< 0.05), whereas the control group was 264.2g

The following statistically significant body weight changes were determined in male animals:
- Test group 8: day 0-> 4: 4.7 (p< 0.01), whereas the control group was 10.4g
- Test group 8: day 11-> 18: 17.1 (p< 0.05), whereas the control group was 22.0g
- Test group 8: day 18-> 25: 12.6 (p< 0.01), whereas the control group was 17.5g
- Test group 8: day 25-> 32: 11.7 (p< 0.05), whereas the control group was 16.1g
- Test group 8: day 74-> 81: 9.6 (p< 0.01), whereas the control group was 5.7g
- Test group 8: day 88-> 92: -0.1 (p< 0.01), whereas the control group was 4.8g
- Test group 8: day 102-> 109: 12.1 (p< 0.05), whereas the control group was 7.6g

- Test group 18: day 74-> 81: 7.3 (p< 0.05), whereas the control group was 0.9g

The following statistically significant body weight changes were determined in female animals:
- Test group 28: day 0-> 4: 5.3 (p< 0.01), whereas the control group was 10.3g
- Test group 28: day 11-> 18: 7.3 (p< 0.01), whereas the control group was 17.7g

-> Retarded body weight development in male/female animals as treatment-related, adverse effects

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in male animals:
•Test group 8: day 0 - 4: +21.3 g (p≤ 0.01), whereas the control group was +24.4 g
• Test group 8: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The following statistically significant changes of mean food consumption were determined in female animals:
Test group 8: day 4 - 11: +16.5 g (p≤ 0.01), whereas the control group was +17.6 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse. The finding in test group 8 was considered not biologically relevant due to its transient nature.

HAEMATOLOGICAL FINDINGS:
The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges:
Increased hemoglobin values in males of test group 8 (22 mg/m3 Zinc sulfate)

CLINICAL CHEMISTRY:
At the end of administration in males of test group 8 (22 mg/m3 Zinc sulfate) total bilirubin values were significantly increased, but this was the only relevantly changed clinical chemistry parameter among these individuals. This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test groups 8 (22 mg/m3 Zinc sulfate)
After the 8-week recovery period, in males of test group 28 (10 mg/m3 Zinc oxide 22 mg/m3 Zinc sulfate) creatinine values were significantly lower compared to controls. However, total bilirubin values were within historical control ranges
(males, total bilirubin 1.34-2.07 μmol/L) whereas creatinine values were marginally below this range (males; creatinine 31.8-37.0 μmol/L). Therefore, total bilirubin increase was regarded as incidental and not treatment related whereas creatinine decrease was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).

NEUROBEHAVIOUR:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
Decrease of activity in the female animals of test group 7 (10 mg/m³, reference item 1)
at interval 5 on day 87 (p ≤ 0.05).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

ORGAN WEIGHTS
When compared with control group 0 (=100%),
Reference item 2- Test group 8 (22mg/m3) (Zinc sulfate) :
Increase of absolute/relative lung weights in males (125%/138%) and females
(114%/119%)
--> These effects were observed as treatment-related, adverse effects

GROSS PATHOLOGY

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
•Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 5 males and 8 females--> These effects were observed as treatment-related, adverse effects
Test group 28 (Recovery group , Zinc sulfate monohydrate)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
One female animal of test group 8 (reference item 2, 22 mg/m³) revealed an erosion/ulcer at the base of the epiglottis. All male and all female animals of the same test group revealed squamous metaplasia of the respiratory epithelium, mainly in the region of the base of the epiglottis. This finding is characterized by flattening of cells, increase of cellular layers, and keratinization on the surface. Furthermore, one male and nine females showed mixed (macrophages, neutrophils, lymphocytes) inflammatory cell infiltrates in this region. These findings were regarded to be treatment-related.
Recovery animals:
In the larynx mainly males and females of the reference test groups were affected. The same findings as described for the main group animals were still observed in the recovery animals.
These findings were regarded to be treatment-related.


Lungs:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

In males and females of the two reference items, similar findings were observed as described for test group 3 and 6 (test item 1 and 2, 2 mg/m³). Only the severity was slightly higher when compared with the other test items, especially in test group 7 animals.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

The males and females exposed to the reference items showed similar findings as the animals exposed to the test substances.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.


Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
Females of test group 7 (reference item 1, 10 mg/m³) and males and females of test group 8 (reference item 2, 22 mg/m³) revealed the same findings in the nasal cavity.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period in BAL of males and females in test group 8 (22 mg/m3 Zinc sulfate) total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts were significantly increased whereas relative macrophage counts were significantly decreased. Additionally, in BAL of males in this test group absolute macrophage and eosinophil cell (not significantly) counts were significantly increased. These alterations were regarded as treatment related and adverse.
Recovery animals:
After the 8-week recovery period, in BAL cytology of males and females of test group 28 (22 mg/m3 Zinc sulfate) no changes were observed.

Proteins/enzymes:

Parental animals:
After the administration period, in BAL of males and females of test group 8 (22 mg/m3 Zinc sulfate) total protein levels as well as lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity were moderately, significantly increased whereas γ -Glutamyl-transferase (GGT) activity was only marginally but also significantly increased. These alterations were regarded as treatment related and adverse. In BAL of males of test group 8 (22 mg/m3 Zinc sulfate) β -N-Acetyl glucosaminidase (NAG) activity was marginally, significantly increased in males of this test group, but the change was below 2fold. Therefore, this alteration was regarded as maybe treatment related but nonadverse.
Recovery animals:
After the 8-week recovery period, no changes of total protein and enzyme activities in BAL of males and females in test group 28 (22 mg/m3 Zinc sulfate) were observed.


OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. In all other examined organs, the zinc level was comparable with the control.

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

21.92 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the target concentration of 22 mg/m³

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

21.92 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology at the target conc of 22 mg/m3

Critical effects observed:

yes

Lowest effective dose / conc.:

21.92 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.

To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 

Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.

The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).

All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.

Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).

To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Reference 7

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-30 to 2015-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the OECD Guideline and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2013-12-18

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approx. 7 weeks
- Housing: up to 5 rats per cage (Polysulfon cages). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding. For enrichment wooden gnawing blocks were added.
- Diet: mouse/rat laboratory diet GLP, 10 mm pellets; ad libitum, but not during exposure
- Water: tap water; ad libitum, but not during exposure
- Acclimation period: 3 days

DETAILS OF FOOD AND WATER QUALITY: Food and drinking water analyses showed the food and drinking water used to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15/h ; fully airconditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2012-06-13 To: 2012-07-11

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.8 - <= 2.1 µm

Remarks on MMAD:

MMAD / GSD: please refer to: 'Any other information on materials and methods incl. tables'.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1L/min
- System of generating particulates/aerosols: dust aerosol was generated with the dust generator and compressed air mixed with conditioned dilution air and passed via the cyclonic separator and the dilution tube into the inhalation system.
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 50 ± 20%,
- Air flow rate: 3L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Scattered light photometers (Visguard (Sigrist, Germany) in test group 1 and (RAM1 (Mie, USA); Gravimetrically by filter samples
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
- A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust/aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling with reference to the sample volume of the inhalation atmosphere.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 consecutive days per week, 6 h per day

Dose / conc.:

0.47 mg/m³ air (analytical)

Remarks:

SD: ± 0.20 mg/m³; target concentration: 0.5 mg/m³

Dose / conc.:

1.63 mg/m³ air (analytical)

Remarks:

SD: ± 0.65 mg/m³; target concentration: 1.5 mg/m³

Dose / conc.:

3.01 mg/m³ air (analytical)

Remarks:

SD: ± 1.19 mg/m³; target concentration: 3.0 mg/m³

Dose / conc.:

4.37 mg/m³ air (analytical)

Remarks:

SD: ± 1.31 mg/m³; target concentration: 4.5 mg/m³

No. of animals per sex per dose:

5 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available data, on request of sponsor, concentrations were selected for the study:
4.5 mg/m³: as high concentration causing toxic effects
3.0 mg/m³: as high intermediate concentration
1.5 mg/m³: as mid concentration
0.5 mg/m³: as low concentration and expected NOAEC
- Fasting period before blood sampling for clinical biochemistry: no fasting

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day and once on weekends during exposure period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: At the start of the pre-exposure, at the start of the exposure period and then, as a rule, twice a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Study termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Study termination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: study termination
- Dose groups that were examined: all
- Number of animals: 5 males per test group
- Parameters checked: Cytology: Total cell count, macrophages, lymphocytes, eosinophils, monocytes, non-classified cells. Protein and enzymes: Total protein, GGT, lactate dehydrogenase, ALP, N-acetyl-β-glucosaminidase (NAG BAL).

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- The animals (main groups) were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.
- Organ weights determined: adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, spleen, testes, thymus, and thyroid glands.

HISTOPATHOLOGY: Yes
- Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings in all gross lesions, nasal cavitiy (4 levels), larynx (3 levels), trachea, lungs (5 lobes), tracheobronchial lymph nodes, mediastinal lymph nodes, adrenal glands, bone marrow (femur), brain, oesophagus, heart, kidneys, liver, ovaries, seminal vesicles, spinal cord (cervical, thoracic, and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thyroid glands, thymus, and uterus.
- A correlation between gross lesions and histopathological findings was performed.

Statistics:

- Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means. Differences were considered statistically significant if p ≤ 0.05.
- Blood parameters and BAL: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. Differences were considered statistically significant if p ≤ 0.05.
- Weigth parameters in pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. Differences were considered statistically significant if p ≤ 0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the high concentration (4.37 mg/m³), in 4 of the 5 females alopecia was seen, in two of them it was accompanied by injury of the same region, probably caused by scratching. In females, alopecia and injury were observed first on study day 19 in two animals, followed by another two animals on study day 22 and 23. All findings in male and female animals continued until the animals were sacrificed at the end of the study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight (please refer to: 'Attached background material')::
- At 4.37 mg/m³: Males of the main group showed decreased mean body weights on study day 19 (-10 %, p< 0.05) through to study day 28 (-13 %, p<0.01; terminal body weight).

Body weight changes (please refer to: 'Attached background material')::
- At 4.37 mg/m³: Males of the main group showed a reduced body weight gain from study day 19 to 23 (0.6 g while it was 6.5 g in the control, p<0.05) and from study day 26 to 27 (-10.8 g while it was -1.6 g in the control, p<0.01)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- At 4.37 mg/m³: The absolute and relative lung weights were statistically significantly increased in males (118% and 135%, respectively) and females (127% and 130%, respectively), when compared to the negative control group (100%) (please refer to: 'Attached background material').
- At 3.01 mg/m³: The absolute and relative lung weights were statistically significantly increased in males (120% and 126%, respectively) and females (131% and 134%, respectively), when compared to the negative control group (100%).
- At 1.63 mg/m³: The relative lung weight was statistically significantly increased in females (111%), when compared to the negative control group (100%).

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nasal cavity:
- The inhalation of the test substance led to single small (minimal) to multiple large areas (severe) of degeneration/ regeneration in the olfactory epithelium at the septum, the nasoturbinate and/or ethmoid turbinate (please refer to: 'Attached background material'). This finding was observed in 4 out of 5 males and 3 out of 5 females of test group 2 (1.63 mg/m³) as well as in all males and females of test groups 3 (3.01 mg/m³) and 4 (4.37 mg/m³). The severity increased with concentration. The occurrence of degeneration/ regeneration of the olfactory epithelium in males and females of test groups 2 (1.63 mg/m³) to 4 (4.37 mg/m³) was considered to be a consequence of irritant effects of the test substance and adverse.

Lungs:
- In the lungs, alveolar histiocytosis was observed in all male and female animals of test groups 3 (3.01 mg/m³) and 4 (4.37 mg/m³) (please refer to: 'Attached background material'). In contrast to the spontaneously occurring histiocytosis (one/ few foci of alveolar histiocytosis), in these animals, single or few alveolar macrophages were seen in some or many alveoli and were distributed multifocally in all lobes over the whole lung. The alveolar histiocytosis was associated with single or few inflammatory cells showing the same distribution pattern. In addition, granular material, probably test substance, was apparent in alveolar lumina. These findings were correlated with the increased lung weights in these treatment groups. The occurrence of alveolar histiocytosis and inflammatory infiltrates in animals of test groups 3 (3.01 mg/m³) and 4 (4.37 mg/m³) was considered a response to irritant effects of the test substance and was regarded as adverse.
Because the distribution pattern of alveolar histiocytosis and inflammatory cells in one female (no. 64) of test group 2 (1.63 mg/m³) was comparable to that seen in animals of test groups 3 and 4, the occurrence of these findings was also considered to be treatment-related and adverse.
- Although there was no clear histopathological correlate for the increased mean relative weight of the lungs in females of test group 2 (1.63 mg/m³), the weight increase was regarded to be treatment-related considering the changes found in BAL. The minimal or slight activation of the tracheobronchial lymph nodes (lympho-reticulocellular hyperplasia) in 2 out of 5 males and all females of test group 3 (3.01 mg/m³) and in all males and females of test group 4 (4.37 mg/m³) was considered as reaction to the inflammatory process in the lungs.

Tracheobronchial lymph nodes:
A minimal or slight lympho-reticulocellular hyperplasia was observed in 2 males and all females of test group 3 (3.01 mg/m³), as well as in all males and females of test group 4 (4.37 mg/m³). In males and females of test groups 3 and 4, the lympho-reticulocellular hyperplasia might be seen as response to the inflammatory process observed in the lungs and was therefore regarded to be treatment-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL):
- After 4 weeks of inhalation, in males of the satellite test groups 31 and 41 (3.01 and 4.37 mg/m³) total BAL cell counts (not statistically significant in test group 31) as well as absolute neutrophil, lymphocyte and monocyte cell counts were increased (please refer to: 'Attached background material'). Absolute monocyte and neutrophil counts were not dose-dependently altered between these two test groups. Additionally, in males of the satellite test group 21 (1.63 mg/m³) absolute neutrophil and monocyte cell counts were relevantly higher compared to controls. Absolute macrophage counts were increased only in males of the satellite test group 41 (4.37 mg/m³). Higher non classified cells in the satellite test groups 21 up to 41 (1.63; 3.01, 4.37 mg/m³) were degraded cells which could not be categorized.
- The alterations in the absolute cell counts were also reflected in changes of the relative BAL cell counts: increased relative lymphocyte, neutrophil, monocyte and non-classified cell counts in the satellite test groups 31 and 41 (3.01 and 4.37 mg/m³) and additionally increased relative neutrophil, monocyte and non-classified cell counts in the satellite test group 21 (1.63 mg/m³). Relative monocyte and neutrophil counts were not dose-dependently altered between the satellite test group 31 and 41. Relative macrophages counts were decreased in the satellite test groups 21 up to 41 (1.63, 3.01 and 4.37 mg/m³), but also not dose-dependently between test groups 31 and 41.
- In rats of the satellite test groups 21 up to 41 (1.63; 3.01 and 4.37 mg/m³), total protein content as well as lactate dehydrogenase (not statistically significant in test group 31) and alkaline phosphatase activities in BAL were increased. However, the increases of all three parameters in the satellite test groups 21 (1.63 mg/m³) were below a 2-fold increase of the historical control ranges (total protein: 18-64 mg/L; LDH BAL 0.19-0.50 μkat/L; ALP BAL 0.23-0.87 μkat/L. Therefore, these alteration in test group 21 were regarded as treatment-related, but not adverse.
- N-acetyl-β-glucosaminidase and γ-glutamyltransferase activities in BAL of rats of the satellite test groups 31 and 41 (3.01 and 4.37 mg/m³) were increased. However, the increases were not dose-dependent and the means were below or only about 2-fold higher compared to historical controls (NAG BAL 7-45 nkat/L; GGT BAL 0-41 nkat/L, PART III). Therefore, these alteration in test groups 31 and 41 (3.01 and 4.37 mg/m³) were regarded as treatment-related but not adverse.

Details on results:

MORTALITY:
- No deaths were recorded throughout the study

CLINICAL SIGNS:
- During the pre-exposure period the animals showed no clinical signs and findings different from normal.
- During the exposure period the male and female animals of the control group, low concentration (0.47 mg/m³) and intermediate concentration (3.01 mg/m³) groups showed no clinical signs and findings different from normal. In 2 of the total 10 male animals (5 main and 5 satellite group animals) of the low intermediate group (1.5 mg/m³), alopecia was observed in the ear region starting from study day 20 and 21. In one of them injury in the same region was seen, probably due to scratching. As this finding in males was only observed in test group 2, it is considered incidental due to lack of relation-ship to the exposure concentrations.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight:
- At 0.47 mg/m³: mean body weights decreased on study day 19, (-9 %, p< 0.05), 27 (-10 %, p< 0.05) and 28 (-10 %, p< 0.05). The significantly decreased body weight in test group 1 male animals is considered as incidental because the body weight of the satellite animals exposed to the same concentration of ZnO, as well as those of group 2 and 3 were all comparable to the control.
- The mean body weights of the test substance exposed female animals and male satellite group animals were not statistically significantly different from the control group 0.

Body weight gain:
In male animals of the main groups, following statistically significant changes were observed, when compared with the control.
- Test group 1 (0.47 mg/m³) from study day 5 to 9 (-1.1 g while it was 7.7 g in the control,
p<0.05)
- Test group 2 (1.63 mg/m³) from study day 5 to 9 (-2.4 g while it was 7.7 g in the control,
p<0.05)
- Test group 2 (1.63 mg/m³) from study day 19 to 23 (0.0 g while it was 6.5 g in the control,
p<0.05)
The changes in test group 1 and 2 are considered as incidental due to lack of concentration response relationship.

FOOD CONSUMPTION
No substance-related changes of food consumption were observed during the whole study period.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

ORGAN WEIGHT INCLUDING ORGAN / BODY WEIGHT RATIOS:
- The terminal body weight was significantly decreased in males of test group 4 (4.37 mg/m³) resulting in increased relative brain and testes weights.
- The reduced terminal body weight in males of test group 1 (0.47 mg/m³) resulted in increased relative weights of adrenal glands, brain, and testes. Because there was no concentration-response relationship, the reduced terminal body weight in test group 1 was considered to be incidental.
- The increased relative weights of adrenal glands and testes in males of test group 2 (1.63 mg/m³) were related to the slightly decreased terminal body weight (-6%) in this test group.
- For the increased weights of thyroid glands in females of test groups 2 (1.63 mg/m³), 3 (3.01 mg/m³), and 4 (4.37 mg/m³), a treatment related effect could not be ruled out.

GROSS PATHOLOGY
No test item-related findings were observed.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- A minimal or slight lympho-reticulocellular hyperplasia was observed in 2 males of test group 2 (1.63 mg/m³). Because the 2 males (animal nos. 12 and 15) of test group 2 with minimal lympho-reticulocellular hyperplasia of the tracheobronchial lymph nodes did not show any finding in the lungs, a treatment-related effect seemed rather unlikely.
- For the increased weights of thyroid glands in females of test group 2 (1.63 mg/m³), 3 (3.01 mg/m³) and 4 (4.37 mg/m³), a treatment related effect could not be ruled out, but because there were no histopathological correlates, the weight increase was regarded as nonadverse.

Key result

Dose descriptor:

NOAEC

Effect level:

0.47 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

A 28-day repeated dose inhalation toxicity study was conducted to evaluate the effects of microscaled ZnO in rats using nose-only exposure according to the OECD Guideline 412 in compliance with GLP. In this study, male/female Wistar rats were exposed (nose only) at 0.5, 1.5, 3.0 and 4.5 mg/m³ (analytical concentration: 0.47, 1.63, 3.01, and 4.37 mg/m³) microscaled ZnO. Fresh air treated animals served as concurrent control.
Inhalation exposure of 4.37 mg/m³ ZnO for 28 days (20 exposure days) caused alopecia in ear region of female animals and impaired the body weight development in males. In bronchoalveolar lavage fluid, neutrophils and other cytological and biochemical parameters were changes significantly in animals exposed to 1.63 mg/m³ and higher. At 3.01 and 4.37 mg/m³ significantly increased absolute and relative lung weight was found. Histological examination revealed degeneration/regeneration of the olfactory tract in nasal cavity (level II to IV). In accordance with the findings in lavage fluid and the increased lung weight, histology of the lung reveals multifocal alveolar histiocytosis which were associated with single or few inflammatory cells.

Based on the above-mentioned findings, the No Observed Adverse Effect Concentration (NOAEC) was 0.47 mg/m³ under the current study condition.

The study was conducted according to the OECD Guideline and is considered to be reliable without restriction.

Reference 8

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-05 to 2013-12-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study with no restriction performed under GLP conditions. The deficiencies were restricted to the exclusive use of male rats only and missing ophthalmoscopic examinations.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

(additional endpoints additional endpoints (bronchoalveolar lavage, electron microscope analysis, toxicokinetics, genotoxicity) to address nanoparticle-specific aspects of toxicity)

Deviations:

yes

Remarks:

only male rats were used; no ophthalmoscopy

Principles of method if other than guideline:

Additional endpoints: bronchoalveolar lavage, electron microscope analysis (non-GLP), toxicokinetics (non-GLP); genotoxicity; three doses of the test substance (coated ZnO nanomaterial) were compared to one dose of the reference substances (non-coated and nanoscaled ZnO)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2011-08-15

Limit test:

no

Species:

rat

Strain:

other: Crl:WU

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfill the criteria stated by a U.S. EPA Workshop (Vu et al., 1996) such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs which might interfere with the scheduled examinations.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: Approx. 8 weeks
- Weight at study initiation: Approx. 230 g (males), approx. 165 g (females)
- Fasting period before study: No
- Housing: 2 rats per cage in Makrolon® (polycarbonate) cages type III; absorbing softwood bedding
- Diet: commercial chow in pellet form; ad libitum
- Water: tap water; ad libitum
- Acclimation period: 1 d followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 15%
- Air changes: Fully airconditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2009-10-13 To: 2010-02-12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.64 - <= 0.75 µm

Remarks on MMAD:

MMAD / GSD: Particle size: MMAD was checked using a Marple impactor. The MMAD (0.64-0.75 µm) of the aerosol entering the exposure units was < 3.0 μm (the
critical value for respirability of an aerosol in rats) and suggest agglomeration or aggregation of the nanoparticles. Z-COTE HP1: 0.5 mg/m³ (MMAD: 0.75; GSD: 2.87); 2 mg/m³ (MMAD: 0.64; GSD: 3.22), and 8 mg/m³ (MMAD: 0.70; GSD: 3.71).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1L/min
- System of generating particulates/aerosols: Feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 55 ± 15%,
- Air flow rate: 1L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrically by filter samples, feed back loop to actual aerosol concentrations measured by an aerosol photometer
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Computerized control with feed back loop to actual aerosol concentrations measured by an aerosol photometer, comparison to gravimetric concentrations estimated from filter samples at breathing zone. The target aerosol concentrations of 0.5, 2 and 8 mg Z-COTE HP1/m³ were achieved to 98%, 98%, and 103%, respectively.

Duration of treatment / exposure:

14 days (10 days exposure)

Frequency of treatment:

5 consecutive days per week, 6 h per day

Dose / conc.:

0.5 mg/m³ air (analytical)

Remarks:

SD: ± 0.05 mg/m³ (gravimetric analysis); 0.49 ± 0.06 mg/m³ (photometer); target concentration: 0.5 mg/m³

Dose / conc.:

1.96 mg/m³ air (analytical)

Remarks:

SD: ± 0.38 mg/m³ (gravimetric analysis); 1.95 ± 0.38 mg/m³ (photometer); target concentration: 2 mg/m³

Dose / conc.:

8.83 mg/m³ air (analytical)

Remarks:

SD: ± 0.77 mg/m³ (gravimetric analysis); 8.27 ± 0.73 mg/m³ (photometer); target concentration: 8 mg/m³

No. of animals per sex per dose:

45 males per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on dose range finding study (Creutzenberg 2009)
- Rationale for animal assignment: The animals were allocated to groups on a body weight basis. The animals were weighed, randomized and grouped by the PROVANTIS system (management of toxicology laboratory data, Instem Computer System Ltd., Walton Industrial Estate, Stone, Staffs, ST 15 OLT, Great Britain, ProvantisTM version 8.2.0.1). This process ensures that all animals in the male and female groups had body weights deviating not more than approx. 10% from the mean weight across those groups, at that time.
- Rationale for selecting satellite groups: Additional endpoints genetic toxicology, bronchoalveolar lavage, toxicokinetics, electron microscope analysis
- Post-exposure recovery period in satellite groups: 14 days
- Fasting period before blood sampling for clinical biochemistry: 16-hour fasting period

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day and once on weekends during exposure period

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during exposure period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption for each dose group determined and mean daily diet consumption calculated as g food/day, weekly observations

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: First day after end of exposure period
- Anaesthetic used for blood collection: Yes (Halothane)
- Animals fasted: Yes (16 hours, water ad libitum)
- How many animals: 10 animals per dose group
- Parameters checked: Red blood cells (RBC), total white blood cells (WBC), haemoglobin (HB), differential white cell count (% and absolute), haematocrit (HCT), platelets (PTL), mean cell volume (MCV), mean haemoglobin/erythrocyte (MCH), mean haemoglobin concentration/erythrocyte (MCHC), and prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: First day after end of exposure period
- Animals fasted: Yes (16 hours, water ad libitum)
- How many animals: 10 animals per dose group
- Parameters checked: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), urea, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB (A/G), glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K), and phosphate (PO4).

URINALYSIS: Yes
- Time schedule for collection of urine: First day after end of exposure period
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes (16 hours, water ad libitum)
- Parameters checked: Appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, and leukocytes were measured semi-quantitatively (5 males per dose group).

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1 and 14 d after end of exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats per time point and group
- Parameters checked: Cytological parameters: total cell count, viability test (giving percentage of alive leukocytes among the total number of cells), differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions); Biochemical parameters: lactic dehydrogenase (LDH), β-glucuronidase, and total protein; Reactive oxygen intermediates (ROI); Cytokines: TNF-α, IL-6, IL-8, and TGF-β).

LUNG BURDEN: Yes
- Time schedule for analysis: 1 and 14 d after end of exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats per time point and group
- Parameters checked: zinc level

OTHER:
- Toxicokinetics according to OECD TG 417, chemical Zn analysis in organs and urine (1 and 14 d after end of exposure period)
- Electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kidney, liver, spleen, and erythrocytes (1 and 14 d after end of exposure period)

Sacrifice and pathology:

GROSS PATHOLOGY:
- Day 1 and Day 14 after end of exposure
- All animals
- The following organs were trimmed, and wet weights were recorded: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, lung, and heart.
- The respiratory tract was preserved as follows: Nasal passages (including nasal -associated lymphoid tissue-NALT), larynx, trachea, lungs, and LALN (mediastinal and tracheobronchial). All tissues listed in OECD Guideline no. 412 including the [bracketed] organs in Table 2 of this guideline (the eyes will be examined in situ [within the orbital]) and additionally the epididymides were prepared for histopathology.

HISTOPATHOLOGY:
- Day 1 and Day 14 after end of exposure
- Full histopathology on the respiratory tract and other organs and tissues, as listed in OECD 412 (adopted on 07-Sep-09) of all animals in the clean air control group (dose group 1), the Z-COTE HP1 high dose group (dose group 4), and of all animals that died or were killed during the study.
- Histopathology of lung lobes, including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of all groups.
- Trimming of lungs: 3 sections; nose 4 sections
- In the clean air control and the high dose groups, the other organs listed above (Gross histopathology) were also included in the histopathological examination. In the low and mid dose groups (no. 2 and 3), other organs than the respiratory tract were not included as no treatment related macroscopical findings occurred.
- Lungs were fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with haematoxylin and eosin (H & E).

Other examinations:

- Bronchoalveolar lavage (BAL) (1 and 14 days after end of exposure period: cell count, biochemical parameters, lung wet weight)
- Toxicokinetics according OECD TG 417, chemical Zn analysis in organs and urine (1 and 14 days after end of exposure period)
- Electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kidney, liver, spleen, and erythrocytes (1 and 14 d after end of exposure period)
- Oxidative damage: Immunohistochemical detection of 8-OH-dG in lung tissue (1 and 14 days after end of exposure period)
- Genotoxicity: Mammalian erythrocyte micronucleus test using bone marrow of exposed rats (1 d after end of exposure period); modified Comet assay in BAL cells (1 and 14 d postexposure)
- Measurement of cytokines in BAL cells (1 and 14 d postexposure)

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's Test. The statistical evaluation of the histopathological findings was done with the two-tailed Fisher Test by Provantis system.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Results of the 1-day post-exposure observation period:
- Nasal and Paranasal Cavities: Two of 5 males of the high-dose (8.83 mg/m³) Z-COTE HP1 showed (multi)focal very slight to slight degeneration of the olfactory epithelium (level 2 to 5 of the nasal cavity sections).
- Lungs: (Multi)focal very slight accumulation of particle-laden macrophages was observed in 2/5 and 1/5 males of the 0.5 mg/m³ and 1.96 mg/m³ Z-COTE HP1 group, respectively, while all (5/5) males of the 8.83 mg/m³ Z-COTE HP1 showed this change at a slight degree of severity. (Multi)focal very slight to slight bronchiolo-alveolar hyperplasia, mainly of the bronchiolar type (=alveolar bronchiolisation) was observed in a single male of the 0.5 mg/m³ Z-COTE HP1 group as well as in 3/5 males of the 8.83 mg/m³ Z-COTE HP1. All males (5/5) of the 8.83 mg/m³ exposure group and a single rat of the 0.5 mg/m³ Z-COTE HP1 group showed (multi)focal very slight to slight interstitial mononuclear or (mixed) inflammatory cell infiltration. In addition, all males of the 8.83 mg/m³ exposure group were affected by (multi)focal very slight alveolar granulocyte infiltration. A further test substance-related finding, multifocal very slight to slight alveolar lipoproteinosis which was mainly caused by decay of alveolar macrophages, occurred exclusively in all (5/5) males of the 8 mg/m³ Z-COTE HP1 group.

Results of the 14-day post-exposure observation period:
- Lungs: (Multi)focal very slight (minimal) accumulation of particle-laden macrophages was diagnosed in a single male (1/5) of the 0.5 mg/m³ Z-COTE HP1 group, in 5/5 males each of the 1.96 mg/m³ Z-COTE HP1 and the 8.83 mg/m³ Z-COTE HP1, respectively. All of the adverse effects seen at the 1-day postexposure timepoint (bronchiolo-alveolar hyperplasia, inflammatory cell infiltration and alveolar lipoproteinosis) had reversed completely.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
- At 8.83 mg/m³ Z-COTE HP1: On post-exposure day 1, the proportion of polymorphonuclear neutrophils was statistically significantly increased (146.3-fold) and the proportion of macrophages decreased (-59.2%), when compared to the control group (please refer to: 'Attached background material'). Moreover, the absolute macrophage count was statistically significantly increased.

Biochemical parameters:
- At 8.83 mg/m³ Z-COTE HP1: On post-exposure day 1, the activity of lactic dehydrogenase (7.4-fold), and ß-glucuronidase (11.3-fold) as well as the total protein concentration (4.2-fold) were statistically significantly increased, when compared to the control group.

ROI:
- Comparing the treatment groups, no significant difference was observed in the animals. Reflecting the results at day 1, significant increased ROI secretions were observed in the 0.5 and 1.96 mg/m³ Z-COTE HP1 treated animals compared to the clean air treated control group. A significantly decreased ROI production was measured in the 8.83 mg/m³ ZCOTE HP1 group compared to the controls. At day 14, no significant changes were observed between all groups. However, a slight increase of the spontaneous ROI secretion was measured in those groups treated with 1.96 and 8.83 mg/m³ Z-COTE HP1 in an exposure dose related manner. Zymosan induction did not result in statistically significantly effects observed between groups.

Cytokines:
- At day 1, the CINC-1 concentration in BALF of particle exposed animals showed a dose related increase compared to clean air exposure in the 1.96 and 8.83 mg/m³ Z-COTE HP1 treatment groups which was significant at the higher dose. 14 days after end of exposure, no differences between all groups were observed.
- The results show a significant increased secretion of IL-6 into BALF after exposure to 1.96 or 8.83 mg/m³ Z-COTE® HP1 at day 1. This result was also observed at day 14 of the recovery period indicating an ongoing inflammatory reaction.
- TGF-β was significantly increased in all three groups treated with Z-COTE HP1 in concentrations of 0.5, 1.96, or 8.83 mg/m³ at day 1. Measurement at day 14 also showed this result.
- TNF- β was significantly increased in BALF of animals treated with Z-COTE HP1 in concentrations of 1.96 or 8 mg/m³ at day 1 and day 14. Measurement at day 14 also showed this result.

Details on results:

CLINICAL SIGNS AND MORTALITY
- In several rats of all treatment groups and in a few rats of the control group very slight red-brown coloured noses and brown-red encrusted eyelids were seen on some days directly after the 6 h exposure period (short time findings). These are temporary - probably stress related - findings, often seen in rodent nose-only inhalation studies, which disappear directly after the end of the daily restrainment/exposure.
No further clinical effects were reported. No mortality was observed.

BODY WEIGHT AND WEIGHT GAIN
- Statistically significant changes were not observed in the treatment groups as compared to controls.

FOOD CONSUMPTION
- Statistically significant changes as compared to controls were observed only in the low dose Z-Cote HP1 group (+7.4% - +14.1%) increase after end of exposure (please refer to: 'Attached background material'). These significant changes are considered as incidental findings.

HAEMATOLOGY
- The only statistically significant change was observed for the prothrombin time (-13.1%) in the Z-COTE HP1 (please refer to: 'Attached background material'). This finding is considered as an incidental, not-treatment related finding.

CLINICAL CHEMISTRY
No statistically significant changes relative to control detected.

URINALYSIS
No statistically significant changes relative to control detected.

ORGAN WEIGHTS
The absolute and relative organ wet weights did not show statistically significant changes as compared to controls.

GROSS PATHOLOGY
Upon necropsy, test substance- or dose-related macroscopical findings were not observed.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
Results of the 1-day post-exposure observation period:
- Nasal and Paranasal Cavities: Sporadic findings of the nasal and paranasal cavities which were unrelated to exposure included (multi)focal very slight to slight mucous (goblet) cell hyperplasia (level 1 of the nasal cavity sections) affecting mainly the respiratory epithelial lining of the nasal septum, epithelial mononuclear/inflammatory cell infiltration (level 3 to 4 of the nasal cavity sections) and epithelial/subepithelial mineralisation (level 4 to 5 of the nasal cavity sections) in single males of different groups.
- Lung: Spontaneous pulmonary findings which were unrelated to treatment included neuroendocrine cell hyperplasia and osseous metaplasia at low incidences in different groups.
- Other organs: Various sporadic findings were observed in the other organs examined histopathologically. These occurred either incidentally or were similar in distribution pattern and severity in control rats compared to treatment groups. All of them were considered to be without any relation to treatment. The lesions observed in the larynx in up to 2/5 rats per group (squamous-cell metaplasia, granulomatous inflammation, foreign-body granulomas and mononuclear/inflammatory cell infiltration) were related to aspiration and/or inspissation of plant fibres derived from food or bedding material.
Results of the 14-day post-exposure observation period:
- Lung: Spontaneous pulmonary findings which were unrelated to treatment included slight aspiration pneumonia and osseous metaplasia in single males of two groups.
- Other organs: Various sporadic findings were observed in the other organs examined histopathologically. These occurred either incidentally or were similar in distribution pattern and severity in control rats compared to treatment groups. All of them were considered to be without any relation to treatment.

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
- At 0.5 and 1.96 mg/m³ Z-COTE HP1: No statistically significantly effects were observed.

Biochemical parameters:
- At 0.5 and 1.96 mg/m³ Z-COTE HP1: No statistically significantly effects were observed.
- At 8.83 mg/m³ Z-COTE: The total protein level was statistically significantly decreased (-26%) on post-exposure day 14. The decrease was considered to be not toxicologically relevant.

ROI:
- Zymosan induction did not result in statistically significantly effects observed between groups.

TOXICOKINETCS:
- One day after the end of the exposure period, the absolute Zn content was statistically significantly increased to 367% in the lung of the high dose group of Z-COTE HP1 as compared to the clean air control group (please refer to: ‘Attached background material’). In all other organs the Zn levels were very close to the control values. The deposited mass of Z-COTE HP1 in the 14-d exposure period was approx. 290 µg/lung, the analytical results, thus, demonstrating a practically complete dissolution of the retained test item.
- After 14 d of recovery, the Zn content was found to be statistically significantly increased in the lung associated lymph nodes (in the 0.5 mg/m³ Z-COTE HP1 group: +89.3%), brain (in all of the three Z-COTE HP1 groups: +60.4%, +14,4%, and +8.6%), kidney (in the 1.96 mg/m³ Z-COTE HP1: +29.5%), and the lung (in the mid and high dose of Z-COTE HP1: +25.9% and +13.2%). These values were close to controls and not dose-dependent and therefore considered as incidental findings. Overall, no relevant amounts of increased Z-COTE HP1 were detected in any body compartment demonstrating the rapid elimination.

Key result

Dose descriptor:

NOAEC

Effect level:

1.96 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

In this sub-acute nose-only aerosol inhalation study, according to OECD TG 412, eight-week-old male Wistar (Crl:WU) rats (45/group) were acclimatised for one day followed by three weeks of training in nose-only tubes without exposure. The test animals were exposed to Z-COTE HP1 (coated with triethoxycaprylylsilane) aerosol concentration levels of 0.5, 1.96, and 8.83 mg/m³ (analytical) for 6 hours per day and 5 days per week over a period of 14 days (10 exposure days). A clean air control group was concurrently. The test animals were checked once or twice per day for clinical signs. The body weights and food consumption were measured weekly. One day after euthanasia, haematology and clinical biochemistry as well as urinalysis were performed. Gross pathology and comprehensive histopathological examinations were included. Additionally, bronchoalveolar lavage fluid (BALF) analyses were performed 1 and 14 days after the end of the exposure period. Moreover, the study included zinc level measurements in several organs, toxicokinetics, and TEM/EDS analysis.

All test animals survived treatment and were euthanized at scheduled dates. Effects indicating systemic toxicity were not observed. Body weight development did not show any statistically significant changes as compared to concurrent controls. The food consumption showed no relevant changes after exposure to any of the two nanomaterials. The organ weights, clinical biochemistry, and haematology did not show any statistically significant changes as compared to concurrent controls. The bronchoalveolar lavage fluid (BALF) analysis revealed statistically significant increases of polymorphonuclear neutrophils and lactic dehydrogenase, ß-glucuronidase, and total protein levels and also an increase of absolute numbers of macrophages in the Z-COTE HP1 high dose group 1 day after end of exposure. However, all these effects were reversible and had returned to control levels at the 14-day post-exposure sacrifice date. Thus, the Z-COTE HP1 dust showed a strong acute response, however, rapid recovery upon cessation of exposure. The analysis of the oxidative stress related secretion of reactive oxygen intermediates (ROI) and cytokines showed an increased amount of the several endpoints indicating an ongoing inflammatory situation in the lung of the Z-COTE HP1 exposed animals. The secretion of ROI was enhanced in the 0.5 and 1.96 mg/m³ Z-COTE HP1-treated animals as compared to clean air controls. An increased concentration of the stimulatory cytokines CINC-1, tumor necrosis factor-α, interleukin-6 and the more deregulating mediator transforming growth factor-β was measured in the Z-COTE HP1 treated animals. This was observed for all cytokines at day 1 and day 14 except for CINC-1. In TEM analyses of the nanosized particle Z-COTE HP1 treated groups, structures resembling nanoparticles were rarely observed within the cytoplasm of predominantly macrophages in the lung. EDX-analysis could not verify existing ZnO particles. Histopathological examination 1 day after end of exposure revealed in the nasal and paranasal cavities a (multi)focal very slight to slight degeneration of the olfactory epithelium in males of the high-dose Z-COTE HP1 group. In lungs, (multi)focal very slight to slight bronchiolo-alveolar hyperplasia was observed in rats exposed to 8.83 mg/m³. In the same group, (multi)focal very slight to slight interstitial mononuclear or (mixed) inflammatory cell infiltration and (multi)focal very slight alveolar granulocyte infiltration was observed. As a further test substance-related finding which was mainly caused by decay of alveolar macrophages, exclusively in all males of the 8.83 mg/m³ Z-COTE HP1 group, multifocal very slight to slight alveolar lipoproteinosis occurred. Histopathological examination 14 day after end of exposure revealed a full recovery of all treatment-related effects observed before. Chemical analysis of the test item in various organs showed a slight increase of the absolute Zn content (statistically not significant: 110% in liver, kidneys and brain as compared to the clean air control group in the Z-COTE HP1 high dose group (some statistical increases are considered as non-relevant). Only in lungs, a statistically significant increase of the absolute Zn content was found in the Z-COTE HP1 high dose group on day 1 (51 μg ZnO/lung). Statistically significant increases observed on day 14 are very close to control group levels and thus not considered as relevant.

Based on the results, the NOAEC and LOAEC for nanoscale ZnO in male Wistar rats were established at 1.96 and 8.83 mg/m³, respectively.

The study presented herein is a guideline study with no restriction performed under GLP conditions. The deficiencies were restricted to the exclusive use of male rats only and missing ophthalmoscopic examinations.

Reference 9

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-23 to 2013-08-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study without restrictions performed under GLP conditions. The deficiencies of the study are restricted to the lack of female exposure groups and the lack of ophthalmoscopic examinations.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

- adopted on 2009-09-07
- additional endpoints investigated

Deviations:

yes

Remarks:

only male rats were used; ophthalmology not performed

Principles of method if other than guideline:

Additional endpoints: bronchoalveolar lavage, cell proliferation, electron microscope analysis (non-GLP), toxicokinetics (non-GLP); three doses of the test substance (coated ZnO nanomaterial) were compared to one dose of a reference substance (non-coated microscaled ZnO)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(WU)

Details on species / strain selection:

Wistar rats are commonly used in subchronic and chronic inhalation toxicity studies. They fulfill the criteria stated by a U.S. EPA Workshop (Vu et al., 1996) such as (i) a low background rate of neoplasia, (ii) a low background rate of pulmonary disease, (iii) longevity, and (iv) a history of laboratory use. In this study the specified Wistar strain is preferred to the Fischer strain because young Fischer rats available in Germany sporadically show a slight latent inflammation of lungs
which might interfere with the scheduled examinations.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland (Sulzfeld, Germany)
- Age at study initiation: Approx. 8 weeks
- Weight at study initiation: Approx. 230g
- Fasting period before study: No
- Housing: 2 rats per cage, absorbing softwood bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 1 day followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 55 ± 15%
- Air changes: fully airconditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2010-07-26 To: 2011-03-24

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.64 - <= 1.17 µm

Remarks on MMAD:

MMAD / GSD (please refer to: 'Any other information on material and methods incl. tables'): Particle size: MMAD was checked using a Marple impactor. The MMAD (0.64-1.17 µm) of the aerosol entering the exposure units was < 3.0 μm (the critical value for respirability of an aerosol in rats) and suggest agglomeration or aggregation of the nanoparticles.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1L/min
- System of generating particulates/aerosols: Feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 55 ± 15%,
- Air flow rate: 1L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: i) Gravimetrically by filter samples; ii) Actual aerosol concentrations measured by an aerosol photometer. The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant. The ratio between photometer signal and actual concentration was determined throughout the study by comparing to gravimetric concentrations.
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The target aerosol concentrations of 0.3, 1.5 and 4.5 mg Z-COTE HP1/m³ were achieved to 103%, 99%, and 99%, respectively.

Duration of treatment / exposure:

90 days (65 exposure days)

Frequency of treatment:

5 consecutive days per week, 6 hours per day

Dose / conc.:

0.31 mg/m³ air (analytical)

Remarks:

SD: ± 0.03 mg/m³; target concentration: 0.3 mg/m³

Dose / conc.:

1.48 mg/m³ air (analytical)

Remarks:

SD: ± 0.12; target concentration: 1.5 mg/m³

Dose / conc.:

4.45 mg/m³ air (analytical)

Remarks:

SD: ± 0.45 mg/m³; target concentration: 4.5 mg/m³

No. of animals per sex per dose:

65 male rats per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of the DRF study (Fraunhofer ITEM study no. 02G09005) nominal aerosol concentrations of 0.3, 1.5 and 4.5 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment: The animals were allocated to groups on a body weight basis. The animals were weighed, randomized and grouped by the PROVANTIS system (management of toxicology laboratory data, Instem Computer System Ltd., Walton Industrial Estate, Stone, Staffs, ST 15 OLT, Great Britain, ProvantisTM version 8.2.0.8).
- Fasting period before blood sampling for clinical biochemistry: 16-hour fasting period
- Post-exposure recovery period in satellite groups: 1 month recovery period

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed in their cages once a day (twice a day during the exposure period).

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during exposure period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption was recorded weekly during the study period (including post-exposure observation period) using 10 male animals per dose group.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: first day after end of exposure period
- Anaesthetic used for blood collection: Yes (Halothane)
- Animals fasted: Yes (16 hours; water ad libitum)
- How many animals: 10 animals per dose group
- Parameters checked: Red blood cells (RBC), total white blood cells (WBC), haemoglobin (HB), differential white cell count (% and absolute), haematocrit (HCT), platelets (PTL), mean cell volume (MCV), mean haemoglobin/erythrocyte (MCH), mean haemoglobin concentration/erythrocyte (MCHC), prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: first day after end of exposure period
- Animals fasted: Yes (16 hours; water ad libitum)
- How many animals: 10 animals per dose group
- Parameters checked: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT), urea, creatinine (CREA), total protein (TP), albumin (ALB), globulin (GLB), ALB/GLB (A/G), glucose (GLUC), cholesterol (CHOL), sodium (Na), calcium (Ca), potassium (K), phosphate (PO4).

URINALYSIS: Yes
- Time schedule for collection of urine: first day after end of exposure period
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes (16 hours; water ad libitum)
- Parameters checked: Urine specific gravity, urine pH, urine protein, urine glucose, urine ketones, urine bilirubin, urine leukocytes, urine blood, urine nitrite, urobilinogene, and urine weight.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: 1, 8, and 29 d after end of exposure period
- Dose groups that were examined: all
- Number of animals: 5 male rats per time point and group
- Parameters checked: Cytological parameters: total cell count, viability test (giving percentage of alive leukocytes among the total number of cells), differential cell count (inflammatory (PMNs) or immunological (lymphocytes) reactions); Biochemical parameters: lactic dehydrogenase, β-glucuronidase, and total protein.

LUNG BURDEN: Yes
- Time schedule for analysis: 1 and 29 d after end of exposure period
- Dose groups that were examined: all
- Number of animals: 5 rats per group
- Parameters checked: Zn content

OTHER:
- Lung cell proliferation (9 and 29 d after end of exposure period)
- Toxicokinetics according to OECD TG 417, chemical Zn analysis in organs, blood, and urine (1 and 29 d after end of exposure period)
- Electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kidney, liver, spleen, and erythrocytes (1, 8, and 29 d after end of exposure period)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- All animals were subjected to a complete necropsy, which includes careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
- Organ weights: liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, lung, and heart

HISTOPATHOLOGY: Yes
- Full histopathology on the respiratory tract and other organs and tissues, as listed in OECD 413 (adopted on 07-Sep-09) of all animals in the clean air control group (dose group 1), the Z-COTE® HP1 high dose group (dose group 4), and of all animals that died or were killed during the study.
- Histopathology of lung lobes, including bronchi and the lung-associated lymph nodes (LALN, mediastinal and tracheobronchial), trachea, larynx, pharynx and the nasal cavities (including NALT) in all animals of all groups.
- Trimming of lungs: 3 sections; nose 4 sections.
- In the low and mid dose groups (no. 2 and 3), other organs than the respiratory tract were not included as no treatment related macroscopical findings occurred.
- For the animals sacrificed 1-month post-exposure, all tissues were preserved but only those showing changes on day 1 were examined histopathologically.
- Lungs were fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E).

Other examinations:

- Bronchoalveolar lavage (1, 8, and 29 d after end of exposure period: cell count, biochemical parameters, cytokines)
- Lung cell proliferation (9 and 29 d after end of exposure period)
- Toxicokinetics according OECD TG 417, chemical Zn analysis in organs, blood, and urine (1 and 29 d after end of exposure period)
- Electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kideny, liver, spleen, and erythrocytes (1, 8, and 29 d after end of exposure period)

Statistics:

Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's Test. The statistical evaluation of the histopathological findings was done with the two-tailed Fisher Test by Provantis system.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Inorganic phosphate was significantly decreased in males exposed to 1.48, and 4.45 mg/m³ (-8.2% and -7.4%), when compared to controls (please refer to: 'Attached background material'). The decrease was considered to be not toxicologically relevant.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology 1 day after end of exposure revealed in the nasal and paranasal cavities the following test item-related findings (please refer to: 'Any other information on results incl. tables'):
- Between 1/10 and 2/10 males of the ZnO exposure groups only (not observed in the control group) showed (multi)focal very slight to slight mucous cell hyperplasia affecting mainly the respiratory epithelial lining of the nasal septum and the ventral nasal meatus in levels 2 to 3 of the nasal cavity sections.
- Very slight (multi)focal epithelial hyaline (eosinophilic) droplets were markedly increased in the high dose Z-COTE HP1 group (6/10) while in the other groups (including the control group) the incidences of this finding ranged between 2/10 and 3/10 rats per groups. The occurrence/increased severity of the above findings is considered to be test item-related.
- At the end of the recovery period all these lesions were diagnosed as fully reversible.

Histopathology 1 day after end of exposure revealed in lungs the following test item-related findings:
- (Multi)focal very slight to slight accumulation of particle-laden macrophages was observed dose-dependently (all very slight/slight) in the Z-COTE HP1 groups, in 4/10 males (very slight) of the high dose group.
- (Multi)focal very slight bronchiolo-alveolar hyperplasia, mainly of the bronchiolar type (=alveolar bronchiolisation) was observed exclusively in 4/10 males of the Z-COTE HP1 high dose group.
- (Multi)focal very slight alveolar granulocyte infiltration and (multi)focal very slight to slight interstitial mononuclear cell infiltration was diagnosed as exposure-related in the Z-COTE HP1 high dose.
- At the end of the recovery period all these lesions were reduced in severity or fully reversible.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Biochemical parameters (please refer to: 'Attached background material'):
- At 4.45 mg/m³: On post-exposure day 1, the LDH was statistically significantly increased (2.2-fold), when compared to the control group. The effect was reversible and had returned to control levels at the 8-day post-exposure sacrifice.
ROI:
- The measurement of the reactive oxygen species (ROI) produced by alveolar macrophages showed a maximum activation status of the respiratory burst in alveolar macrophage cultures. On PE-1, significantly decreased ROI secretions were observed in the 1.48 (-46%) and 4.45 mg/m³ (-55.9%) Z-COTE HP1 treated animals compared to the control. Including Zymosane stimulation significant increases were detected in the 1.48 and 4.45 mg/m³ Z-COTE HP1 groups after 1 (+19.6% and 17.6%, respectively) and 8 d (15.5% and 24.8%, respectively) with a normalization after 29 d.

TOXICOKINETICS:
On the first day postexposure the Zn content in lungs of animals treated with the Z-COTE HP1 high dose was increased to 180% compared to the control (please refer to: 'Attached background material'). The deposited mass of the test substance in the 90-day exposure period was approx. 2000 µg/lung and the analytical results demonstrated a practically complete dissolution of the retained test substance. No significantly increased amounts of the test item were detected in any other body compartment demonstrating the rapid elimination.

CELL PROLIFERATION:
- The unit length labelling index of the terminal bronchiolar epithelium was significantly decreased at 9 (-50.5%) and 29 (-40.8%) days post-exposure in the
high dose group (please refer to: 'Attached background material'). No indication of an induction of a hyperplastic effect of the test substance or the reference substance was observed.

Details on results:

CLINICAL SIGNS AND MORTALITY
Due to a technical defect of the clean air supply 15/65 rats of group 3 died during the exposure. One animal died during narcosis performed for the administration of BrdU. 4 animals died during the study or were killed in a moribund condition. 2 of them (group 1 and 5) died due to a severe urogenital infection (group 5) and one due to malignant lymphoma while the cause of death remained unclear for the fourth rat (group 1). Therefore, no animal died due to test substance related effects.
Several rats of the treatment group and a few rats of the control group showed very slight red-brown coloured noses and brown-red encrusted eyelids on some days directly after the 6-hour exposure period. These are temporary - probably stress-related - findings often seen in rodent nose-only inhalation studies which disappeared directly after the end of the daily exposure. Generally, the clinical health of all animals was within the normal range seen in rats of this strain and age.

BODY WEIGHT AND WEIGHT GAIN
Group 3 showed statistically significant reduced body weights on Day 28 (-3.1%) and 35 (-2.7%) compared to control (please refer to: 'Attached background material').

FOOD CONSUMPTION
Statistically significant decrease of food consumption as compared to controls was observed on several dates in groups 2 to 4 (please refer to: 'Attached background material').

HAEMATOLOGICAL FINDINGS:
- No test item related findings were observed.
- At 4.45 mg/m³: The segmented neutrophil calculation and proportion (+42% and 26%, respectively) were statistically significantly increased, when compared to controls (please refer to: 'Attached background material'). The proportion of lymphocytes was statistically significantly decreased (-7.8%).
- At 1.48 mg/m³: The haemoglobin concentration was statistically significantly increased (+2.7%), when compared to the control group.
- At 0.31 mg/m³: The haemoglobin concentration was statistically significantly increased (+3%), when compared to the control group.

CLINICAL CHEMISTRY
- No other statistically significant changes were observed .

URINALYSIS
No significant changes were observed.

ORGAN WEIGHTS
- At 0.31 mg/m³: On post-exposure day 1, the absolute and relative weight of the left epididymides (+16.1% and +17.3%, respectively) and left testes (+21.4% and +22.6%, respectively) were statistically significantly increased, when compared to the control group (please refer to: 'Attached background material').
- At 1.45 mg/m³: On post-exposure day 1, the relative weight of the left epididymides (+14.8%) and left testes (+20.4%) were statistically significantly increased, when compared to the control group. Moreover, the absolute lest testes weight was statistically significantly increased by 19%.
- At 4.48 mg/m³: On post-exposure day 1, the absolute and relative weight of the left epididymides (+19.5% and +18.2%, respectively) and left testes (+19.1% and +17.7%, respectively) were statistically significantly increased, when compared to the control group.
- The changes were considered to be not substance related. No other changes were found.

GROSS PATHOLOGY
No test item-related findings were observed.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:
1. Animals sacrificed 1 day after end of the 3-month exposure:
- In the kidneys, up to 5/10 males showed (multi)focal very slight to slight tubular basophilia (basophilic tubules) besides other spontaneous changes. A single control rat (no. 1108) showed a severe unilateral purulent pyelonephritis which was associated with moderate necrosis of the renal papilla, slight hyperplasia of the pelvic epithelium and a slight purulent pyelitis of the other kidney.
- A common spontaneous finding in the liver was (multi)focal very slight to slight microgranuloma(s) (incidence between 6/10 and 7/10 males per group).
- A high incidence of inflammatory lesions of the prostate was diagnosed. Up to 5/10 males per group showed (multi)focal slight to severe purulent inflammation, a single control rat had a severe abscess-forming inflammation and 1/10 males each of groups 1 and 4 were affected by a moderate or severe chronic granulomatous inflammation. Moreover, up to 3/10 males per group showed very slight or slight (multi)focal interstitial mononuclear cell infiltration.
- The incidence of testicular atrophy was unusually high for rats of this strain and age. Eight of ten males each of groups 1, and 4 revealed slight to severe, uni- or bilateral tubular atrophy of the testis with a multifocal or diffuse distribution pattern. This degenerative change was often associated with aspermia, oligospermia and/or atrophy of the epididymides.
- Several further findings in various organs were noted which occurred incidentally and at low frequencies and were not unusual for rats of this strain and age. All these findings were unrelated to treatment.
2. Animals sacrificed 29 days after end of the 3-month exposure:
- In the kidneys, up to 8/10 males showed (multi)focal very slight to slight tubular basophilia (basophilic tubules) besides other spontaneous changes.
- Very slight to slight (multi)focal microgranuloma(s) of the liver were also seen at comparable incidences (between 5/10 and 7/10 males per group) as in the first examination time point.
- The high incidence of inflammatory lesions of the prostate persisted until the end of the recovery period. Up to 7/10 males per group showed (multi)focal very slight to moderate purulent inflammation and up to 2/10 males per group were affected by a moderate or severe chronic granulomatous inflammation.
- The incidence of testicular atrophy also remained at a high level: 4/10 to 6/10 males of groups 1, 3, and 4 showed slight to severe, uni- or bilateral tubular atrophy of the testis with a multifocal or diffuse distribution pattern. This degenerative change was often associated with aspermia, oligospermia and/or atrophy of the epididymides.
- Several further findings in various organs were noted which occurred incidentally and at low frequencies and were not unusual for rats of this strain and age. All these findings were unrelated to treatment.
- The findings described for prostate and testes could be related to the daily 6-hr restraint in the nose-only tubes for exposure.


BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Biochemical parameters:
- At: 1.48 mg/m³: On post-exposure day 8, the β-glucuronidase activity was statistically significantly decreased (-57.1%), when compared to the control group.
ROI:
- At 1.48 mg/m³: On PE-29, significantly increased ROI secretions were observed in the 1.5 mg/m³ (+40.8%) Z-COTE HP1 treated animals compared to the control.

Cytokines:
- No relevant effects were observed.

OTHER FINDINGS
- Electron microscopy: Electron dense structures were found one and 8 days postexposure in the cytoplasm of different cells and free in the lung lining fluid of animals treated with ZnO as well as in animals of the control group. These structures were composed of irregular homogenous to fine granular material which measured only a few nanometers. these structures were found in animals treated with clean air as well as in the Z-Cote HP1 group one and eight days after exposure. Furthermore, these structures were composed of irregular homogenous to fine granular material. The granular structure measured only few nanometer. However, these structures did not resemble nanoparticles similar to Z-Cote HP1. Though similar material has also been found in the clean air treated group, some of these structures found in the Z-Cote HP1 treated group might have been nanoparticles which were solved but still led to a higher metal ion concentration at this spot resulting in a higher electron density.
- Toxicokinetics: The zinc level in the kidneys was statistically significantly decreased in animals exposed to 1.48 and 4.45 mg/m³ Z-COTE HP1 (-15.0% and -14.8%, respectively) on post-exposure day 1, when compared to the control group.

Key result

Dose descriptor:

NOAEC

Effect level:

1.48 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: increased activity of lactate dehydrogenase in BAL and the increased numbers of lymphocytes in the BAL at the highest tested concentration.

Key result

Dose descriptor:

LOAEC

Effect level:

4.45 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

other: increased activity of lactate dehydrogenase in BAL and the increased numbers of lymphocytes in the BAL at the highest tested concentration.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

4.45 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

HISTOPATHOLOGY

Animals sacrified 1 d postexposure:

Effect	Group 1 (control)	Group 2	Group 3	Group 4	Group 5
Nasal and Paranasal Cavities
focal degeneration of the olfactory epithelium					1/10 slight
(multi)focal hyperplasia of the olfactory epithelium					3/10 very slight, 1/10 slight
(multi)focal mucous cell hyperplasia affecting mainly the respiratory epithelium lining the nasal septum		1/10 very slight, 1/10 slight	2/10 very slight	1/10 slight	1/10 very slight, 2/10 slight
(multi)focal epithelial hyaline (eosinophilic) droplets	3/10 very slight	3/10 very slight	2/10 very slight	6/10 very slight	6/10 very slight
Lung
(multi)focal accumulation of particle-laden macrophages		1/10 very slight	10/10 very slight	6/10 very slight, 4/10 slight	4/10 very slight, 6/10 slight
(multi)focal bronchiolo-alveolar hyperplasia , (bronchiolar type)				4/10 very slight	9/10 very slight
(multi)focal bronchial/bronchiolar mucous cell hyperplasia		1/10 slight		2/10 slight	1/10 slight
(multi)focal alveolar granulocyte infiltration				2/10 very slight	5/10 very slight
(multi)focal interstitial mononuclear cell infiltration	1/10 very slight, 1/10 slight	2/10 very slight	3/10 very slight	8/10 very slight, 1/10 slight	6/10 very slight, 4/10 slight
Lung associated lymph nodes
(multi)focal accumulation of particle-laden macrophages		4/10 very slight	4/10 very slight	1/10 very slight	2/10 very slight, 6/10 slight
Lymphoid hyperplasia		1/10 slight	1/10 slight	1/10 slight	3/10 slight, 1/10 moderate

Animals sacrified 29 d postexposure:

Effect	Group 1 (control)	Group 2	Group 3	Group 4	Group 5
Nasal and Paranasal Cavities
(multi)focal mucous cell hyperplasia affecting mainly the respiratory epithelium lining the nasal septum	2/10 very slight, 1/10 slight	1/10 slight	1/10 very slight	2/10 very slight	1/10 very slight, 2/10 slight
(multi)focal epithelial hyaline (eosinophilic) droplets	7/10 very slight	4/10 very slight	2/10 very slight	7/10 very slight	4/10 very slight
Lung
(multi)focal accumulation of particle-laden macrophages			3/10 very slight		3/10 very slight, 1/10 slight
(multi)focal bronchiolo-alveolar hyperplasia , (bronchiolar type)	1/10 very slight	1/10 very slight		1/10 very slight	2/10 very slight
(multi)focal bronchial/bronchiolar mucous cell hyperplasia			1/10 slight	1/10 slight
(multi)focal interstitial mononuclear cell infiltration	3/10 very slight		1/10 very slight	3/10 very slight	2/10 very slight, 2/10 slight
Lung associated lymph nodes
(multi)focal accumulation of particle-laden macrophages		1/10 very slight	3/10 very slight	2/10 very slight	5/10 very slight, 2/10 slight
Lymphoid hyperplasia		1/10 slight		1/10 slight	3/10 slight

At the end of the recovery period all the lesions regarding the nasal and paranasal cavities were diagnosed as fully reversible. The lung effects were reduced in severity or fully reversible at the end of the recovery period. Spontaneous changes like tubular basophilia in the kidney, microgranuloma in the liver, inflammatory prostate lesions and testicular atrophy were found in the ZnO treated animals as well as in control animals in the same extent. In part the incidences of these effects were unusually high for rats of this strain and age. However, these changes were considered to be not test substance-related due to similar incidences in animals of the the control and the treatment groups.

Conclusions:

In this sub-chronic nose-only aerosol inhalation study, according to OECD TG 413, eight-week-old male Wistar (Crl:WI(WU)) rats (65/group) were acclimatised for one day followed by three weeks of training in nose-only tubes without exposure. The test animals were exposed to Z-COTE HP1 aerosol target concentration levels of 0.3, 1.5, and 4.5 mg/m³ (analytical concentrations: 0.31, 1.48, and 4.45 mg/m³) for 6 hours per day and 5 days per week over a period of 90 days (65 exposure days). A clean air control group was concurrently. The test animals were checked once or twice per day for clinical signs. The body weights and food consumption were measured weekly. One day after euthanasia, haematology and clinical biochemistry as well as urinalysis were performed. Gross pathology and comprehensive histopathological examinations were included. Additionally, bronchoalveolar lavage fluid (BALF) analyses were performed 1, 8, 29 days after the end of the exposure period. Moreover, the study included zinc level measurements in several organs, lung cell proliferation analysis, toxicokinetics, and TEM analysis in nasal cavities, lung, trachea, larynx, bronchioles, kidney, liver, spleen, and erythrocytes.

Test substance related findings or losses of animals did not occur. Effects indicating systemic toxicity were not observed. Body weight development did not show any relevant statistically significant changes. Food consumption data show some statistically significant changes, however, these are considered as incidental. Haematology, clinical chemistry and urinalysis data did not show any relevant statistically significant changes as compared to concurrent controls. The organ weight changes observed for the left epididymides and left testes were considered to be not test substance related. Gross pathology revealed no relevant changes. The BALF analyses revealed a statistically significantly increased lactic dehydrogenase activity, when compared to the control group. Moreover, significantly decreased ROI secretions were observed in the 1.48 and 4.45 mg/m³ Z-COTE HP1 treated animals as compared to the clean air treated control group. At days 8 and 29 of recovery these effects had more or less normalised. Including zymosane stimulation statistically significant increases were detected in the 1.48 and 4.45 mg/m³ Z-COTE HP1 groups after 1 and 8 days with a normalisation after 29 days. One day after the last exposure, the histopathological examinations revealed (multi)focal very slight (0/10; 1/10; 2/10; 0/10) to slight (0/10; 0/10; 1/10; 0/10; 1/10) mucous cell hyperplasia affecting mainly the respiratory epithelial lining of the nasal septum and the ventral nasal meatus of animals exposed to Z-COTE HP1. Very slight (multi)focal epithelial hyaline (eosinophilic) droplets were markedly increased in the high dose Z-COTE HP1 group (6/10) while in the other groups (including the control group) the incidences of this finding ranged between 2/10 and 3/10 rats per groups. The occurrence/increased severity of the above findings is considered to be test substance-related. At the end of the recovery period all these lesions were diagnosed as fully reversible. Multi)focal very slight to slight accumulation of particle-laden macrophages was observed dose-dependently (all very slight/slight) in the Z-COTE HP1 groups, in 4/10 males (very slight) of the high dose group. (Multi)focal very slight bronchiolo-alveolar hyperplasia, mainly of the bronchiolar type (=alveolar bronchiolisation) was observed exclusively in 4/10 males of the Z-COTE HP1 high dose group. (Multi)focal very slight alveolar granulocyte infiltration and (multi)focal very slight to slight interstitial mononuclear cell infiltration was diagnosed as exposure-related in the Z-COTE HP1 high dose groups. At the end of the recovery period all these lesions were reduced in severity or fully reversible. The cell proliferation analysis revealed no indication of an induction of a hyperplastic effect of the test substance. Slight lymphoid hyperplasia was observed in all dose groups at post-exposure day 1 (1/10 vs 0/10 in controls) and animals exposed at 0.31 and 4.45 mg/m³ at post-exposure day 29 (1/10 vs 0/10 in controls). The toxicokinetics demonstrated a practically complete dissolution of the retained test item. Overall, no relevant amounts of increased Z-COTE HP1 were detected in any body compartment demonstrating the rapid elimination. The TEM analysis did not detect distinct particles at any time point.

Under the study condition, the NOAEC for the nano-scaled ZnO was assessed, and science-justified to be 1.48 mg/m³.

Only in the high dose Z-COTE HP1 (4.45 mg/m³) group increased incidences of interstitial mononuclear cells infiltrates were detected at the end of exposure, which were used as basis for the NOAEC. In addition, the NOAEC of 1.48 mg/m³ is justified by the results revealed in test animals killed 29 days after end of exposure, in which the incidence of interstitial mononuclear cell infiltrates is comparable between controls and treated animals and is even equal between controls and low and mid dose animals of end of exposure groups. Furthermore, the ‘accumulation of particle-laden macrophages’ represents only a physiological reaction of macrophages (phagocytosis of foreign material) and not an inflammatory, adverse process. Based on the results seen 29 days after end of exposure an ongoing activation of alveolar macrophages seems unlikely because the number of animals with accumulation of particle-laden macrophages is clearly lower and no further findings indicative for an ongoing activation or progression (e.g. fibrosis, granulomatous inflammation) are seen.
Therefore, an ongoing activation of lung macrophages is rather unlikely.
Concerning lymphoid hyperplasia: in general, lymphoid hyperplasia in lymph nodes has to be interpreted as physiological response to any immunological stimulus. It is an adaptive reaction and gives no basis for judging as adverse (for setting a NOAEC). In the current study, there is only a very slight lymphoid hyperplasia in the lung associated lymph nodes seen in one single animal/ test group and only in animals directly after end of exposure. In animals 29 days after end of exposure, the number of affected animals is comparable low and even zero in the mid dose test group. Also, a concentration (dose)-response relationship is missing in all sacrifice groups.
Effects indicating systemic toxicity were not observed. Spontaneous changes in the non-respiratory organs (incl. in those organs where the weight was increased), like tubular basophilia in the kidney, microgranuloma in the liver, inflammatory prostate lesions and testicular atrophy were found in the ZnO treated animals as well as in control animals in the same extent. All of the observed lesions were background changes of this particular rat strain [Wistar WU]. These rats showed a high incidence of infectious urogenital inflammation which mainly affected prostate, but also the kidneys, testes and epididymides. The incidence of testicular atrophy leading to aspermia, oligospermia and/or atrophy of the epididymides was unusually high for rats of this strain and age (Communication Fraunhofer ITEM-reproductive toxicology unit: "In 2010, massive fertility problems in Wistar (WU) Rats from Charles River were observed at ITEM, but also in other test facilities using this strain". Therefore, these morphological changes were considered to be not test substance-related due to similar incidences in animals of the control and the treatment groups.

The study presented herein is a guideline study without restrictions performed under GLP conditions. The deficiencies of the study are restricted to the lack of female exposure groups and the lack of ophthalmoscopic examinations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 - .. September 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not performed under GLP conditions. Only male rats were used.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

This study is a dose-range finding study

Deviations:

yes

Remarks:

only male rats were used

Principles of method if other than guideline:

No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.77 - <= 1.99 µm

Geometric standard deviation (GSD):

2.08

Remarks on MMAD:

MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.

Dose / conc.:

10.9 mg/m³ air

Remarks:

Test Group 1
(T 0420, 12 mg/m³)

Dose / conc.:

20.7 mg/m³ air

Remarks:

Test Group 2
(T 0420, 24 mg/m³)

Dose / conc.:

10.8 mg/m³ air

Remarks:

Test Group 3
(T 0421, 12 mg/m³)

Dose / conc.:

21.3 mg/m³ air

Remarks:

Test Group 4
(T 0421, 24 mg/m³)

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

No. of animals per sex per dose:

5 animals were used per group (total of 5 groups and total of 25 animals).

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: /

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly (Monday and Friday) thereafter until one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights of last weighing. Those of the weekends will be calculated as the difference of Monday to the previous Friday.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
Food consumption was determined weekly, from Monday to Friday and from Friday to Monday. Food consumption was calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: all
- Number of animals: 25

LUNG BURDEN: No

OTHER: /

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

No information provided

Clinical signs:

no effects observed

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.

Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively.

Food efficiency:

not specified

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

/

Ophthalmological findings:

not specified

Description (incidence and severity):

/

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.

Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).

In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

/

Endocrine findings:

not specified

Description (incidence and severity):

/

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

not specified

Description (incidence and severity):

/

Immunological findings:

not specified

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.

Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.

The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.

In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

Details on results:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations,.

The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.

During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).

Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).

BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities

In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.

Regarding pathology, the lungs and nasal cavity were the target organs.

In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Effect level:

mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Remarks on result:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Critical effects observed:

not specified

Concentration measurements in the exposure system

Study means and standard deviations of test substance concentrations:

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness dust generation (%)
		Mean	SD
1	12	10.94	0.41	18	59.9 %
2	24	20.67	0.89	28	74.7 %
3	12	10.76	0.37	19	56.7 %
4	24	21.28	1.02	42	50.3 %

Results of the particle size analyses

All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.

Cascade impactor measurements:

T 420			T 0421
	MMAD / GSD	% < 3 µm MMAD		MMAD / GSD	% < 3 µm MMAD
Test group 1 Measurement 1	1.90 µm / 2.03	74.2 %	Test group 3 Measurement 1	1.99 µm / 1.99	72.4 %
Test group 1 Measurement 2	1.79 µm / 2.04	76.5 %	Test group 3 Measurement 2	1.92 µm / 2.17	71.7 %
Test group 2 Measurement 1	1.85 µm / 2.02	75.3 %	Test group 4 Measurement 1	1.77 µm / 1.97	78.2 %
Test group 2 Measurement 2	1.77 µm / 2.17	75.2 %	Test group 4 Measurement 2	1.79 µm / 2.26	73.5 %

In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.

Particle size distribution measured by APS:

	Measurement date	MMAD / GSD		Measurement date	MMAD / GSD
Test group 1	26 Jun 20	2.47 µm/2.24	Test group 3	26 Jun 20	3.23 µm/1.87
		2.39 µm/2.22			3.38 µm/1.98
		2.47 µm/2.34			3.33 µm/1.91
	02 Jul 20	2.28 µm/2.37		02 Jul 20	2.93 µm/1.97
		2.13 µm/2.25			3.11 µm/2.21
		2.10 µm/2.23			3.46 µm/2.39
Test group 2	25 Jun 20	1.94 µm/2.66	Test group 4	25 Jun 20	2.41 µm/2.16
		1.82 µm/2.54			2.40 µm/2.18
		1.82 µm/2.52			2.29 µm/2.09
	03 Jul 20	2.41 µm/2.46		03 Jul 20	2.76 µm/2.25
		2.50 µm/2.37			2.67 µm/2.22
		2.50 µm/2.55			2.59 µm/2.17

 

BAL RESULTS

Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Cells	7.4*	8.9**	4.8**	8.6**
Eosinophils	4.1	16.0	6.7	11.0
Lymphocytes	4.9*	7.4**	4.0*	7.5**
Macrophages	0.6	1.1	1.1	0.7
Neutrophils	620.8*	722.3**	333.3**	734.0**
Monocytes	105.2*	91.2**	48.0**	71.4**
Epithelial cells	+	+	+	+

One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

+ increase could not be calculated because of zero activity in controls

Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Protein	14.4**	18.6**	5.4**	17.5**
GGT	4.6**	4.1**	2.9**	4.3**
LDH	11.0**	11.2**	4.6**	11.7**
ALP	16.6**	23.5**	7.0**	27.4**
NAG	2.7*	2.7*	1.7**	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

 

Histopathology

Treatment-related findings were observed in organs listed in the tables below with incidences and grading:

Incidence and grading of histological findings in larynx:

Larynx (Level I)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Epithelial alteration	1	0	4	1	2
·         Grade 1	1		4	1	2

The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).

Incidence and grading of histological findings in lungs

Lungs	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Infiltration, granulocytic	0	5	5	5	5
·         Grade 1		4		3	2
·         Grade 2		1	5	2	3
Histiocytosis, alveolar	0	5	5	5	5
·         Grade 1				1
·         Grade 2		4		2
·         Grade 3		1	5	2	3
·         Grade 4					2

In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.

Incidence and grading of histological findings in the nasal cavity:

Nasal cavity (Level IV)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Degeneration/regeneration, olfactory epithelium	0	5	5	5	5
·         Grade 1		3
·         Grade 2		2		3
·         Grade 3			5	2	1
·         Grade 4					4

Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.

Incidence and grading of histological findings in the tracheobronchial lymph nodes:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Histiocytosis (m)focal	0	3	3	4	5
·         Grade 1				2	1
·         Grade 2		2	3	2	4
·         Grade 3		1

Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Pathology

Weight parameters

Absolute weights

When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of absolute lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	113%	135%**	114%**	141%**

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of relative lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	117%	138%**	119%**	154%**

*p <= 0.05; **p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross lesions

In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.

Incidence of gross lesions observed during necropsy:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Enlarged	0	2	1	0	2

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:

Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.

Executive summary:

Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.

The following is a summary of the most relevant results:

Test group 4 (T0421, 24 mg/m³)

• Impaired body weight development, reduced food consumption


• Increased absolute and relative neutrophil cell counts in blood


• Decrease relative lymphocyte counts in blood


• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (141%/154%)


• Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals


• Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 3 (T0421 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (114%/119%)


• Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 2 (T0420, 24 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (135%/138%)


• Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

 

Test group 1 (T0420, 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 June 2020 - .. September 2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was not performed under GLP condtions. Only male rats were used.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

This study is a dose-range finding study

Deviations:

yes

Remarks:

only male rats were used

Principles of method if other than guideline:

No testing guidelines exist for this type of study, which is a dose range finding study. The results of this study should facilitate the selection of appropriate concentration for the following 90-day inhalation study.
The study was conducted based on the above test guidelines pertaining to inhalation repeated dose inhalation toxicity studies.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 93.8% for T0421.
- Test substance No.: 20/0051-1 for T0421.
- Batch identification: T0421.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: Aug 2020 for T0421.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing: rats were housed together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany. Bedding in the Polycarbonate cages were dust-free bedding. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). For enrichment wooden Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added. Wooden gnawing blocks (SAFE® block large) J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany.
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland
- Water (ad libitum): tap water
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 1.77 - <= 1.99 µm

Geometric standard deviation (GSD):

2.08

Remarks on MMAD:

MMAD / GSD: MMAD = 1.77-1.99 μm (geometric standard deviation = 1.97-2.26)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Exposure was over 14 days, at a rate of 6 hours per day for 5 days per week. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air.

Dose / conc.:

10.9 mg/m³ air

Remarks:

Test Group 1
(T 0420, 12 mg/m³)

Dose / conc.:

20.7 mg/m³ air

Remarks:

Test Group 2
(T 0420, 24 mg/m³)

Dose / conc.:

10.8 mg/m³ air

Remarks:

Test Group 3
(T 0421, 12 mg/m³)

Dose / conc.:

21.3 mg/m³ air

Remarks:

Test Group 4
(T 0421, 24 mg/m³)

Dose / conc.:

0 mg/m³ air

Remarks:

Control group

No. of animals per sex per dose:

5 animals were used per group (total of 5 groups and total of 25 animals).

Control animals:

yes

Details on study design:

- Dose selection rationale: The doses were chosen based on available data and upon approval of the sponsor. This is a dose range finding study which should facilitate the selection of appropriate concentration for a following 90-day inhalation study.
- Rationale for animal assignment (if not random): /
- Fasting period before blood sampling for clinical biochemistry: not specified
- Rationale for selecting satellite groups: /
- Post-exposure recovery period in satellite groups: /
- Section schedule rationale (if not random): /
- Other: /

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal. During exposure only a group wise examination was possible.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Not specified

BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly (Monday and Friday) thereafter until one day prior to gross necropsy. The body weight change was calculated as the difference of actual body weights and the weights of last weighing. Those of the weekends will be calculated as the difference of Monday to the previous Friday.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
Food consumption was determined weekly, from Monday to Friday and from Friday to Monday. Food consumption was calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: all
- Number of animals: 25

LUNG BURDEN: No

OTHER: /

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

No information provided

Clinical signs:

no effects observed

Description (incidence and severity):

During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.

The body weight change was statistically significantly decreased in the following animals:
- Test group 2 (24 mg/m³ test item 1) from study day 0 to day 1: -7.9 g (p≤ 0.05, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 0 to day 1: -6.7 g (p≤ 0.01, concurrent control was -0.4 g)
- Test group 4 (24 mg/m³ test item 4) from study day 4 to day 8: -1.7 g (p≤ 0.01, concurrent control was 5.5 g)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Due to social housing, there was only once cage (with 5 animals) per test group. No statistical evaluation was possible.

Food consumption of all exposed animals were slightly lower than the concurrent control group in a concentration-related manner. In the high concentrations, the food consumption of test group 2 (test item 1) animals was about 24 % lower than the control, while that of test group 4 animals (test item 2) was 32 % lower than in the controls. At low concentration, the food consumption was 10 % and 20 % lower than the control in test group 2 (test item 1) and test group 4 (test item 2) animals, respectively.

Food efficiency:

not specified

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

/

Ophthalmological findings:

not specified

Description (incidence and severity):

/

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At study day 14, in males of test group 4 (T0421, 24 mg/m3) absolute and relative neutrophil cell counts were slightly, but significantly increased, whereas relative lymphocyte counts were significantly decreased. These changes were regarded as treatment related and adverse.

Absolute and relative neutrophil cell counts were already significantly increased in rats of test group 3 (T0421, 12 mg/m3) The absolute neutrophil mean was marginally above the historical control range, whereas the mean of neutrophil relative counts was within this range (males, absolute neutrophils 0.53-1.09 Giga/L; relative neutrophils 8.5-19.0 %). Because the change was marginal and no other differential blood cell counts were altered, this change was regarded as treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).

In rats of test groups 1 and 2 (T0420, 12 and 24 mg/m3) absolute neutrophil cell counts were significantly increased, and in rats of test group 1 absolute eosinophil cell counts were significantly increased. However, the mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

In rats of test group 4 (T0421, 24 mg/m3) relative, large unstained cell (LUC) counts were significantly decreased and hemoglobin values were significantly increased. Both parameters were within historical control ranges (males, relative LUC 0.2-0.7 %, hemoglobin 8.6-9.6 mmol/L). In males of test group 4 absolute reticulocyte counts were significantly decreased. The mean was below the historical control range (males, absolute reticulocytes 112.7-190.0 Giga/L). However, because of slightly higher hemoglobin and hematocrit values as well as red blood cell (RBC) counts compared to the controls lower reticulocyte counts were a consequence because the bone marrow wanted to adjust the circulating red blood cell counts. This effect is regarded as a physiological feedback regulation and was therefore regarded as treatment related, but non adverse.

Clinical biochemistry findings:

not specified

Description (incidence and severity):

/

Endocrine findings:

not specified

Description (incidence and severity):

/

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

not specified

Description (incidence and severity):

/

Immunological findings:

not specified

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights
When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed.
Relative changes of absolute lung weights:
Lung weights were increased in test group 1 with test item T0420 (113%) and were statistically significantly increased (p <= 0.01) in test groups 2 (135%) with test item T0420, 3 (114%) and 4 (141%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed:
Relative changes of relative lung weights
Lung weights were increased in test group 1 with test item T0420 (117%) and were statistically significantly increased (p <= 0.01) in test groups 2 (138%) with test item T0420, 3 (119%) and 4 (154%) with test item T0421. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged, gray mottled lungs with enlarged and gray coloured lung-associated lymph nodes (high dose Days 28 and 42), enlarged lungs and the lung associated lymph nodes (mid dose) and enlarged lung-associated lymph nodes
(low dose).
Incidence of gross lesions observed during necropsy
None of the animals in the control group nor in Test group 3 showed any gross lesions in the tracheobronchial lymph nodes. 2 males animals of the Test group 1 (12mg/m3) showed enlarged heobronchial lymph nodes, 1 male animal of the Test group 2 (24mg/m3) and 2 male animals of the test group 4 (24mg/m3).
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in organs listed in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the ADDITIONAL RESULTS section below with incidences and grading

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
At study day 14, rats in high concentration groups of both test items (test groups 2 and 4, T0420 and T0421, in each group 24 mg/m3) showed equivalent cell count increases in the bronchoalveolar lavage fluid (BALF): about 9fold significant increase of the total cell counts; highly, significantly increased absolute and relative neutrophil counts (about 700 fold absolute neutrophil increase) and absolute and relative monocyte counts (about 70 to 90 fold absolute monocyte increase) as well as moderately, significantly increased absolute lymphocyte counts (about 7 fold absolute lymphocyte increase). Relative macrophage cell counts were significantly decreased in both mentioned test groups.

Whereas the low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same changes as the high test item concentration groups, low concentration test group of T0421 (test group 3, 12 mg/m3) had considerable lower values: in test group 1 (T420) about 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts; in test group 3 (T0421) about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts. Relative neutrophil and monocyte counts were also significantly increased in the mentioned test groups whereas relative macrophage counts were significantly decreased.
At study day 14, total protein levels as well as enzyme activities of -Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and -N-Acetyl glucosaminidase (NAG) in BALF were significantly increased in all test groups. Quantitatively, the same trend could be observed as described in the BALF cytology. High concentration test groups of both test items (test groups 2 and 4, T0420 and T0421, each 24 mg/m3) showed equivalent changes: about 18 fold increase in total protein levels, 24 to 27 fold increase of ALP, 11 fold increase of LDH, 4 fold increase of GGT and 3 fold increase of NAG activities.

The low concentration test group of T0420 (test group 1, 12 mg/m3) showed nearly the same alterations in BALF compared to the high concentration test groups: 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities.

In the low concentration test group of T0421 (test group 3 (12 mg/m3) considerable lower changes could be observed: 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

Details on results:

In this study, male Wistar rats were whole-body exposed to dust aerosols of T0420 and T0421 at target concentrations of 12 and 24 mg/m³ for 6 hours daily on 5 consecutive days for 14 days. A concurrent control group was exposed to conditioned air. To examine the influence of coating, two substances T0420 and T0421 were tested at comparable concentrations. After the last exposure, animals designated for bronchoalveolar lavage, histological examinations.

The tested atmospheric concentrations were slightly lower than the target concentration. They were maintained throughout the study. Cascade impactor measurement of both substances showed particle sizes that were close to each other. The MMADs ranged from 1.77 to 1.99 µm, which were well within the respirable range. The fraction of particles < 3 µm MMAD was higher than 70 % in all test groups. With regard to particle size distribution, there were no difference between the two substances.

During the exposure period, no clinical signs of toxicity were observed. The body weight, body weight gain was slightly lower than in the concurrent control group, although statistical significance was only in body weight change of test group 4 on single days. Although statistical evaluation could not be performed due to social housing, food consumption was apparently lower in animals exposed to both test substances than in the controls. Overall, the retarded body weight development and food consumption were slightly more severe in animals exposed to test item 2 (T0421) than those exposed to test item 1 (T0420).

Regarding clinical pathology, alterations of bronchoalveolar lavage (BAL) parameters were similar in the high concentration test groups of T0420 and T0421 (test groups 2 and 4, in each group 24 mg/m3): moderately, significantly increased total cell counts (about 9 fold), highly increased neutrophil and monocyte counts (absolute neutrophils about 700 fold, absolute monocyte about 70 to 90 fold), and slightly increased lymphocyte counts (absolute lymphocytes 7 fold). Correspondingly to the neutrophil cell count increase, alkaline phosphatase (ALP) activities were moderately, significantly increased in both test groups (24 to 27-fold). Lactate dehydrogenase (LDH) activities indicating general cell destruction were also moderately, significantly increased (about 11-fold), whereas gamma-Glutamyl-transferase (GGT) and beta-N-Acetyl glucosaminidase (NAG) activities were only marginally, but also significantly increased (GGT about 4 fold, NAG about 3 fold).

BAL values in the low concentration test group of T420 (test group 1, 12 mg/m3) were changed in the same magnitude as in the high concentration test groups: 7 fold significant increase of total cell counts, about 600 fold, significant increase of absolute neutrophil cell counts, about 100 fold significant increase of absolute monocyte cell counts and 5 fold significant increase of absolute lymphocyte counts, 14 fold increase of total protein levels, 17 fold increase of ALP, 11 fold increase of LDH, 5 fold increase of GGT and 3 fold increase of NAG activities

In contrast BAL values of the low concentration group of T0421 (test group 3, 12 mg/m3) showed considerable lower changes compared to controls: about 5 fold significant increase of total cell counts, about 300 fold significant increase of absolute neutrophil cell counts, about 50 fold significant increase of absolute monocyte counts and about 4 fold significant increase of absolute monocyte counts, 5 fold increase of total protein levels, 7 fold increase of ALP, 5 fold increase of LDH, 3 fold increase of GGT and 2 fold increase of NAG activities.

In addition to the mentioned local changes in the BAL, in the high concentration group of T0421 (test group 4, 24 mg/m3), a marginal increase of the absolute and relative blood neutrophil cell counts coupled with a decrease of the relative lymphocyte counts indicated a systemic acute-phase reaction.

Regarding pathology, the lungs and nasal cavity were the target organs.

In the lungs a disseminated infiltration of granulocytes and alveolar histiocytes was observed. This resulted also in an increase of the lung weight in most test groups. Some histiocytes were assumed to be destroyed. This inflammatory reaction was seen as consequence to the inhalation of the ZnO and was considered to be adverse.
In the nasal cavity in all levels, but most severely in level IV, there was degeneration and/or regeneration of the olfactory epithelium seen. This finding was regarded to be treatment-related and adverse.
The increased size of tracheobronchial lymph nodes was caused by alveolar histiocytes, transporting the phagocytosed ZnO particles from the lungs to the regional lymph nodes. As no other findings were observed, this was regarded to be treatment-related but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Effect level:

mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Remarks on result:

other: Dose range finding study - no conclusion on NOAEL or LOAEL at this stage.

Critical effects observed:

not specified

Concentration measurements in the exposure system

Study means and standard deviations of test substance concentrations:

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness dust generation (%)
		Mean	SD
1	12	10.94	0.41	18	59.9 %
2	24	20.67	0.89	28	74.7 %
3	12	10.76	0.37	19	56.7 %
4	24	21.28	1.02	42	50.3 %

Results of the particle size analyses

All measurements of particle size resulted in MMADs between 1.77 and 1.99 µm with GSDs between 1.97 and 2.26. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 74.2 % and 76.5 % for test item 1 (T0420) and between 71.7 % to 78.2 % for test item 2 (T0421). These values were well within the requirement of the test guidelines and showed that the generated test atmospheres contained high fraction of respirable particles. Moreover, all the measured values were close to each other, there were no significant difference between the two test items at comparable concentrations.

Cascade impactor measurements:

T 420			T 0421
	MMAD / GSD	% < 3 µm MMAD		MMAD / GSD	% < 3 µm MMAD
Test group 1 Measurement 1	1.90 µm / 2.03	74.2 %	Test group 3 Measurement 1	1.99 µm / 1.99	72.4 %
Test group 1 Measurement 2	1.79 µm / 2.04	76.5 %	Test group 3 Measurement 2	1.92 µm / 2.17	71.7 %
Test group 2 Measurement 1	1.85 µm / 2.02	75.3 %	Test group 4 Measurement 1	1.77 µm / 1.97	78.2 %
Test group 2 Measurement 2	1.77 µm / 2.17	75.2 %	Test group 4 Measurement 2	1.79 µm / 2.26	73.5 %

In the following table the data of APS measurements were presented. The APS measurement showed slightly higher MMAD. The difference between the two devices are to be explained by the mechanisms the measurements based on.

Particle size distribution measured by APS:

	Measurement date	MMAD / GSD		Measurement date	MMAD / GSD
Test group 1	26 Jun 20	2.47 µm/2.24	Test group 3	26 Jun 20	3.23 µm/1.87
		2.39 µm/2.22			3.38 µm/1.98
		2.47 µm/2.34			3.33 µm/1.91
	02 Jul 20	2.28 µm/2.37		02 Jul 20	2.93 µm/1.97
		2.13 µm/2.25			3.11 µm/2.21
		2.10 µm/2.23			3.46 µm/2.39
Test group 2	25 Jun 20	1.94 µm/2.66	Test group 4	25 Jun 20	2.41 µm/2.16
		1.82 µm/2.54			2.40 µm/2.18
		1.82 µm/2.52			2.29 µm/2.09
	03 Jul 20	2.41 µm/2.46		03 Jul 20	2.76 µm/2.25
		2.50 µm/2.37			2.67 µm/2.22
		2.50 µm/2.55			2.59 µm/2.17

 

BAL RESULTS

Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure):

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Cells	7.4*	8.9**	4.8**	8.6**
Eosinophils	4.1	16.0	6.7	11.0
Lymphocytes	4.9*	7.4**	4.0*	7.5**
Macrophages	0.6	1.1	1.1	0.7
Neutrophils	620.8*	722.3**	333.3**	734.0**
Monocytes	105.2*	91.2**	48.0**	71.4**
Epithelial cells	+	+	+	+

One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

+ increase could not be calculated because of zero activity in controls

Changes in median total protein and enzyme levels in BAL (x-fold of concurrent control) of male rats on study day 14 (1 day after last exposure). Medians instead of means were used because of high individual variation of the values with great impact on the mean values:

Analyte	Gr. 1 T0420 12 mg/m^3	Gr. 2 T0420 24 mg/m^3	Gr. 3 T0421 12 mg/m^3	Gr. 4 T0421 24 mg/m^3
Total Protein	14.4**	18.6**	5.4**	17.5**
GGT	4.6**	4.1**	2.9**	4.3**
LDH	11.0**	11.2**	4.6**	11.7**
ALP	16.6**	23.5**	7.0**	27.4**
NAG	2.7*	2.7*	1.7**	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p £ 0.05; ** : p £ 0.01

 

Histopathology

Treatment-related findings were observed in organs listed in the tables below with incidences and grading:

Incidence and grading of histological findings in larynx:

Larynx (Level I)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Epithelial alteration	1	0	4	1	2
·         Grade 1	1		4	1	2

The finding epithelial alteration is a common observation in inhalation studies and in most cases, the base of the epiglottis is affected. It shows a minimal flattening of the epithelial cells and loss of cilia. It was considered to be treatment-related but with the minimal grading it was not regarded to be adverse (Kaufmann et al., 2009).

Incidence and grading of histological findings in lungs

Lungs	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Infiltration, granulocytic	0	5	5	5	5
·         Grade 1		4		3	2
·         Grade 2		1	5	2	3
Histiocytosis, alveolar	0	5	5	5	5
·         Grade 1				1
·         Grade 2		4		2
·         Grade 3		1	5	2	3
·         Grade 4					2

In general, Lob. cran. dexter, Lob. medius dexter, and Lob. accessorius of the lungs were slightly less severely affected compared to Pulmo sinister and Lob. caudalis dexter. The finding was characterized by disseminated intra-alveolar inflammatory cells (mainly histiocytes and less number of granulocytes), intermingled by foamy roundish structures, containing occasionally a faint, bluish, roundish structures inside (interpreted as nucleus) or finely granular, eosinophilic material inside the alveoli. These structures were considered to be destroyed alveolar macrophages. These findings were regarded to be treatment-related.

Incidence and grading of histological findings in the nasal cavity:

Nasal cavity (Level IV)	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Degeneration/regeneration, olfactory epithelium	0	5	5	5	5
·         Grade 1		3
·         Grade 2		2		3
·         Grade 3			5	2	1
·         Grade 4					4

Level IV of the nasal cavity was the most severely affected level and was here taken representatively for findings in the nasal cavity. There was loss, irregularity or flattening of the olfactory epithelium (interpreted as degeneration). The most affected areas were the septum, the dorsal meatus and the tips of the conchae. In some areas there was in addition an increase of nuclear size and basophilia, mainly of basal cells (interpreted as regeneration). These findings were considered as treatment-related.

Incidence and grading of histological findings in the tracheobronchial lymph nodes:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Histiocytosis (m)focal	0	3	3	4	5
·         Grade 1				2	1
·         Grade 2		2	3	2	4
·         Grade 3		1

Histiocytes with intracytoplasmic material similar to those seen in the lungs, were found in tracheobronchial lymph nodes. This finding is regarded as treatment-related, but not adverse.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Pathology

Weight parameters

Absolute weights

When compared to control group 0 (=100%), the mean absolute weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of absolute lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	113%	135%**	114%**	141%**

*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights

When compared to control group 0 (=100%), the mean relative weights of following organs were significantly changed (statistically significant changes printed in bold):

Relative changes of relative lung weights:

	Male animals
	T0420		T0421
Test group (mg/m³)	1 (12)	2 (24)	3 (12)	4 (24)
Lungs	117%	138%**	119%**	154%**

*p <= 0.05; **p <= 0.01

All other mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

The statistical significantly increased lung weight in test group 2, 3, and 4 was regarded to be treatment-related.

Gross lesions

In some animals, the tracheobronchial lymph nodes were enlarged. This was considered as treatment-related.

Incidence of gross lesions observed during necropsy:

Tracheobronchial lymph nodes	Male animals
	Control	T0420		T0421
Test group (mg/m³)	0 (0)	1 (12)	2 (24)	3 (12)	4 (24)
Enlarged	0	2	1	0	2

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:

Both Zinc oxide T420 and T421 caused lesions in nasal cavity and lung already at the low targeted concentration of 12 mg/m³ (measured concentration about 11 mg/m³). No systemic effect was observed. At comparable atmospheric concentrations, Zinc oxide T421 seemed to cause more severe effects than those caused by T420.

Executive summary:

Groups of male Wistar rats were exposed whole-body to the dust aerosols of Zinc oxide T0420 and Zinc oxide T0421 for 6 hours per day on 5 days per week for two weeks. The target concentrations were 12 and 24 mg/m³ for each of the test items. A concurrent control group was exposed to clean air. Daily clinical observation and body weight were recorded. Animals were sacrificed, assessments including hematology, bronchoalveolar lavage and histopathology (control and high concentration only) of the respiratory tract were carried out.

The following is a summary of the most relevant results:

Test group 4 (T0421, 24 mg/m³)

• Impaired body weight development, reduced food consumption


• Increased absolute and relative neutrophil cell counts in blood


• Decrease relative lymphocyte counts in blood


• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (141%/154%)


• Minimal to slight infiltration of neutrophilic granulocytes and moderate to severe infiltration of alveolar histiocytes in the lungs in all animals


• Slight to severe degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 3 (T0421 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (114%/119%)


• Minimal to slight infiltration of neutrophilic granulocytes and minimal to moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

 

Test group 2 (T0420, 24 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Increase of absolute and relative lung weight (135%/138%)


• Slight infiltration of neutrophilic granulocytes and moderate infiltration of alveolar histiocytes in the lungs in all animals


• Minimal to moderate degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Test group 1 (T0420, 12 mg/m³)

• Increased total cell counts, absolute and relative neutrophil and monocyte counts as well as absolute lymphocyte counts in BAL


• Decreased relative macrophage counts in BALF


• Increased total protein levels as well as g-Glutamyl-transferase (GGT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and b-N-Acetyl glucosaminidase (NAG) activities in BALF


• Minimal to slight infiltration of neutrophilic granulocytes and slight to moderate infiltration of alveolar histiocytes in the lungs in all animals

Minimal to slight degeneration/regeneration of the olfactory epithelium in the nasal cavity of all animals

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2020 - ...June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study without restrictions performed under GLP conditions.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (uncoated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 98.2% for T0420
- Test substance No.: 20/0050-1 for T0420
- Batch identification: T0420

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: May 2022 for T0420.

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.52 - <= 2.01 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.52-2.01 μm (geometric standard deviation = 4.04--2.28)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

0.52 mg/m³ air (analytical)

Remarks:

SD: 0.10 mg/m3; target concentration: 0.5 mg/m³: Test Group 1 (Parental animals F0)

Dose / conc.:

2 mg/m³ air (analytical)

Remarks:

SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 2 (Parental animals F0)

Dose / conc.:

9.97 mg/m³ air (analytical)

Remarks:

SD: 1.23 mg/m3, target concentration: 10 mg/m³: Test Group 3 (Parental animals F0); Test Group 13 (male animals for particle detection); Test Group 23 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ±20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the
control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g

Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

Food efficiency:

not examined

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

/

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.

Immunological findings:

not examined

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
--> These effects were observed as treatment-related, adverse effects
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 9 males and 10 females

Test group 23 (Recovery group R1, 10 mg/m³)
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4
female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal
Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV, exemplarily) in 1 female

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
Test item 1 (Zinc oxide T0420)
The following treatment-related, adverse effects were observed:
Test group 3 (10 mg/m³):
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase
(ALP) and γ -Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:
During pre-exposure period, none of the male and female rats showed any clinical signs and findings different from normal.
Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
Exposure period, test item 1 (test groups 1, 2, 3, 13, and 23):
No clinical signs of toxicity were observed in male and female animals.

BODY WEIGHT AND WEIGHT GAIN
Body weight change:
The following statistically significant body weight changes were determined in male animals:
- Test group 1: day 74 -> 81: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 1: day 92 -> 93: -5.1g (p< 0.05), whereas the control group was 2.3g
- Test group 1: day 93 -> 94: 6.7g (p< 0.05), whereas the control group was -5.3g
- Test group 3: day 11 -> 18: 17.8g (p< 0.05), whereas the control group was 22.0g
- Test group 3: day 25 -> 32: 11.0g (p< 0.05), whereas the control group was 16.1g
- Test group 3: day 94 -> 95: -10.7g (p< 0.05), whereas the control group was 2.0g
- Test group 13: day 18 -> 25: 9.9g (p< 0.01), whereas the control group was 5.7g
- Test group 13: day 74 -> 81: 7.5g (p< 0.01), whereas the control group was 0.9g
- Test group 23: day 0 -> 4: 4.5g (p< 0.01), whereas the control group was 12.6g
- Test group 23: day 18 -> 25: 9.7g (p< 0.01), whereas the control group was 16.9g
- Test group 23: day 53 -> 60: 9.5g (p< 0.05), whereas the control group was 2.6g
- Test group 23: day 109 -> 116: 9.6g (p< 0.01), whereas the control group was 2.2g
The following statistically significant body weight changes were determined in female
animals:
- Test group 23: day 60 -> 67: 12.1g (p< 0.05), whereas the control group was 1.0g
- Test group 23 day 102 -> 109: 3.5g (p< 0.05), whereas the control group was 9.4g
Although the deviations in body weight changes were statistically significant, they did not show any trend with the exposure-duration, as some of the means were higher than the control, on the other days lower, indicating that they were rather biological variations than substance-related changes. Moreover, the mean body weight (as well as the final body weight) did not significantly change, when compared with the concurrent control. These deviations from the control were considered not biologically relevant.

FOOD CONSUMPTION
The following statistically significant changes of mean food consumption were determined in
female animals:
• Test group 1: day 11 - 18: +19.4 g (p≤ 0.05), whereas the control group was +17.6 g
• Test group 1: day 25 - 32: +19.1 g (p≤ 0.05), whereas the control group was +17.5 g
• Test group 1: day 32 - 39: +19.3 g (p≤ 0.05), whereas the control group was +17.8 g
The increased food consumption in female animals was most likely because animals spread out the food from the supply and was considered not adverse.

HAEMATOLOGICAL FINDINGS:
In females of test group 3 (10 mg/m3 Zinc oxide T0420) absolute and relative neutrophil cell
counts were significantly increased whereas relative lymphocyte counts were significantly
decreased. However, total white blood cell counts were not altered among these individuals,
and absolute neutrophil counts were within the historical control range (females, absolute
neutrophils 0.60-0.96 giga/L). Therefore, these changes were regarded as incidental and not
treatment related.

The following significant changes were regarded as incidental and not treatment related,
because the values were within historical control ranges: decreased relative eosinophil cell
counts in males of test groups 2 and 3 (2 and 10 mg/m3 Zinc oxide T0420) prolonged prothrombin time (HQT, i.e., Hepatoquick’s test) in females of test group 3 (10 mg/m3 Zinc oxide T0420)( males, relative eosinophils 1.4-3.1 %; relative basophils 0.1-0.4 %; hemoglobin 8.6-9.3 mmol/L; females, absolute monocytes 0.05-0.11 Giga/L; relative monocytes 1.8-2.8 %; RBC 7.55-8.84 Tera/L; MCV 50.7-55.1 fL; MCH 1.10-1.21 fmol; HQT 34.0-40.2 sec).
The following significant changes were regarded as incidental and not treatment related,
because the alteration was not dose dependent: decreased absolute and relative monocyte counts in females of test group 2 (2 mg/m3 Zinc oxide T0420) as well as absolute monocyte counts in females of test group 3 (10 mg/m3 Zinc oxide T0420).

In females of test group 23 (10 mg/m3 Zinc oxide T0420) absolute monocyte counts were
significantly decreased, but the values were within the historical control range (females,
absolute monocytes 0.05-0.07 Giga/L). Therefore this change was regarded as incidental and
not treatment related.

CLINICAL CHEMISTRY:
The following significant changes were regarded as incidental and not treatment related
because the values were within historical control ranges: increased inorganic phosphate levels
in males of test groups 3 (10 mg/m3 Zinc oxide T0420 )

NEUROBEHAVIOUR:
Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed.
Overall motor activity (summation of all intervals):
Test item T0420 (Test groups 1, 2 and 3):
there were no statistically significant deviations from the control group 0.

ORGAN WEIGHTS
When compared with control group 0 (=100%), Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³): Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)

GROSS PATHOLOGY
Test item 1 (Zinc oxide T0420):
Test group 3 (10 mg/m³):
•Macroscopically observed white foci in the lungs of 5 males and 8 females.
•Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 6 males and 9 females
Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
--> The foci observed in the lungs of males and females of test group 23 (test item 1, 10 mg/m³)
and test group 26 (test item 2, 10 mg/m³) were considered to be treatment-related as similar
findings were observed in the respective main groups. The same comes true for the
enlargement of the mediastinal lymph nodes in one female of test group 23 (test item 1,
10 mg/m³) and one male of test group 26 (test item 2, 10 mg/m³). These findings were regarded
to be treatment-related.


HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC:

Larynx (level I):
In the larynx, the most severe findings were observed in level I, therefore only findings in level I of the larynx are given

Parental animals:
Minimal epithelial alteration was observed in several test groups treated with test item 1 or test item 2 as well as in control animals. This finding is characterized by an increase of cell layers and replacement of respiratory epithelium by squamous epithelial cells, which may exhibit slight nuclear polymorphism and cellular atypia. The site most susceptible for this lesion, is the base of the epiglottis as it was observed in the present study. This finding was regarded to be treatment-related (inhalation).

Recovery animals: no findings observed

Lungs:

Parental animals:
Mainly, high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. Within alveoli, mainly in the bronchio-alveolar transition region, a multifocal accumulation of alveolar macrophages with vacuolar (foamy) cytoplasm was seen. The alveolar macrophages often revealed nuclei of increased size and occasionally multiple nuclei.
Intermingled with the foamy macrophages, cellular debris of presumable fragmented
macrophages and neutrophils were observed. In the region of these cellular accumulations, proliferation (hyperplasia) of type II pneumocytes was observed.
Males of test group 2 and 4 (test item 1 and 2, 2 mg/m³) revealed also an accumulation of foamy macrophages, only.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Lymph nodes (mediastinal):
Parental animals:
The mediastinal and tracheobronchial lymph nodes revealed comparable findings.
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were more severely affected. A lympho-reticular cell hyperplasia was observed, which can be explained by an activation of the draining lymph nodes of the lungs. Furthermore, aggregates of macrophages were seen within the lymph nodes. These findings were considered as treatment-related.
Single animals of test group 1, 2 (test item 1, 0.5 and 2 mg/m³), and test group 5 (test item 2, 2 mg/m³) revealed similar findings.

Recovery animals:
The same findings as described for the main group animals were observed in the recovery animals. These findings were regarded to be treatment-related.

Nasal cavity:

Parental animals:
The nasal cavity was investigated in four levels. The most severely affected levels were level III and IV
In general, the high dose group males and females (test group 3 and 6 [test item 1 and 2, 2 mg/m³]) were affected. The finding was characterized by loss of olfactory epithelial cells and occasionally regeneration. Mainly the dorsal meatus and areas on the nasal septum were affected. This finding was regarded to be treatment-related.
One female of test group 1 (test item 1, 0.5 mg/m³) and three males and one female of test group 5 (test item 2, 2 mg/m³) showed minimal to slight degeneration of the olfactory epithelium. As this finding normally does not occur as a background lesion, it was assumed to have been most likely caused by the test substances.

Recovery animals:
Findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Trachea
In the trachea, two male animals of test group 8 (reference item 2, 22 mg/m³) revealed a flattening of the respiratory epithelium at the carina. This finding was considered to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.



BRONCHOALVEOLAR LAVAGE FLUID (BALF):
Cytology:
Parental animals:
After the administration period, in the bronchoalveolar lavage (BAL) of males and females of
test group 3 (10 mg/m3 Zinc oxide T0420) total cell counts, as well as absolute and relative
lymphocyte, neutrophil cell (PMN) and monocyte counts as well as absolute eosinophil cell
counts (not significantly) were increased. Relative macrophage counts were significantly
decreased. These alterations were regarded as treatment related and adverse.
In the BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) absolute and relative lymphocyte, neutrophil cell and monocyte counts were already significantly increased whereas
relative macrophage counts were significantly decreased. In males of test group 1 (0.5 mg/m3
Zinc oxide T0420) absolute and relative lymphocyte counts were significantly increased. In
females of test group 2 absolute and relative monocyte counts as well as relative neutrophil
counts were significantly increased whereas relative macrophage counts were significantly
decreased. However, in the BAL of both sexes of test group 2 as well as in BAL of males of
test group 1 total cell counts were not altered, and the differential cell counts were only
marginally changed (below 10fold). Therefore, the cell count changes in BAL of both sexes in
test group 2 and in BAL of males of test group 1 were regarded as treatment related but non adverse.

Recovery animals:
After the 8-week recovery period, no significant changes in BAL cytology were observed in
BAL of both sexes of test group 23 (10 mg/m3 Zinc oxide T0420).
Proteins/enzymes:
Parental animals:
After the administration period, in BAL of males and females of test group 3 (10 mg/m3 Zinc
oxide T0420) total protein levels as well as lactate dehydrogenase (LDH) and alkaline
phosphatase (ALP) activity were moderately, significantly increased whereas β-N-Acetyl
glucosaminidathe zinc content in lungs, liver, heart and brain of parse (NAG) in males of this test group and γ-Glutamyl-transferase (GGT) activity
in both sexes were marginally but also significantly increased. These alterations were regarded
as treatment related and adverse.
Additionally, in BAL of females of test group 3 (10 mg/m3 Zinc oxide T0420) NAG activity was
significantly increased, and in BAL of males of test group 2 (2 mg/m3 Zinc oxide T0420) LDH,
ALP and GGT activities and in females of this test group ALP and GGT activities were
significantly increased. However, the changes were marginally (below 2fold). Therefore, these
alterations were regarded as treatment related but non-adverse.
Recovery animals:
After the 8-week recovery period, in BAL of males and females of test group 23 (10 mg/m3
Zinc oxide T0420) no protein level and enzyme activity changes were observed.


OTHER FINDINGS:
organ burden:
ICP-OES analysis Zinc content in lungs, liver, heart and brain of parental male/female animals
As an essential element, the data showed high biological background level of zinc element (very high endogenous background). In the high concentration exposure groups, slightly increased zinc concentration was only found in lung. In all other examined organs, the zinc level was comparable with the control.

- Electron microscopy:
Electron microscope analysis of particulate matter in organs and tissues: (see section overall remarks/attachments)

Dose descriptor:

NOAEC

Remarks:

local toxicity

Effect level:

0.52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: At the target mid concentration of 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal

Dose descriptor:

LOAEC

Remarks:

local toxicity

Effect level:

0.52 mg/m³ air (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: At the target low concentration of 0.5 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one female animal

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

9.97 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

histopathology: non-neoplastic

Remarks on result:

other: No systemic toxicity was observed in hematology, clinical chemistry and histopathology

Critical effects observed:

yes

Lowest effective dose / conc.:

2 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Executive summary:

This study was a 90-Day Study (OECD test guideline (TG) 413) combined with the Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) in rat with neurotoxicity and developmental (neuro)toxicity evaluation, including detailed clinical observations addressing potential neurobehavioral effects, histological and morphological evaluations of the brains of the pups on post-natal day 22.

To compare the toxicity of uncoated and coated nano Zinc oxide, these two materials (Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated) were tested at each three concentrations. In addition, micronsize Zinc oxide T0242 and a soluble salt zinc sulfate monohydrate was tested as reference items. 

Groups of male and female Wistar rats were whole-body exposed to the aerosols of ZnO nano materials, Zinc oxide T0420 and Zinc oxide T0421, for 6 hours daily, at least 90 days. Zinc oxide T0420 was uncoated, Zinc oxide T0421 was coated.

The target concentrations for Zinc oxide T0420 and T0421 were 0.5, 2 and 10 mg/m³ referring to the non-volatile fraction. For the reference item 1 microscale Zinc oxide T0242, 10 mg/m³ was tested. For the reference item 2, Zinc sulfate monohydrate a target concentration of 22 mg/m³ was tested because this is equimolar to zinc ion of the ZnO materials. Concurrent control groups were exposed to humidified air (control group 0, 10 and 20).

All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3)). Control animals were exposed to conditioned air. Male and female rats aged about 6 or 7 weeks when supplied, were used as F0 generation parental animals. The animals were exposed for 43 days before mating. The mating period were maximal 2 weeks. After the mating period, the exposure of all male F0 animals were continued until they are exposed for total minimal 90 days. After the mating period, the female F0 animals were exposed further until gestation day 19. To allow them to deliver and rearing their pups (F1 generation), they were not exposed from gestation day 20 to postnatal day (PND) 3. From PND 4 through to PND 21, the dams were exposed with their pups in exposure cages containing beddings. During the exposure food was withdrawn. Water was provided in form of hydrogel pads from PND 14 to 16 onward. The first parental female animals were in gestation stage already after the first few mating days, therefore, the post-weaning period were adjusted in such a way, that a total of minimum 90 exposure will be achieved for females.

Daily clinical observations, body weights, food consumption, ophthalmology, detailed clinical observation and FOB/MA were recorded. Moreover, male and female fertility were determined. Additional assessments including hematology and clinical chemistry in blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of exposure period. In addition, recovery groups of male and female animals were included; after an exposure period of about 90 days, these animals were kept for an additional period of ca. 60 days without exposure (control group 20, and test groups 23, 26, 27 and 28, respectively).

To assess the reproductive/developmental toxicity of the test substances (incl. reference substances), estrus cycles, male and female reproduction, delivery data were collected. In the pups, open field observations were performed on PND 13 and 21, motor activity measurements were performed on PND 13, 17 and 21. On PND 22, thyroid hormones, brain weights, neuropathology, general histopathology were examined in separate subsets of animals.

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 1 (Zinc oxide T0420)
Test group 3 (10 mg/m³)

• Decreased food consumption during gestation and lactation of parental females
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (140%/150%) and females (128%/130%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 6 males and 9 females
• Slight to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 9 males and all females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 23 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 1 male and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 female
• Minimal to moderate numbers of foamy macrophages in the lungs in 3 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male and 2 female animals
• Minimal infiltration of neutrophils of alveoli of the lungs in 1 male and 2 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females

Test group 2 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in one male animal

Test group 1 (0.5 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium in 1 female

Conclusion for adult animals exposed to test item 1 (Zinc oxide T0420):
Inhalation exposure to Zinc oxide T0420 caused changes in lung, lung-draining lymph nodes and nasal cavity at the high concentration of 10 mg/m³. These findings were almost, though not completely resolved during the post-exposure observation period. At 2 mg/m³, minimal degeneration/regeneration in the nasal cavity was noted in one male animal, and at 0.5 mg/m³ in one female animal. Due to findings in nasal cavity, the NOAEC for local toxicity at the respiratory tract was 0.5 mg/m³ for male rats. a No Observed Adverse Effect Concentration (NOAEC) for local toxicity for females could not be unequivocally determined.

No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0420.

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
• Decreased food consumption during gestation and lactation of parental females
• Decreased body weights/body weight gain during gestation and lactation of parental females
• Increased total white blood cell (WBC) as well as absolute neutrophil and lymphocyte counts in blood of males
• Slightly increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males in BAL
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities in BAL of both sexes
• Increased  γ-Glutamyl-transferase (GGT) activity in BAL of males
• Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 10 males and 5 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium 

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage and histopathology

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Conclusion for adult animals exposed to test item 2 (Zinc oxide T0421):
Inhalation exposure to Zinc oxide T0421 caused changes several lavage parameters, as well as histological changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. All these effects were completely resolved after the postexposure observation period. In blood, increased neutrophils and lymphocyte was notice at the concentration of 10 mg/m³, which is considered secondary to the inflammation in the lung.
At the mid concentration of 2 mg/m³, histological findings were still observed in the nasal cavity of three male and two female rats. Thus, the No Observed Adverse Effect Concentration (NOAEC) for local toxicity was 0.5 mg/m³ under the current study conditions. 
Besides the increased neutrophils and lymphocytes in blood, no other changes were observed in hematology, clinical chemistry. No histopathological changes were observed in any organs and tissues that are not part of the respiratory tract. The NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity, that were not attributed to the local effect, was 10 mg/m³ for Zinc oxide T0421.

Reference item 1 (Zinc oxide T0242)
Test group 7 (10 mg/m³)
• Retarded body weight development in male animals
• Increased total cell counts as well as absolute and relative neutrophil cell and monocyte counts in BAL of both sexes
• Increased absolute lymphocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increased β-N-Acetyl glucosaminidase (NAG) activity in BAL of males
• Increase of absolute/relative lung weights in males (130%/141%) and females (137%/141%)
• Macroscopically observed white foci in the lungs of 5 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 7 males and 10 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to moderate infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 4 males and 6 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 6 males and 8 females
• Minimal degeneration/regeneration of the olfactory epithelium 

Test group 27 (Recovery group R1, 10 mg/m³)
• Macroscopically observed white foci in the lungs of 3 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal cellular debris in the lungs in 1 male animal
• Minimal infiltration of neutrophils of lung alveoli in 1 male animal
• Minimal hyperplasia of type II pneumocytes in 4 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 2 males and 3 females

Conclusion for adult animals exposed to reference item 1 (Zinc oxide T0242):
Inhalation exposure to Zinc oxide T0242 caused changes in lung, lung-draining lymph nodes and nasal cavity at the highest tested concentration of 10 mg/m³. These findings were greatly, though not completely, resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 10 mg/m³ for Zinc oxide T0242.

Reference item 2 (Zinc sulfate monohydrate)
Test group 8 (22 mg/m³)
• During exposure period, salivation and respiration sounds were detected in several male and female animals.
• Retarded body weight development in all male and female animals. 
• Decreased food consumption during gestation and lactation of parental female animals
• Recreased body weights/body weight gain during gestation and lactation of parental female animals
• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute macrophage and eosinophil cell counts in BAL of males
• Increased total protein levels lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and γ-Glutamyl-transferase (GGT) activities in BAL of both sexes
• Increase of absolute/relative lung weights in males (125%/138%) and females (114%/119%)
• Macroscopically observed white foci in the lungs of 3 males and 7 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial, highest number is given) in 5 males and 8 females
• Erosion/ulcer of the laryngeal epithelium at the base of the epiglottis in 1 female
• Minimal to slight squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in all males and all females
• Minimal to slight inflammatory cell infiltrates of the laryngeal epithelium in 1 male and 9 females
• Minimal to severe numbers of foamy macrophages in the lungs in all male and all female animals
• Minimal to moderate cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female animals
• Minimal to slight hyperplasia of type II pneumocytes in 6 males and 8 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes (exemplarily) in 5 males and 5 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes (exemplarily) in 8 males and 8 females
• Minimal to moderate degeneration/regeneration of the olfactory epithelium in all males and all females

Test group 28 (Recovery group R1: 22 mg/m³)
• Macroscopically observed white foci in the lungs of 2 males and 2 females
• Macroscopically enlarged draining lymph nodes (mediastinal) in 1 male and 1 female
• Minimal squamous metaplasia of the laryngeal epithelium at the base of the epiglottis in 1 male and 1 female animal
• Minimal to slight inflammatory cell infiltrates in the laryngeal epithelium in 4 males and 3 females
• Minimal to slight numbers of foamy macrophages in the lungs in 2 male and 4 female animals
• Minimal hyperplasia of type II pneumocytes in 3 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes in 1 male and 1 female animal
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes in 4 males and 4 females
• Minimal degeneration/regeneration of the olfactory epithelium in 1 male and 1 female 

Conclusion for adult animals exposed to reference item 2 (Zinc sulfate monohydrate):
Inhalation exposure to Zinc sulfate monohydrate caused changes in lung, lung-draining lymph nodes, larynx and nasal cavity at the highest tested concentration of 22 mg/m³. These findings were partly resolved during the post-exposure observation period.
No systemic toxicity was observed in hematology, clinical chemistry and histopathology the NOAEC (No Observed Adverse Effect Concentration) for systemic toxicity was 22 mg/m³ for Zinc sulfate monohydrate.

Overall assessment for adult animals:

With regards to systemic toxicity, none of the test or reference substances caused any systemic toxicity that were not triggered by the local toxicity.

Comparing the local effects of the two nano Zinc oxide materials, the overall finding in the lungs, mediastinal lymph nodes, in the nasal cavity were comparable at the tested concentrations, as well as the changes of lavage parameters. The small differences are considered biological variations. There were no considerable differences between the effects caused by zinc oxide nanoparticles and those caused by micron-size zinc oxide particle.

For reference substance 2 (zinc sulfate monohydrate), lower incidence and severity was found in the lungs than in the other zinc oxide treated groups, but higher incidence and severity in nasal cavity and larynx. This difference is considered being related to the different deposition pattern, caused by the different aerodynamic diameter. The aerodynamic diameter of zinc sulfate monohydrate was larger than the different types of zinc oxide. The mean MMAD of zinc sulfate monohydrate was with 2.3 µm considerably higher than those measured at the high concentrations of the test items 1 (1.19 µm) and 2 (0.97 µm). The deposited dose at the upper respiratory tract was higher, while those deposited in the lung was lower.

After the recovery period, all parameters in lavage fluid returned to the control level in all animals, irrespective of the exposed test and reference substance. With regards of histological findings in the respiratory tract, all changes reduced greatly in incidence and severity. Only single animals showed still some mild effects.

Reference 4

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2020 - ...June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study presented herein is a guideline study without restrictions performed under GLP conditions.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

- adopted on 2018-06-25
- additional endpoints investigated

Deviations:

yes

Principles of method if other than guideline:

Additional endpoints (covered in IUCLID 7.8.1): reproduction toxicology , developmental toxicity, (developmental) neurotoxicity
three doses of the test substance (coated ZnO nanomaterial) were compared to one dose of 2 reference substances (non-coated microscaled ZnO and Zinc sulfate monohydrate)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Purity, including information on contaminants, isomers, etc.: 93.8% for T0421.
- Test substance No.: 20/0051-1 for T0421.
- Batch identification: T0421.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at room temperature. The stability under the storage condition over the exposure period is guaranteed by the sponsor, and the sponsor holds this responsibility. Expiry date of the test substance: Aug 2020 for T0421.

INFORMATION ON NANOMATERIALS
- Chemical Composition:
- Density:
- Particle size & distribution:
- Specific surface area:
- Isoelectric point:
- Dissolution (rate):

Test substance preparation:
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: homogenous, white solid.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats, Crl:WI(Han)
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks (female), about 8 weeks (male)
- Weight at study initiation: The weight variation of the animals used did not exceed +/- 20 percent of the mean weight of each sex.
- Fasting period before study: The animals did not have access to food or water during exposure.
- Housing:
From delivery until mating and male animals after mating: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals
During mating: type III polycarbonate cages, 1 male/1 female per cage
During rearing: up to PND 22: type III polycarbonate cages, 1 dam with her litter
After weaning the females from study day 90 after exposure onward until sacrifice: Typ 2000P: ca. 2065 cm2 (polysulfone cages) / up to 5 animals. Remaining females with litters will be
maintained in type III cages until weaning.
For Motor Activity Measurement: Typ III polycarbonate cages (floor area about 800 cm²) / 1 animal
During Exposure: Wire cages, type DK III / up to 2 animals Females from PND 4 until study day 94 (and females without litter from the same time period onwards): perforated polycarbonate cages type II. From study day 95 onward wire cages, type DK III
- Diet (ad libitum): mouse and rat maintenance diet, GLP, 12 mm pellets, Granovit AG, Kaiseraugst, Switzerland before and after exposure. Food was withdrawn during exposure.
- Water (ad libitum): tap water
- Acclimation period: 11 days

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. In view of the aim and duration of the study, the contaminants occurring in commercial food should not influence the results. The drinking water is regularly assayed for chemical contaminants both by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for bacteria by a contract laboratory. The Drinking Water Regulation will serve as the guideline for maximum tolerable contaminants. In view of the aim and duration of the study, there are no special requirements exceeding the specification of drinking water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: beginning of experiment To: end of experiment

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Remarks:

whole-body exposure for the reasons explained see IUCLID section 13.2 'Human health requirements Final Decision: protocol deviations and rationale'

Vehicle:

other: unchanged (no vehicle)

Mass median aerodynamic diameter (MMAD):

>= 0.6 - <= 2.53 µm

Remarks on MMAD:

MMAD / GSD: MMAD = 0.60-2.53 μm (geometric standard deviation = 3.08-2.23)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Generation of the inhalation atmospheres via a solid particle generators (brush-generator; BASF SE, Ludwigshafen, Germany) & Aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany). Whole body exposure systems were used. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE).
- Method of holding animals in test chamber: Whole body exposure systems. The animals were kept singly in wire cages located in a glass steel inhalation chamber, volume of 1.1 m³ (BASF SE). The chambers were located in exhaust hoods in an air conditioned room.
- Source and rate of air: Conditioned air from the central air conditioning system, compressed and exhaust air. Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG, Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter (SMF 5/3, 108 mm, Donalson), the compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is conducted to the laboratories via pipes, where the pressure is reduced to 5 - 6 bar. In the laboratory, the compressed air can be taken as required.
- Method of conditioning air: Conditioned air from the central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The particles/aerosol was generated via a solid particle generator (brush-generator; BASF SE, Ludwigshafen, Germany) and an aerosol mixing tube (stainless steel; BASF SE, Ludwigshafen, Germany), according to the following method: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage; mixed with conditioned dilution air and passed into the inhalation system.
- Temperature, humidity, pressure in air chamber: Daily mean relative humidities in the inhalation systems ranged between 41.6 and 60.8 %. Daily mean temperatures in the inhalation systems ranged between 21.4 and 23.7°C. They are within the range suggested by the respective testing guidelines.
- Air flow rate: The air flows were constantly maintained in the desired range.
- Air change rate: An air change of about 24 to 25 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.Equipment for particle size analysis: Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA) ; Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA) ; Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA) ; Sampling probe internal diameter 7 mm ; Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany). The calculation of the particle size distribution was carried out in the Laboratory for Inhalation Toxicology of the Experimental Toxicology and Ecology of BASF SE on the basis of mathematical methods for evaluating particle measurements (OECD guidance document No. 39). Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: On two days during the exposure period, with 3 repeats on each day. To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The instrument measures particles in the size range from 0.011 to 1.083 µm. Using a conductive sample hose, the SMPS sampled at 0.3 liters per minute (LPM) with a sheath flow of 3 LPM. At this setting the single-stage, inertial impactor incorporated into the inlet of the SMPS to remove larger particles had a 50% cut size of 1.082 µm according to the software calculation. The sampling duration was about 7 minutes. As a rule 10 repeats were measured for each exposure concentration.
- Treatment of exhaust air: Exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration could be/was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were determined by gravimetrical measurements of filter samples in all test groups. Control group was not sampled. This analytical method was judged to be valid because the test substances did not possess an appreciable vapor pressure.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 consecutive days per week, 6 hours per day (exception: no exposure on the day of FOB/MA and parental females from GD20 – PND 3).

Dose / conc.:

0 mg/m³ air

Remarks:

Test Group 0 (Parental animals F0) - air control; Test Group 10 (male animals for particle detection); Test Group 20 (Recovery animals) - air control

Dose / conc.:

0.52 mg/m³ air (analytical)

Remarks:

SD: 0.16 mg/m3; target concentration: 0.5 mg/m³: Test Group 4 (Parental animals F0)

Dose / conc.:

2.01 mg/m³ air (analytical)

Remarks:

SD: 0.20 mg/m3; target concentration: 2.0 mg/m³: Test Group 5 (Parental animals F0)

Dose / conc.:

10.07 mg/m³ air (analytical)

Remarks:

SD: 0.96 mg/m3, target concentration: 10 mg/m³: Test Group 6 (Parental animals F0); Test Group 16 (male animals for particle detection); Test Group 26 (Recovery animals)

No. of animals per sex per dose:

16/sex/dose group (parental animals)
5/sex at the high dose (recovery animals)
3 males at the high dose (for particle detection)

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on the results of the 14-day range finding study (BASF study no 36I0050/20I005 - Ma- Hock 2021), upon approval of the sponsor, nominal aerosol concentrations of 0.5, 2.0 and 10.0 mg/m³ were used for the test substance in the low, mid and high dose groups, respectively.
- Rationale for animal assignment:
Prior to the pre-exposure period, the animals were distributed according to weight among the
individual test groups, separated by sex. The weight variation of the animals used did not
exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions
was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists
were created. The list of randomization instructions were compiled with a computer (Laboratory
data processing, Experimental Toxicology and Ecology, BASF SE).
- Fasting period before blood sampling for clinical biochemistry: not specified
- Post-exposure recovery period in satellite groups: 45 days recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On non-exposure days a cage-side examination will be conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overall toxicity.

MORTALITY: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: YES
- Time schedule: All parental animals and recovery group animals were subjected to detailed clinical observations (DCO) outside their cages once before the beginning of the administration period and once during the first two weeks of the exposure, once monthly thereafter. DCO was performed in the morning before exposure. For observation, the animals were removed from their cages and placed in a standard arena (50 x 37.5 cm with a lateral border of 25 cm) for at least 20 seconds/animal.

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of the animals was determined at the start of the pre-exposure, at the start of
the exposure period and then, as a rule, once a week as well as prior to gross necropsy. The
body weight of the recovery animals were determined at the start of the recovery period, and
once a week during the recovery period.
The following exceptions were notable for the female parental animals:
• During the mating period, the females were weighed on the day of positive evidence of
sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 14, and 21.
• In the females without positive evidence of sperm, body weight was determined once a week during mating and gestation periods and in the females without litter during lactation period.

As a rule, the animals were weighed at the same time of the day (in the morning).

Body weight change was calculated as the difference between body weight on the respective exposure day and body weight and the weight of previous weighing. Group means were derived from the individual differences.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams
per animal and day.
Generally, food consumption was determined once a week for the male and female animals
and post mating period (males), with the following exceptions:
• Food consumption was not determined during the mating period (male and female
parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-
14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-
4, 4-7, 7-13.
During recovery period, food consumption was determined in the animals of test groups 20 –
28 of the recovery animals. It was determined at the start of the recovery period and once a
week during the recovery period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: YES
Before the beginning of exposure, the eyes of all parental animals were examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Dr. Gerhard Mann chem.-pharm. Fabrik GmbH and Bausch & Lomb GmbH, Germany). At the end of the exposure period, only animals selected for examinations according to OECD 413, 10 males and 10 females per group, were subjected to ophthalmological examination. In the first step, only control (test group 0) and high concentration groups (test groups 3, 6, 7 and 8) were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: 10 M + 10 F per dose group
-Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes, preparation of blood smears, prothrombin time (PT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: 10 M + 10 F per dose group
- Parameters checked: alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-glutamyl transpeptidase (GGT), sodium (Na), potassium (K), chloride (CL), Inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine (CREA), glucose (GLUC), total biluribin (TBIL), total protein (TP), albumin (ALB), globulin (GLB), triglycerides (TRIG), cholesterol (CHOL)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the 90days exposure period
- Dose groups that were examined: 10 M + 10 F per dose group
- Battery of functions tested: sensory activity / grip strength / motor activity / reflexes


IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Not specified
- Dose groups that were examined: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Number of animals: 10 M + 10 F per dose group and recovery groups (highest dose: 5M + 5F)
- Parameters checked: Cytological parameters: total cell count, cell differential analysis of cytospin preparations; protein; Enzymes: lactate dehydrogenase, alkaline phosphatase, N-acetyl-beta-D-Glucosaminidase (NAG BAL), gamma−Glutamyltransferase

ORGAN (lung, liver, heart, brain, olfactory bulb) BURDEN: Yes
- Time schedule for analysis: at the end of the exposure period and after the recovery period (45days post exposure)
- Dose groups that were examined: all
- Number of animals: 3 /sex / group
- Parameters checked: Zn content

OTHER: - Electron microscope analysis of particulate matter in organs and tissues: 3 male animals of the highest dose group

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule:
1.Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lung
9. Ovaries
10. Prostate (ventral and dorsolateral part together, fixed)
11. Seminal vesicles with coagulating glands (fixed)
12. Spleen
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
Organs and tissues of F0 animals histologically processed:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Eyes with optic nerve
14. Extraorbital lacrimal gland
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum
20. Kidneys
21. Larynx (3 levels)
22. Liver
23. Lungs
24. Lymph nodes (tracheobronchial and mediastinal)
25. Lymph nodes (mesenteric)
26. Mammary gland (female)
27. Nasal cavity (4 levels)
28. Olfactory bulb
29. Ovaries
30. Oviducts
31. Pancreas
32. Pharynx
33. Parathyroid glands
34. Peyer’s patches
35. Pituitary gland
36. Prostate
37. Rectum
38. Salivary glands
(mandibular and sublingual glands)
39. Sciatic nerve
40. Seminal vesicles
41. Skeletal muscle
42. Skin
43. Spinal cord
(cervical, thoracic and lumbar cord)
44. Spleen
45. Sternum with marrow
46. Stomach
(forestomach and glandular stomach)
47. Teeth
48. Testes
49. Thymus
50. Thyroid glands
51. Trachea
52. Urinary bladder
53. Uterus
54. Vagina

Statistics:

Statistical evaluation for test groups low, mid, high in comparison with air control group
- Food consumption (parental animals), body weight and body weight change (parental animals
and pups (for the pup weights, the litter means were used)), gestation days, anogenital distance,
anogenital index
--> DUNNETT test (two-sided)
- Male and female mating indices, male and female fertility indices, gestation index, females mated, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
--> FISHER'S EXACT test (one-sided)
-Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
--> WILCOXON test (one-sided+) with BONFERRONI-HOLM
-Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, lactation index
--> WILCOXON test (one sided-) with BONFERRONI-HOLM
-% live male day x, %live female day x
--> WILCOXON test (two-sided)
- Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
--> KRUSKAL-WALLIS and WILCOXON test (two-sided)
-Number of cycles and Cycle Length
--> KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided)
-Blood parameters
--> For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the
control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Broncho-alveolar lavage fluid (BALF)
--> Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
-Weight parameters in pathology
-->Non-parametric one-way analysis using KRUSKAL-WALLIS H test (two-sided).

Clinical signs:

no effects observed

Description (incidence and severity):

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period
on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these
groups. The findings in the eye was considered incidental due to missing concentration response
relationship.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.


The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control group was 446.1g
- Test group 26: day 92: 409.9g (p< 0.01), whereas the control group was 449.2g
- Test group 26: day 102: 415.4g (p< 0.01), whereas the control group was 450.4g
- Test group 26: day 109: 425.0g (p< 0.01), whereas the control group was 457.6g
Test group 26: day 116: 431.1g (p< 0.01), whereas the control group was 459.8g
- Test group 26: day 123: 437.3g (p< 0.01), whereas the control group was 465.5g
- Test group 26: day 130: 441.0g (p< 0.01), whereas the control group was 470.0g
- Test group 26: day 137: 447.8g (p< 0.05), whereas the control group was 472.3g
- Test group 26: day 144: 450.7g (p< 0.05), whereas the control group was 475.3g
- Test group 26: day 146: 452.6g (p< 0.05), whereas the control group was 475.9g

The following significant deviations from the control were observed in male recovery group animals exposed to high concentration of test item 2:
- Test group 26: day 0 -> 4: 5.5 (p< 0.01), whereas the control group was 12.6g
- Test group 26: day 11 -> 18: 10.7 (p< 0.01), whereas the control group was 21.7g
- Test group 26: day 18 -> 25: 10.6 (p< 0.01), whereas the control group was 16.9g
- Test group 26: day 67 -> 74: 8.2 (p< 0.05), whereas the control group was 4.4g
- Test group 26: day 130 -> 137: 6.8 (p< 0.01), whereas the control group was 2.3g
The mean body weights of the recovery group animals were statistically lower than the concurrent control group throughout the whole exposure and post-exposure period. The mean body weight changes were only significantly decreased during the initial period of the exposure period. This showed that the body weight development of the male animals of this groups was impaired at the initial time of the exposure. During the course of continuous exposure, as well as the recovery period, the body weight did not increase in such an extent that could compensate the initially reduced body weight gain. The retarded body weight development was also observed in main group animals exposed at the same concentration in the same chamber. Thus, the effect was considered treatment-related. As the final mean body weight was only about 5 % lower than the control, the body weight effect was considered not biologically relevant and not adverse.

Further, the following significant body weight changes were observed in male animals of test group 16 (10 mg/m³, Zinc oxide T0421)
- Test group 16: day 0 -> 4: 4.4g (p< 0.05), whereas the control group was 11.6g
- Test group 16: day 18 -> 25: 9.8g (p< 0.05), whereas the control group was 20.6g
- Test group 16: day 67 -> 7 4: -1.1g (p< 0.05), whereas the control group was 8.5g
- Test group 16: day 74 -> 81: 10.0g (p< 0.01), whereas the control group was 0.9g
As discussed above, the findings were considered treatment-related, but of no biological relevance and not adverse.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant changes of mean food consumption were determined in
male animals:
• Test group 6: day 0 - 4: +22.6 g (p≤ 0.05), whereas the control group was +24.4 g
• Test group 6: day 18 - 25: +22.5 g (p≤ 0.05), whereas the control group was +25.2 g
The lowered food consumption in test group 6 coincidenced with lower mean body weights and mean body weight change of these groups in the same time range. They may be related to the daily inhalation exposure to the test and reference substance. They were of transient nature, thus, they were considered not of biological relevance.

Food efficiency:

not examined

Description (incidence and severity):

/

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

/

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were
observed in several animals of all test groups and the control group without any concentration response relationship.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in males of test group 6 (10 mg/m3 Zinc oxide T0421) total white blood cell (WBC) counts as well as absolute neutrophil and lymphocyte counts were slightly but significantly increased. These changes were regarded as treatment-related and adverse.

The following significant changes were regarded as incidental and not treatment related, because the values were within historical control ranges: decreased relative eosinophil cell counts in males of test groups 4 and 6 (0.5 and 10 mg/m3 Zinc oxide T0421)

The following significant changes were regarded as incidental and not treatment related, because the alteration was not dose dependent: increased hematocrit value in males of test group 5 (2 mg/m3 Zinc oxide T0421); increased absolute monocyte counts in females of test group 4 (0.5 mg/m3 Zinc oxide T0421).

After the recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) absolute large unstained cell (LUC) counts were significantly increased. This was the only change of the differential blood cell counts among these individuals. Therefore, it was regarded as if at all treatment related as non-adverse.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly increased potassium values in males of test group 6 (10 mg/m3 Zinc oxide T0421). This was regarded if at all treatment related as non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were regarded as incidental and not treatment related because the values were within historical control ranges: increased inorganic phosphate levels in males of test group 6 and 8 (10 mg/m3 Zinc oxide T0421
; decreased albumin values in females of test group 6 (10 mg/m3 Zinc oxide T0421);
Significantly increased alkaline phosphatase activities in females of test groups 5 and 6 (2 and 10 mg/m3 Zinc oxide T0421) were regarded as incidental and not treatment related, because the alteration was not dose dependent.
After the 8-week recovery period, in males of test group 26 (10 mg/m3 Zinc oxide T0421) total bilirubin values were significantly increased.

Endocrine findings:

no effects observed

Description (incidence and severity):

After the administration period, in parental males and in male and female pups at PND22 of all
test groups, no treatment-related alterations of T4 and TSH levels were observed.

Urinalysis findings:

not specified

Description (incidence and severity):

/

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observational battery:
Quantitative parameters: no substance-related findings were observed.
Home cage observations: no substance-related findings were observed.
Open field observations: no substance-related findings were observed.
Sensorimotor tests/reflexes: no substance-related findings were observed

Overall motor activity (summation of all intervals):
Test item 2 (Zinc oxide T0421) (Test groups 4, 5 and 6):
there were no statistically significant deviations from the control group 0.

Single intervals:
Comparing the single intervals with the control group, the following statistically significant
deviations were seen:
• Decrease of activity in the male animals of test group 6 (10 mg/m³, test item 2) at interval
9 on day 87 (p ≤ 0.01).
-->No other abnormalities were detected.
These changes were considered incidental because they were of transient nature and the
overall motor activity was not changed in the respective group.

Immunological findings:

not examined

Description (incidence and severity):

/

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

When compared with control group 0 (=100%), Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³): Increase of absolute/relative lung weights in males (136%/143%) and females (131%/137%)
--> These effects were observed as treatment-related, adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item 2 (Zinc oxide T0421):
Test group 6 (10 mg/m³):
• Macroscopically observed white foci in the lungs of 6 males and 8 females
• Macroscopically enlarged draining lymph nodes (mediastinal or tracheobronchial,
highest number is given) in 10 males and 5 females
--> These effects were observed as treatment-related, adverse effects
Test group 26 (Recovery group R1, 10 mg/m³)
No treatment-related adverse findings

Neuropathological findings:

not examined

Description (incidence and severity):

neuropathological examinations of pups on PND22 (developmental neurotoxicity cohort) are examined (see IUCLID section 7.8.1)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in in the larynx, lungs, nasal cavity and the tracheobronchial lymph nodes. These are further described in the details on results section

The following treatment-related, adverse effects were observed:
Main group (F0)
Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)
Minimal to severe numbers of foamy macrophages in the lungs in all male and all female
animals
• Minimal to severe cellular debris in the lungs in all male and all female animals
• Minimal to slight infiltration of neutrophils of alveoli of the lungs in all male and all female
animals
• Minimal to slight hyperplasia of type II pneumocytes in 8 males and 9 females
• Minimal to slight lympho-reticular cell hyperplasia in the mediastinal lymph nodes
(exemplarily) in 8 males and 3 females
• Minimal to slight increased macrophage aggregates in the mediastinal lymph nodes
(exemplarily) in 4 males and 2 females
• Minimal to slight degeneration/regeneration of the olfactory epithelium (nasal cavity,
level IV, exemplarily) in 6 males and 10 females

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings

Test group 5 (2 mg/m³)
• Minimal degeneration/regeneration of the olfactory epithelium (nasal cavity, level IV,
exemplarily) in 1 male and 1 female
Test group 4 (0.5 mg/m³)
No treatment-related adverse findings

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BAL
Main group (F0)
The following treatment-related, adverse effects were observed:

Test item 2 (Zinc oxide T0421)
Test group 6 (10 mg/m³)

• Increased total cell counts as well as absolute and relative lymphocyte, neutrophil cell
and monocyte counts in BAL of both sexes
• Decreased relative macrophages counts in BAL of both sexes
• Increased absolute eosinophil cell counts in males
• Increased total protein levels lactate dehydrogenase (LDH) and alkaline phosphatase
(ALP) activities in BAL of both sexes
• Increased β-Glutamyl-transferase (GGT) activity in BAL of males

Test group 26 (Recovery group R1, 10 mg/m³)
• No treatment-related adverse findings in lavage

Details on results:

CLINICAL SIGNS AND MORTALITY:
Mortality:
No deaths were recorded throughout the study.
Clinical observations:

-During the pre-exposure period and the exposure period the animals showed no clinical signs and findings different from normal.
-Exposure period, control group animals (test groups 0, 10, 20):
There were no clinical signs and findings different from normal.
-Exposure period, test item 2 (test groups 4, 5, 6, 16 and 26):
One male animal (No.: 82) of test group 5 showed protruding eyeball during exposure period on study days 26 – 31. No clinical signs of toxicity were noted in any other animals of these groups. The findings in the eye was considered incidental due to missing concentration response relationship.

BODY WEIGHT AND WEIGHT GAIN
In animals exposed to low (0.5 mg/m³) and mid concentration (2 mg/m³) of test item 2 (Zinc oxide T0421), there were no statistically significant deviation from the concurrent control group was observed in body weight.

The following statistically significant body weight changes were determined in male animals of group 4 and 5:
- Test group 4: day 60 -> 67: 1.3g (p< 0.05), whereas the control group was 7.8g
- Test group 4: day 67 -> 74: 10.7g (p< 0.05), whereas the control group was 6.6g
- Test group 5: day 74 -> 81: 10.2g (p< 0.01), whereas the control group was 5.7g
- Test group 5: day 81 -> 88: 8.2g (p< 0.05), whereas the control group was 5.4g
These values were mostly higher than the control value and were of transient nature. They did not influence the mean body weight. Thus, they were considered incidental.


The following statistically significant body weight changes were determined in male animals:
- Test group 6: day 18: 329.8g (p< 0.05), whereas the control group was 345.9g
- Test group 6: day 25: 340.2g (p< 0.01), whereas the control group was 363.4g
- Test group 6: day 32: 351.3g (p< 0.01), whereas the control group was 379.4g
- Test group 6: day 39: 365.7g (p< 0.01), whereas the control group was 390.9g
- Test group 6: day 46: 373.3g (p< 0.05), whereas the control group was 394.9g

The following statistically significant body weight changes were determined in male animals:

- Test group 6: day 0 -> 4: 6.9 g (p< 0.05), whereas the control group was 10.4g
- Test group 6: day 11 -> 18: 17.2g (p< 0.05), whereas the control group was 22.0g
- Test group 6: day 18 -> 25: 10.4g (p< 0.01), whereas the control group was 17.5g
- Test group 6: day 25 -> 32: 11.1g (p< 0.05), whereas the control group was 16.1g
- Test group 6: day 81 -> 88: 8.1g (p< 0.05), whereas the control group was 5.4g

The mean body weights of test group 6 were lower than the concurrent control group animals. On study days 18, 25, 32 and 39 they co-incidence with statistically significantly lowered mean body weight change, they are likely attributed to the exposure to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421). The lower body weight change from study day 0 to study day 4 could be considered initial response to the exposure. The changes were very minor, and the mean body weight at the end of the exposure period of this group was not statistically lower than the control. Thus, they were considered not biologically relevant and not adverse.

The following statistically significant changes of body weight were determined in female
animals:
- Test group 6: day 32: 210.9g (p< 0.05), whereas the control group was 221.0g
- Test group 6: day 93: 234.4g (p< 0.01), whereas the control group was 251.8g
The following statistically significant body weight changes were determined in female
animals:
- Test group 6: day 11-> 18: 9.1 (p< 0.05), whereas the control group was 13.8g
- Test group 6: day 102 -> 109: -1.4 (p< 0.05), whereas the control group was 14.3g
- Test group 6: day 116 -> 123: 3.7 (p< 0.05), whereas the control group was -3.2g

The significant changes of body weight and body weight changes in female animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421) were all of transient nature. As there were already transient effects observed in male animals of these group, these changes in females may be also attributed to the test substance. As the mean body weight at the end of the exposure period of this group was not statistically lower than the control, these effects in body weights and body weight changes were considered not biologically relevant and not adverse.

The following significant changes were observed in recovery group male animals exposed to high concentration of test item 2 (10 mg/m³, Zinc oxide T0421):
- Test group 26: day 18: 316.6g (p< 0.01), whereas the control group was 344.0g
- Test group 26: day 25: 327.2g (p< 0.01), whereas the control group was 360.9g
- Test group 26: day 32: 338.7g (p< 0.01), whereas the control group was 375.0g
- Test group 26: day 39: 353.3g (p< 0.01), whereas the control group was 390.9g
- Test group 26: day 46: 365.0g (p< 0.01), whereas the control group was 399.8g
- Test group 26: day 53: 373.3g (p< 0.01), whereas the control group was 411.9g
- Test group 26: day 60: 378.8g (p< 0.01), whereas the control group was 414.4g
- Test group 26: day 67: 386.4g (p< 0.01), whereas the control group was 425.4g
- Test group 26: day 74: 394.6g (p< 0.01), whereas the control group was 429.7g
- Test group 26: day 81: 400.7g (p< 0.01), whereas the control group was 439.5g
- Test group 26: day 88: 404.5g (p< 0.01), whereas the control