Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with p-phenylenediamine and sodium methoxide

Endpoint summary

Description of key information

- Oral: according to OECD 422, GLP, rat, NOAEL (for general systemic toxicity) 1000 mg/kg bw/d

- Inhalation: according to OECD 412, GLP, rat, 5-day treatment with 21 days recovery, NOAEC (for systemic effects) 30 mg/m^3 air, NOAEC (for local effects) 10 mg/m^3 air

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jun 2020 - 10 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2020

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Not specified
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g (project 19L00260)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (room temperature)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water over a period of at least 7 days at room temperature was given. As the mixtures were stored no longer than this time period, the stability was guaranteed. The homogeneous distribution of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water was demonstrated.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final concentration of a dissolved solid, stock liquid or gel: Please, see section "Doses/concentrations"

FORM AS APPLIED IN THE TEST: Suspension in vehicle

OTHER SPECIFICS
- Physical state/ appearance: Solid/ orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: About 11 - 12 weeks (males); About 10 weeks (females)
- Weight at study initiation: 374.6 g (mean males); 219.6 g (mean females)
- Fasting period before study: No
- Housing: Up to 5 animals per sex and cage during pretreatment; up to 2 animals per sex and cage during premating; up to 1 animal per sex and cage with exception during mating (1 male/1 female per cage) and rearing up to PND 13 (1 dam with her litter); for motor activity (MA) measurements the animals were housed individually.
- Diet (e.g. ad libitum): Mouse and rat maintenance diet “GLP” (ad libitum)
- Water (e.g. ad libitum): water bottles (ad libitum)
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
- The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings the drinking water was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours (light from 06.00-18.00 h, dark from 18.00-06.00 h)

IN-LIFE DATES: From: 16 Jun 2020 To: 09 Sep 2020 (Sacrifice of parental females)

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Sodium carboxymethyl cellulose in deionized water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the preparation of the administration suspensions the test substance was weighed in a graduated flask depending on the dose group, topped up with 0.5% Sodium carboxymethyl cellulose in deionized water and intensely mixed with magnetic stirrer until it was completely homogenized. From 14 Aug 2020 onwards the test substance preparations were homogenized with an Ultraturrax instead of a magnetic stirrer. The portions of the test substance preparations for the daily administration were stored at room temperature. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 0.67, 2.00, 6.67 g/100mL
- Amount of vehicle (if gavage): 15 mL/kg bw/d
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration control analysis was assessed by UV/VIS spectrospcopy.
The samples for concentration control analysis, which were taken at the start of the administration period (premating), were not measured in time directly after sampling as expected. Therefore, these samples were discarded without measurement.
Therefore, an additional sample set were taken during gestation on 03 Aug 2020 and send to the Analytical Laboratory for analysis in time. This sample set demonstrated equivocal results with values outside of the desired range of the test facility. It was assumed that these results were due to the sampling process and did not represent the actual test substance preparations.
Therefore two further sample sets, taken during the lactation period on 14 Aug 2020 and 18 Aug 2020 were sent to the Analytical Laboratory and analyzed in time.The results of the sample sets during the lactation period demonstrated the correctness of the concentrations of the test substance in 0.5% Sodium carboxymethyl cellulose in deionized water because all samples were clearly found to be in the range of 90 - 110%. The mean of all three mean values of the high-dose group (including the questionable analysis during gestation) is close to the desired range of the test facility (mean: 86%). Therefore, in general, the concentration control analyses of the samples were assessed to be in an acceptable range.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. The treatment lasted up to one day prior to sacrifice.
- males: 31 days
- females: 62 days

Frequency of treatment:

Daily at the same time in the morning

Dose / conc.:

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

Remarks:

low-dose level (Test group 1)

Dose / conc.:

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

Remarks:

mid-dose level (Test group 2)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high-dose level (Test group 3)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on a previous range-finding study and at the request of the Sponsor.
- Fasting period before blood sampling for clinical biochemistry: About 16 to 20 hours

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters examined: Abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). During the mating period all females with negative evidence of sperm were weighed on mating days 1, 7 and 14. 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 of parturition (PND 0), PNDs 4, 7, 10 and 13. Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume, body weight data of these individuals are reported in the individual tables). Females without litter and after weaning (PND 13) were weighed once a week.

FOOD CONSUMPTION: Yes
- Time schedule: Once a week (except for the 2nd premating week (male parental animals) and 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, 7-10 and 10-13.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals:5 males and 5 females
- Parameters checked in table 1 were examined (Please, see section "Any other information on materials and methods incl. tables")

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination (males); at PND 14 (females)
- Animals fasted: Yes
- How many animals: 5 males and 5 females
- Parameters checked in table 2 were examined (Please, see section "Any other information on materials and methods incl. tables")

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection and number of animals: Blood samples from all males at termination and one male and one female pup per litter at PND 13 were assessed for serum levels for thyroid hormones (T4 and TSH).
- Animals fasted: Yes (adults)
- Parameters checked: Total thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: At the end of the administration period starting at about 10.00h (FOB) and from 14:00 h onwards (MA).
- Dose groups that were examined: The first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups.
- Battery of functions tested: Motor activity (MA); The functional observational battery (FOB) started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The observations were performed at random. For details see "Other".

IMMUNOLOGY: No

OTHER:
- Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Impairment of gait, Other findings

- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypes, Gait abnormalities, Activity/arousal level, Feces (consistency/color) excreted during examination (two minutes), Urine excreted within 2 minutes (amount/color), Rearing within 2 minutes, Other findings

- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), Touch sensitivity (touch response), Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (auditory startle response), Coordination of movements (righting response), Behavior during handling, Vocalization, Pain perception (tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings

- Motor activity assessment: The examinations were performed using the TSE Labmaster System. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Sacrifice and pathology:

All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. All paired organs were weighed together (left and right).

GROSS PATHOLOGY: Yes
- Organ weights:
All animals: final body weight, epididymides ovaries, prostate (ventral and dorsolateral part together, fixed), seminal vesicles with coagulating glands (fixed), testes, thyroid glands (with parathyroid glands) (fixed), uterus with cervix
5 animals per sex/test group: adrenal glands (fixed), brain, heart, kidneys, liver, spleen, thymus (fixed)
- Organ/tissue fixation: The following organs or tissues of all parental animals were fixed in in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, esophagus, epididymides (modified Davidson’s solution), extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

HISTOPATHOLOGY: Yes (see table 3 in section "Any other information on materials and methods incl. tables")

Other examinations:

- Estrous cycle: In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.

- Male reproduction data: The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. The mating and fertility indices were calculated for F1 litters.

- Female reproduction and delivery data: The pairing partners, the number of mating days until vaginal sperm were detected as well as the gestational status were recorded for F0 females. The mating, fertility and gestation indices were calculated for F1 litters. The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

Statistics:

- 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 and index: Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided).

- Male and female mating and fertility indices, females mated, females delivering, gestation index, females with stillborn pups, females with all stillborn pups: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided).

- Mating days until day 0 p.c., % post-implantation loss, pups stillborn, % perinatal loss, nipple development, implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival Index: Pairwise comparison of the dose group with the control group using the WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment.

- % live male day x, % live female day x: Comparison of the dose group with the control group was performed using the WILCOXON test (two-sided).

- Number of cycles and cycle length, rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity, organ weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was ≤0.05, a pair-wise comparison of the dose groups with the control group was performed using the 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 ≤0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided). For parameters with unidirectional changes, pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Discolored (orange) feces were observed in all cages of male and female animals of test group 3 (1000 mg/kg bw/d) and in all cages of males and five cages of females of test group 2 (300 mg/kg bw/d). This finding was considered to be related to treatment but not adverse. The test substance is used as a pigment and the color of feces is the same color as the test substance.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse findings were observed on mean body weight and body weight change values in male and female animals in any test group. The following findings were assessed to be not treatment-related since they occurred without a relation to dose. Mean body weight was significantly reduced in female animals of test group 2 (300 mg/kg bw/d) on PND 7. Body weight change values were significantly decreased in female animals of test group 1 (100 mg/kg bw/d) during pre-mating days 0 – 7 as well as 0 - 13.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in food consumption were observed in male and female animals of test groups 1 to 3 (100, 300 and 1000 mg/kg bw/d, respectively). The significantly lower mean value in female animals of test group 2 (300 mg/kg bw/d) on PND 4-7 was assessed as spontaneous in nature and not related to treatment since there was no relation to dose.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At the end of the administration period, in males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) sodium levels were significantly decreased but the values were within the historical control range (males, sodium 140.1-145.8 mmol/L). Therefore, this change was regarded as incidental and not treatment related.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Home cage observations: No test substance-related effects were observed.
- Open field observations: No test substance-related effects were observed. Discolored feces, orange, were observed in two male animals of test group 3 (1000 mg/kg bw/d).
- Sensorimotor tests/reflexes: No test substance-related effects were observed.
- Quantitative parameters: No test substance-related effects were observed.
- Motor activity measurement: Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group. Comparing the single intervals with the control groups, significantly increased values were measured for male animals of test group 2 (300 mg/kg bw/d) at interval 4 and for male animals of test group 1 (100 mg/kg bw/d) at intervals 4 and 6. These differences were regarded to be incidental and not related to treatment as these intervals occurred without relation to dose and the overall motor activity was not affected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to control group 0 (=100%), the mean absolute weight of the thyroid glands was significantly increased in male animals of test group 3 (120%). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared to control group 0 (=100%), the mean relative weight of the liver was significantly decreased in male animals of test group 3 (93%). All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The significant absolute thyroid gland weight increase in males of test group 3 (24.3 mg) was within the historical control range (19.300 – 27.600 mg). The same was true for the significant decrease of the relative liver weight in females of test group 3 (2.392%) which was also within the historical control values (2.4 – 3.312%). No histopathological correlates were seen in both organs. Therefore, these weight changes were not considered treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

An orange discoloration of the gastrointestinal content in the glandular stomach, jejunum, cecum, colon and rectum was noted in general in male and female animals of test groups 2 and 3. This color reflects the presence of the test substance which was also observed microscopically as gold-brown pigment particles in the lumen of these organs. These findings were not considered adverse. All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The female animal, which was not pregnant as well as the male mating partner did not show relevant gross lesions.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All histopathological 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. The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals. The female animal, which was not pregnant as well as the male mating partner did not show relevant histopathological findings.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- The male mating index calculated after the mating period to produce F1 litter was 100% in all test groups. Fertility was proven in nearly all of the F0 parental males of test groups 0, 1 and 3 (0, 100 and 1000 mg/kg bw/d) within the scheduled mating interval to produce F1 litter. Only male animal of test group 2 (300 mg/kg bw/d), which was mated with a female of test group 2 (300 mg/kg bw/d), did not generate pregnancy. The apparently infertile male rat did not show relevant gross lesions. Thus, the male fertility index was 100% in test groups 0, 1 as well as test group 3 and 90% in test group 2. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
- The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 1.9 days for test group 0 (control), 2.9 days for test group 1 (100 mg/kg bw/d) and 2.8 days for test groups 2 and 3 (300 and 1000 mg/kg bw/d) without any relation to dose. All sperm positive females of test groups 0 to 3 (control; 100, 300 and 1000 mg/kg bw/d) delivered pups with the exception of one female animal of test group 2, which did not deliver pups and showed no implantation sites. Thus, the female fertility index was 100% in test groups 0, 1 and 3, 90% in test group 2 without showing any relation to dose. The non-pregnant females had no relevant gross lesions or microscopic findings. The mean duration of gestation was 22.0 days in test group 0 (control) and 3 (1000 mg/kg bw/d), 22.1 days in test group 1 (100 mg/kg bw/d) and 22.4 days in test group 2 (300 mg/kg bw/d). The latter one showed statistical significance. However, the mean value of test group 2 is well within the range of the historical control data (GD range of 21.8 – 22.6 days). Furthermore, the finding was not related to dose. Therefore, it was assessed as incidental and not related to treatment. The gestation index was 100% in all test groups 0 – 3. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.8 / 13.9 / 13.7 and 12.4 implants/dam in test groups 0 - 3, respectively). The postimplantation loss was 7.0% in test group 0 (control), 6.4% in test group 1 (100 mg/kg bw/d), 14.7% in test group 2 (300 mg/kg bw/d) and 6.3% in test group 3 (1000 mg/kg bw/d). The mean number of F1 pups delivered per dam remained unaffected (12.8 / 13.0 / 11.9 and 11.6 pups/dam in test groups 0 - 3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 / 99.2 / 100 and 99.1% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
- Estrous cycle data revealed regular cycles in the rearing F1 female animals of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was between 3.9 and 4.0 days.

Dose descriptor:

NOAEL

Remarks:

for general systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to and including the highest dose level of 1000 mg/kg bw/d.

Critical effects observed:

no

Conclusions:

Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test, the oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d for male and female Wistar rats.

Executive summary:

The test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). 0.5% Sodium carboxymethyl cellulose in deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

Regarding clinical examinations, no treatment-related, adverse findings were observed up to a dose level of 1000 mg/kg bw/d. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Concerning pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. All 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.

Concerning fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. Mating behavior, conception, implantation and parturition were not affected. Concerning developmental toxicity, no treatment-related, adverse findings were observed. Pup status, viability, survival and growth  remained unaffected by the test substance treatment. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

According to OECD TG 422, GLP, Klimisch 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 Oct 2019 - 04 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

COMMISSION REGULATION (EU) No 260/2014 of 24 January 2014

Deviations:

not specified

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group (Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance under storage conditions over the test period was guaranteed until 29 December 2027 by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: Please, see section "Details on inhalation exposure"

FORM AS APPLIED IN THE TEST: dust aerosol

OTHER SPECIFICS
- physical state: solid
- color: orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat was the most frequently used laboratory animal, and there is extensive experience with this species. The rat is also proposed as a suitable test animal by OECD and the EPA. The Wistar strain was selected because a huge amount of historical control data was available for this strain. Furthermore, concerning pulmonary lavage in rats there is a considerable database for this species available in published literature (Henderson 1984, Henderson et al., 1985, Driscoll et al., 1990, Warheit et al., 1991 and 1995) and rats were sensitive in pulmonary toxicity studies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation (mean): 307g (on day -3)
- Fasting period before study: not specified
- Housing: The rats were housed together (up to 5 animals per cage)
- Diet: Mouse/rat laboratory diet; ad libitum
- Water: tap water, ad libitum
- Acclimation period: about 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- The food used in the study was assayed for chemical as well as for microbiological contaminants. On the basis of the duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants as well as for bacteria. On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 – 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark)

IN-LIFE DATES: From: 01 Oct 2019 (Arrival) To: 24 Oct 2019

Route of administration:

other: inhalation: dust aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.23 - <= 1.87 µm

Geometric standard deviation (GSD):

2.28

Remarks on MMAD:

The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 70 %.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-onlyaerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The exposure systems were located in exhaust hoods in an air conditioned room.
- Method of holding animals in test chamber: rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Source and rate of air: Compressed air was produced by an oil-free compressor. For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter, 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: 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. The so generated conditioned air was used to generate inhalation atmospheres.
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass dilution tube, Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. 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 via the cyclonic separator into the inhalation system.
- Temperature, humidity, pressure in air chamber: The daily mean relative humidities in the inhalation systems ranged between 38.9 and 60.2 %. The daily mean temperatures in the inhalation systems ranged between 22.2 and 23.4°C. Compressed air was filtered air pressurized to about 5-6 bar.
- Air flow rate and Air change rate were recording with the automated measuring system. The air flows were constantly maintained in the desired range. An air change between 66 and 68 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: gravimetrical measurement
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
Brief description of analytical method used:
- Calulation of nominal concentrations: The nominal concentration was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
- Analytical determination of concentrations: The concentrations of the inhalation atmospheres were determined by gravimetric measurement of filter samples in test groups 1 to 3. In these groups, the constancy of concentrations in each inhalation system was continuously monitored using scattered light photometers. In the control group (test group 0) no samples were taken.
- Particle size analysis was carried out with a cascade impactor.
- 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. During some daily exposures oscillations and higher variability occurred in the mid and high concentration. The reason for this behaviour could not be identified, because generation and exposure procedure were not changed all over the study. It was not reflected in the analytically determined concentrations, which integrate the variations over the sampling time.
Please see table 6 in section "Any other information on materials and methods incl. tables"

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, for five consecutive days

Dose / conc.:

3 mg/m³ air

Dose / conc.:

10 mg/m³ air

Dose / conc.:

30 mg/m³ air

No. of animals per sex per dose:

5/dose/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not specified
- Fasting period before blood sampling for clinical biochemistry: Before blood sampling and necropsy food will be withdrawn from the animals overnight (about 16-20 hours).
- Rationale for selecting recovery groups: not specified (random)
- Post-exposure recovery period : In order to check for any reversibility, progression or delay of toxic effects the following post-exposure observation groups were used, these being observed for about 3 weeks after the exposure period (5 animals per group).
- Other: By using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is with about 4 min shorter as compared to whole-body chambers with a higher chamber volume.

Positive control:

No

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: 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.

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: Yes
- Time schedule: The animals were weighed prior to the pre-exposure period (study day -3), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 9, 16, 23 and 25.
The body weight change was calculated as the difference of actual body weights and the
weights of last weighing.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure), in the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/group/time point
- Parameters checked in table 1 were examined (please see section "Any other information on materials and methods incl. tables")

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure), in the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Animals fasted: Yes
- How many animals: 5/group/time point
- Parameters checked in table 2 were examined (please see section "Any other information on materials and methods incl. tables")

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure). The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.
- Dose groups that were examined: All dose groups and the control group
- Number of animals: 5/group/time point
- Parameters checked in table 3 and 4 were examined (please see section "Any other information on materials and methods incl. tables")
- Antigens measured in BALF by ELISA: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Macrophage colony stimulating factor (M-CSF), Rodent osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. After the lavage procedure, the left and right lung lobes were instilled with formalin for weight assessment and further histopathological assessment. Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

GROSS PATHOLOGY: Yes
- Organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands. All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
- Organ/tissue fixation: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder.
Fixation was followed by histotechnical processing, examination by light microscopy and
assessment of findings according to the table 5 (plese see section "Any other information on materials and methods incl. tables").

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters.
- Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means.
- Blood parameters: For parameters with bidirectional changes, non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue 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). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity in animals of control and test group 1 through out the study period. In test group 2 (10 mg/m³), substance contaminated fur was observed on study day 3 and day 4 after exposure in one main group and one recovery group animal, respectively. In test group 3 (30 mg/m³), substance contaminated fur was observed for 7 animals on study day 2 after exposure, for 2 animals on study day 3 after exposure and for 4 animals on study day 4 after exposure. No other signs of toxicity were observed in test groups 2 and 3.

Mortality:

no mortality observed

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 through out the whole study period. The mean body weight change of the animals exposed to the test substance up to the highest concentration of 30 mg/m³ were comparable to the concurrent control animals throughout the study period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At study day 5, in males of test group 2 (10 mg/m3) platelet counts were significantly increased, but the alteration was not dose dependent. Therefore, it was regarded as incidental and not treatment related. After a 3-week recovery, in males of test group 1 (3 mg/m3) mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. Additionally, in males of test group 2 (10 mg/m3) MCH was significantly lower compared to controls. These changes were not dose dependent. In males of test groups 2 and 3 (10 and 30 mg/m3) relative basophil counts were increased (in test group 3 not statistically significantly), but the values were within the historical control range (males, relative basophils 0.1-0.6 %). Therefore, these alterations were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At study day 5, in males of test groups 2 and 3 (10 and 30 mg/m3) globulin values were decreased (in test group 3 not statistically significantly), but the values were within the historical control range (males, globulins 21.58-29.13 g/L). Therefore, this alteration was regarded as incidental and not treatment related. After the 3-week recovery, in males of test group 2 (10 mg/m3) total protein, globulin, glucose and cholesterol values were significantly decreased. However, the values were not dose dependently changed. Therefore, these alterations were regarded as incidental and not treatment related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, all mean absolute and relative weight parameters did not show significant differences when compared to the control group.
- In the recovery group, the mean relative kidney weight of males in test group 1 was significantly increased, however this change was not related to the exposure concentration and was considered as incidental and not treatment-related. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, two males in test group 2 had foci within the epididymis. These findings occurred individually and were therefore considered to be incidental or spontaneous in originband without any relation to treatment.
- In the recovery group, one male in test group 1 had focal liver constrictions. This finding occurred individually and was therefore 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):

- Histological findings in the lungs of main group animals: macrophages, which contained a brown to goldish pigment (presumably inhaled test compound)
were multifocally distributed within the lumen of alveoli and terminal bronchioles, with a concentration-dependent increase in number of macrophages and amount of pigment storage within each macrophage, starting from minimal in test group 1 (3 mg/m3) up to mild in test group 3 (30 mg/m3). In test group 1 few pigment particles were within macrophages and therefore could only be visualized at a 100x magnification. No extracellular pigment or signs of inflammation were detected. Single intravascular pigment-laden macrophages were present in animals of all test groups. Furthermore, a minimal number of pigment-laden macrophages were present in the bronchus-associated lymphoid tissue (BALT) in animals of all test groups, with a dose dependent increase in affected animals.
- Histological findings in the lungs of recovery group animals: Pigment-laden macrophages were multifocally distributed in the lumen of the alveoli of all animals of all test groups exposed to the test substance. In general, the number of pigmentladen alveolar macrophages was lower in all exposed recovery group animals compared to exposed animals of the corresponding main groups. In test group 1 (3 mg/m3) macrophages were so inconspicuously laden with pigment that this could only be visualized at 200x. In 3 out of 5 animals of test group 1 (3 mg/m3) the number of alveolar macrophages was not increased compared to the control group 0, but the present macrophages contained pigment. The remaining 2 animals of test group 1 (3 mg/m3) and all animals of test group 2 (10 mg/m3) displayed a minimal increase of alveolar macrophages laden with pigment. All animals of test group 3 (30 mg/m3) displayed a minimal to mild increase of pigment-laden alveolar macrophages, in 3 animals the macrophages formed aggregates, which showed a tendency to concentrate in the bronchiolo-alveolar junction. Single intravascular macrophages were present in animals of all test groups. Examination of the bronchus-associated lymphoid tissue (BALT) revealed a minimal to mild number of pigment-laden macrophages in animals of all test groups.

- Histological findings in the mediastinal lymph nodes of main group animals: In test group 3 (30 mg/m3), 2 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages.
- Histological findings in the mediastinal lymph nodes of recovery group animals: In test group 2 (10 mg/m3), 3 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages, whereas in test group 3 (30 mg/m3) in 3 out of 5 animals macrophages formed multifocal aggregates with a minimal to mild severity. For details please see table 5 in section “Any other information on results incl. Tables”.

- Histological findings in the tracheobronchial lymph nodes of main group animals: In test group 3 (30 mg/m3), 3 of 5 animals showed a minimal number of multifocally distributed single pigment-laden macrophages.
- Histological findings in the tracheobronchial lymph nodes of recovery group animals: In test group 2 (10 mg/m3), 1 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages, whereas in test group 3 (30 mg/m3) in 3 out of 5 animals macrophages formed multifocal aggregates with a minimal to mild severity.

- Histological findings in the larynx of main group animals: A minimal epithelial alteration, characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers) was present at level I in 1 animal of test group 1 (3 mg/m3), 1 animal of test group 2 (10 mg/m3) and 3 animals of test group 3 (30 mg/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.
- Histological findings in the larynx of recovery group animals: A minimal epithelial alteration characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers) was present at level I in 2 animals of test group 2 (10 mg/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.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

At study day 5, in males of test group 3 (30 mg/m3) absolute neutrophil counts were 13.7-fold higher compared to controls and absolute monocyte counts were increased, whereas total cell counts were not greater compared to controls. Relative neutrophil and monocyte counts were also increased. Therefore, higher neutrophil and monocyte counts may be regarded as a marginal adverse effect. After the 3-week recovery, in males of test group 3 (30 mg/m3) absolute macrophage counts were significantly increased, and in males of test group 1 (3 mg/m3) total cell counts in BAL were significantly higher compared to controls. However, the alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

At study day 5, in males of test group 3 (30 mg/m3) alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) activities were significantly increased. However, ALP mean was within the historical control range and GGT activities were marginally above this range (BAL, ALP 0.25-0.51 μkat/L; GGT 20-64 nkat/L). Therefore, these alterations were regarded as incidental and not treatment related. After the 3-week recovery in males of test group 1 (3 mg/m3) lactate dehydrogenase (LDH) and N-acetyl-β-glucosaminidase (NAG) activities were significantly increased, but the changes were not dose dependent. Therefore, these alterations were regarded as incidental and not treatment related.

At study day 5, in males of test groups 2 and 3 (10 and 30 mg/m3) IL-8/CINC-1 levels were significantly increased. Although these increases were marginal, in combination with significant higher neutrophil counts in the BAL of males of test group 3, it can be regarded as treatment related and adverse, whereas the IL-8/CINC-1 increase in test group 2 was isolated and therefore, it is regarded as treatment related but non-adverse. At study day 5 in males of test group 1 (3 mg/m3) osteopontin values were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

For details, please see table 1-3 in section "Any other information on results incl. tables"

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

10 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: findings in lavage fluid

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Critical effects observed:

no

Table 1: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study days 26 (3 weeks after last exposure). At study day 5, mean of differential cell counts in control group (test group 0) is based on only two individuals due to bad cell quality. Therefore, for these parameters, statistics could not be performed.

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
Total Cell count	0.9	0.9	0.9	1.8*	1,2	1.4
Eosinophils	0.0	4.9	0.5	0.8	0.0	0.0
Lymphocytes	1.0	0.3	0.0	+	+	+
Macrophages	0.7	0.7	0.6	1.9	1.2	1.4*
Neutrophils	1.2	0.6	13.7	0.7	1.9	1.2
Monocytes	+	+	+	+	+	+
Epithelial cells	0.0	0.1	0.0	+	+	+

* : p <= 0.05; ** : p <= 0.01 (One-sided Wilcoxon-test)

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

 

Table 2: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure)

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
Total Protein	1.0	1.0	0.9	1.2	0.9	0.8
GGT	1.0	1.1	1.5**	1.4	1.0	1.2
LDH	0.6	0.9	0.6	1.9*	1.1	1.3
ALP	1.1	1.0	1.3*	0.9	1.1	0.9
NAG	0.7	0.8	0.7	3.2**	1.0	1.9

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = N-Acetyl-β-glucosaminidase

* : p <= 0.05; ** : p <= 0.01 (One-sided Wilcoxon-test)

 

Table 3: Changes in antigen levels in BAL (x-fold of concurrent control means) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
MCP-1	1.0	1.0	1.1	0.8	0.8	0.7
CINC-1/IL-8	1.3	1.5*	1.8*	0.6	0.9	0.9
M-CSF	1.0	1.0	1.0	1.0	1.0	1.0
Osteopontin	2.1*	1.1	1.4	0.5	0.6	0.5

BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1; MCP-1 = monocyte chemoattractant protein-1; M-CSF = macrophage colony stimulating factor

* : p <= 0.05; ** : p <= 0.01 (One-sided Wilcoxon-test)

Conclusions:

Inhalation exposure of rats to 30 mg/m³ of the test substance on 5 consecutive days caused Increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in bronchoalveolar lavage. There was no histological correlate. All effects were reversible within 3 weeks exposure-free recovery period. Based on the findings in lavage fluid, the no observed adverse effect concentration (NOAEC) for local effects was 10 mg/m³ under current study conditions. The systemic NOAEC is above 30 mg/m³ (high concentration group).

Executive summary:

The purpose of this study was to determine the pulmonary toxicity in rats using a short term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of the test substance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects. For this purpose nine week old male Wistar rats (10 rats per concentration group) were noseonly exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) on 5 days for 6 hours per day. Body weight, mortality and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and histopathological changes in the target organs. During the exposure period the target concentrations were reached.

The particle size resulted in MMADs between 1.23 and 1.87 μm with GSDs between 2.08 and 2.45. The calculated mass fractions of particles below 3 μm aerodynamic size was greater than 70 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Main group:

- Test group 3 (30 mg/m³): Increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in BAL

- Test group 2 (10 mg/m³), test group 1 (3 mg/m³): No treatment-related adverse effects

Recovery group:

- Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³): No treatment-related adverse effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

According to OECD TG 412, GLP, Klimisch 1

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 Oct 2019 - 04 Jan 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

Deviations:

yes

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

COMMISSION REGULATION (EU) No 260/2014 of 24 January 2014

Deviations:

not specified

Principles of method if other than guideline:

5-day inhalation exposure with 21 days recovery group (Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van Ravenzwaay B, Landsiedel R. 2009. Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance Inhal Toxicol 21, 102-118)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): not specified
- Lot/batch number of test material: 0018514410
- Purity, including information on contaminants, isomers, etc.: 99.0 g/100 g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The stability of the test substance under storage conditions over the test period was guaranteed until 29 December 2027 by the sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: Please, see section "Details on inhalation exposure"

FORM AS APPLIED IN THE TEST: dust aerosol

OTHER SPECIFICS
- physical state: solid
- color: orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The rat was the most frequently used laboratory animal, and there is extensive experience with this species. The rat is also proposed as a suitable test animal by OECD and the EPA. The Wistar strain was selected because a huge amount of historical control data was available for this strain. Furthermore, concerning pulmonary lavage in rats there is a considerable database for this species available in published literature (Henderson 1984, Henderson et al., 1985, Driscoll et al., 1990, Warheit et al., 1991 and 1995) and rats were sensitive in pulmonary toxicity studies.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, 97633 Sulzfeld, Germany
- Age at study initiation: about 7 weeks
- Weight at study initiation (mean): 307g (on day -3)
- Fasting period before study: not specified
- Housing: The rats were housed together (up to 5 animals per cage)
- Diet: Mouse/rat laboratory diet; ad libitum
- Water: tap water, ad libitum
- Acclimation period: about 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
- The food used in the study was assayed for chemical as well as for microbiological contaminants. On the basis of the duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants as well as for bacteria. On the basis of the analytical findings the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 – 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark)

IN-LIFE DATES: From: 01 Oct 2019 (Arrival) To: 24 Oct 2019

Route of administration:

other: inhalation: dust aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.23 - <= 1.87 µm

Geometric standard deviation (GSD):

2.28

Remarks on MMAD:

The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 70 %.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-onlyaerodynamic exposure systems consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The exposure systems were located in exhaust hoods in an air conditioned room.
- Method of holding animals in test chamber: rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Source and rate of air: Compressed air was produced by an oil-free compressor. For this purpose, air is filtered by an inlet air strainer and introduced into the compressor. After passing through an second ultra filter, 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: 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. The so generated conditioned air was used to generate inhalation atmospheres.
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass dilution tube, Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. 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 via the cyclonic separator into the inhalation system.
- Temperature, humidity, pressure in air chamber: The daily mean relative humidities in the inhalation systems ranged between 38.9 and 60.2 %. The daily mean temperatures in the inhalation systems ranged between 22.2 and 23.4°C. Compressed air was filtered air pressurized to about 5-6 bar.
- Air flow rate and Air change rate were recording with the automated measuring system. The air flows were constantly maintained in the desired range. An air change between 66 and 68 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
- Method of particle size determination: gravimetrical measurement
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
Brief description of analytical method used:
- Calulation of nominal concentrations: The nominal concentration was calculated from the study means of the test-substance flow and the supply air flows used during exposure to generate the respective concentrations.
- Analytical determination of concentrations: The concentrations of the inhalation atmospheres were determined by gravimetric measurement of filter samples in test groups 1 to 3. In these groups, the constancy of concentrations in each inhalation system was continuously monitored using scattered light photometers. In the control group (test group 0) no samples were taken.
- Particle size analysis was carried out with a cascade impactor.
- 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. During some daily exposures oscillations and higher variability occurred in the mid and high concentration. The reason for this behaviour could not be identified, because generation and exposure procedure were not changed all over the study. It was not reflected in the analytically determined concentrations, which integrate the variations over the sampling time.
Please see table 6 in section "Any other information on materials and methods incl. tables"

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily, for five consecutive days

Dose / conc.:

3 mg/m³ air

Dose / conc.:

10 mg/m³ air

Dose / conc.:

30 mg/m³ air

No. of animals per sex per dose:

5/dose/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not specified
- Fasting period before blood sampling for clinical biochemistry: Before blood sampling and necropsy food will be withdrawn from the animals overnight (about 16-20 hours).
- Rationale for selecting recovery groups: not specified (random)
- Post-exposure recovery period : In order to check for any reversibility, progression or delay of toxic effects the following post-exposure observation groups were used, these being observed for about 3 weeks after the exposure period (5 animals per group).
- Other: By using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is with about 4 min shorter as compared to whole-body chambers with a higher chamber volume.

Positive control:

No

Observations and examinations performed and frequency:

MORTALITY: Yes
- Time schedule: 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.

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during the pre-exposure period and on post-exposure observation days on working days. On exposure days, clinical observation was performed at least 3 times daily, before, during and after exposure. During exposure only a group wise examination was possible.

BODY WEIGHT: Yes
- Time schedule: The animals were weighed prior to the pre-exposure period (study day -3), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 9, 16, 23 and 25.
The body weight change was calculated as the difference of actual body weights and the
weights of last weighing.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure), in the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/group/time point
- Parameters checked in table 1 were examined (please see section "Any other information on materials and methods incl. tables")

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure), in the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Animals fasted: Yes
- How many animals: 5/group/time point
- Parameters checked in table 2 were examined (please see section "Any other information on materials and methods incl. tables")

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: On study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure). The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.
- Dose groups that were examined: All dose groups and the control group
- Number of animals: 5/group/time point
- Parameters checked in table 3 and 4 were examined (please see section "Any other information on materials and methods incl. tables")
- Antigens measured in BALF by ELISA: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Macrophage colony stimulating factor (M-CSF), Rodent osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

NECROPSY: All animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. After the lavage procedure, the left and right lung lobes were instilled with formalin for weight assessment and further histopathological assessment. Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

GROSS PATHOLOGY: Yes
- Organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (final body weight), Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands. All paired organs were weighed together (left and right).

HISTOPATHOLOGY: Yes
- Organ/tissue fixation: The following organs or tissues were fixed in 4% buffered formaldehyde or modified Davidson’s solution: All gross lesions, Adrenal glands, Brain with olfactory bulb, Bone marrow (femur), Epididymides, Eyes with optic nerve, Heart, Kidneys, Larynx/Pharynx, Liver, Lungs, Lymph nodes (tracheobronchial and mediastinal lymph nodes), Nose (nasal cavity), Oesophagus, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Stomach (forestomach and glandular stomach), Spleen, Testes, Thyroid glands, Thymus, Trachea, Urinary bladder.
Fixation was followed by histotechnical processing, examination by light microscopy and
assessment of findings according to the table 5 (plese see section "Any other information on materials and methods incl. tables").

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters.
- Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means.
- Blood parameters: For parameters with bidirectional changes, non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue 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). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using WILCOXON-test (two-sided) for the equal medians.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity in animals of control and test group 1 through out the study period. In test group 2 (10 mg/m³), substance contaminated fur was observed on study day 3 and day 4 after exposure in one main group and one recovery group animal, respectively. In test group 3 (30 mg/m³), substance contaminated fur was observed for 7 animals on study day 2 after exposure, for 2 animals on study day 3 after exposure and for 4 animals on study day 4 after exposure. No other signs of toxicity were observed in test groups 2 and 3.

Mortality:

no mortality observed

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 through out the whole study period. The mean body weight change of the animals exposed to the test substance up to the highest concentration of 30 mg/m³ were comparable to the concurrent control animals throughout the study period.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At study day 5, in males of test group 2 (10 mg/m3) platelet counts were significantly increased, but the alteration was not dose dependent. Therefore, it was regarded as incidental and not treatment related. After a 3-week recovery, in males of test group 1 (3 mg/m3) mean corpuscular volume (MCV) and mean corpuscular hemoglobin content (MCH) were significantly decreased. Additionally, in males of test group 2 (10 mg/m3) MCH was significantly lower compared to controls. These changes were not dose dependent. In males of test groups 2 and 3 (10 and 30 mg/m3) relative basophil counts were increased (in test group 3 not statistically significantly), but the values were within the historical control range (males, relative basophils 0.1-0.6 %). Therefore, these alterations were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed. At study day 5, in males of test groups 2 and 3 (10 and 30 mg/m3) globulin values were decreased (in test group 3 not statistically significantly), but the values were within the historical control range (males, globulins 21.58-29.13 g/L). Therefore, this alteration was regarded as incidental and not treatment related. After the 3-week recovery, in males of test group 2 (10 mg/m3) total protein, globulin, glucose and cholesterol values were significantly decreased. However, the values were not dose dependently changed. Therefore, these alterations were regarded as incidental and not treatment related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, all mean absolute and relative weight parameters did not show significant differences when compared to the control group.
- In the recovery group, the mean relative kidney weight of males in test group 1 was significantly increased, however this change was not related to the exposure concentration and was considered as incidental and not treatment-related. All other mean absolute and relative weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- In the main group, two males in test group 2 had foci within the epididymis. These findings occurred individually and were therefore considered to be incidental or spontaneous in originband without any relation to treatment.
- In the recovery group, one male in test group 1 had focal liver constrictions. This finding occurred individually and was therefore 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):

- Histological findings in the lungs of main group animals: macrophages, which contained a brown to goldish pigment (presumably inhaled test compound)
were multifocally distributed within the lumen of alveoli and terminal bronchioles, with a concentration-dependent increase in number of macrophages and amount of pigment storage within each macrophage, starting from minimal in test group 1 (3 mg/m3) up to mild in test group 3 (30 mg/m3). In test group 1 few pigment particles were within macrophages and therefore could only be visualized at a 100x magnification. No extracellular pigment or signs of inflammation were detected. Single intravascular pigment-laden macrophages were present in animals of all test groups. Furthermore, a minimal number of pigment-laden macrophages were present in the bronchus-associated lymphoid tissue (BALT) in animals of all test groups, with a dose dependent increase in affected animals.
- Histological findings in the lungs of recovery group animals: Pigment-laden macrophages were multifocally distributed in the lumen of the alveoli of all animals of all test groups exposed to the test substance. In general, the number of pigmentladen alveolar macrophages was lower in all exposed recovery group animals compared to exposed animals of the corresponding main groups. In test group 1 (3 mg/m3) macrophages were so inconspicuously laden with pigment that this could only be visualized at 200x. In 3 out of 5 animals of test group 1 (3 mg/m3) the number of alveolar macrophages was not increased compared to the control group 0, but the present macrophages contained pigment. The remaining 2 animals of test group 1 (3 mg/m3) and all animals of test group 2 (10 mg/m3) displayed a minimal increase of alveolar macrophages laden with pigment. All animals of test group 3 (30 mg/m3) displayed a minimal to mild increase of pigment-laden alveolar macrophages, in 3 animals the macrophages formed aggregates, which showed a tendency to concentrate in the bronchiolo-alveolar junction. Single intravascular macrophages were present in animals of all test groups. Examination of the bronchus-associated lymphoid tissue (BALT) revealed a minimal to mild number of pigment-laden macrophages in animals of all test groups.

- Histological findings in the mediastinal lymph nodes of main group animals: In test group 3 (30 mg/m3), 2 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages.
- Histological findings in the mediastinal lymph nodes of recovery group animals: In test group 2 (10 mg/m3), 3 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages, whereas in test group 3 (30 mg/m3) in 3 out of 5 animals macrophages formed multifocal aggregates with a minimal to mild severity. For details please see table 5 in section “Any other information on results incl. Tables”.

- Histological findings in the tracheobronchial lymph nodes of main group animals: In test group 3 (30 mg/m3), 3 of 5 animals showed a minimal number of multifocally distributed single pigment-laden macrophages.
- Histological findings in the tracheobronchial lymph nodes of recovery group animals: In test group 2 (10 mg/m3), 1 of 5 animals displayed minimal number of multifocally distributed single pigment-laden macrophages, whereas in test group 3 (30 mg/m3) in 3 out of 5 animals macrophages formed multifocal aggregates with a minimal to mild severity.

- Histological findings in the larynx of main group animals: A minimal epithelial alteration, characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers) was present at level I in 1 animal of test group 1 (3 mg/m3), 1 animal of test group 2 (10 mg/m3) and 3 animals of test group 3 (30 mg/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.
- Histological findings in the larynx of recovery group animals: A minimal epithelial alteration characterized by a focal loss of cilia and flattening of the cells (three to four stratified layers) was present at level I in 2 animals of test group 2 (10 mg/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.

Histopathological findings: neoplastic:

not specified

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

At study day 5, in males of test group 3 (30 mg/m3) absolute neutrophil counts were 13.7-fold higher compared to controls and absolute monocyte counts were increased, whereas total cell counts were not greater compared to controls. Relative neutrophil and monocyte counts were also increased. Therefore, higher neutrophil and monocyte counts may be regarded as a marginal adverse effect. After the 3-week recovery, in males of test group 3 (30 mg/m3) absolute macrophage counts were significantly increased, and in males of test group 1 (3 mg/m3) total cell counts in BAL were significantly higher compared to controls. However, the alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

At study day 5, in males of test group 3 (30 mg/m3) alkaline phosphatase (ALP) and γ-glutamyl transferase (GGT) activities were significantly increased. However, ALP mean was within the historical control range and GGT activities were marginally above this range (BAL, ALP 0.25-0.51 μkat/L; GGT 20-64 nkat/L). Therefore, these alterations were regarded as incidental and not treatment related. After the 3-week recovery in males of test group 1 (3 mg/m3) lactate dehydrogenase (LDH) and N-acetyl-β-glucosaminidase (NAG) activities were significantly increased, but the changes were not dose dependent. Therefore, these alterations were regarded as incidental and not treatment related.

At study day 5, in males of test groups 2 and 3 (10 and 30 mg/m3) IL-8/CINC-1 levels were significantly increased. Although these increases were marginal, in combination with significant higher neutrophil counts in the BAL of males of test group 3, it can be regarded as treatment related and adverse, whereas the IL-8/CINC-1 increase in test group 2 was isolated and therefore, it is regarded as treatment related but non-adverse. At study day 5 in males of test group 1 (3 mg/m3) osteopontin values were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

For details, please see table 1-3 in section "Any other information on results incl. tables"

Dose descriptor:

NOAEC

Remarks:

for local effects

Effect level:

10 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: findings in lavage fluid

Dose descriptor:

NOAEC

Remarks:

for systemic effects

Effect level:

30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects observed up to and including the highest dose tested.

Critical effects observed:

no

Table 1: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study days 26 (3 weeks after last exposure). At study day 5, mean of differential cell counts in control group (test group 0) is based on only two individuals due to bad cell quality. Therefore, for these parameters, statistics could not be performed.

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
Total Cell count	0.9	0.9	0.9	1.8*	1,2	1.4
Eosinophils	0.0	4.9	0.5	0.8	0.0	0.0
Lymphocytes	1.0	0.3	0.0	+	+	+
Macrophages	0.7	0.7	0.6	1.9	1.2	1.4*
Neutrophils	1.2	0.6	13.7	0.7	1.9	1.2
Monocytes	+	+	+	+	+	+
Epithelial cells	0.0	0.1	0.0	+	+	+

* : p <= 0.05; ** : p <= 0.01 (One-sided Wilcoxon-test)

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

 

Table 2: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure)

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
Total Protein	1.0	1.0	0.9	1.2	0.9	0.8
GGT	1.0	1.1	1.5**	1.4	1.0	1.2
LDH	0.6	0.9	0.6	1.9*	1.1	1.3
ALP	1.1	1.0	1.3*	0.9	1.1	0.9
NAG	0.7	0.8	0.7	3.2**	1.0	1.9

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = N-Acetyl-β-glucosaminidase

* : p <= 0.05; ** : p <= 0.01 (One-sided Wilcoxon-test)

 

Table 3: Changes in antigen levels in BAL (x-fold of concurrent control means) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure).

Analyte	Study day 5			Study day 26
	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3	Gr. 1 3 mg/m3	Gr. 2 10 mg/m3	Gr. 3 30 mg/m3
MCP-1	1.0	1.0	1.1	0.8	0.8	0.7
CINC-1/IL-8	1.3	1.5*	1.8*	0.6	0.9	0.9
M-CSF	1.0	1.0	1.0	1.0	1.0	1.0
Osteopontin	2.1*	1.1	1.4	0.5	0.6	0.5

BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1; MCP-1 = monocyte chemoattractant protein-1; M-CSF = macrophage colony stimulating factor

* : p <= 0.05; ** : p <= 0.01 (One-sided Wilcoxon-test)

Conclusions:

Inhalation exposure of rats to 30 mg/m³ of the test substance on 5 consecutive days caused Increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in bronchoalveolar lavage. There was no histological correlate. All effects were reversible within 3 weeks exposure-free recovery period. Based on the findings in lavage fluid, the no observed adverse effect concentration (NOAEC) for local effects was 10 mg/m³ under current study conditions. The systemic NOAEC is above 30 mg/m³ (high concentration group).

Executive summary:

The purpose of this study was to determine the pulmonary toxicity in rats using a short term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of the test substance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects. For this purpose nine week old male Wistar rats (10 rats per concentration group) were noseonly exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) on 5 days for 6 hours per day. Body weight, mortality and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and histopathological changes in the target organs. During the exposure period the target concentrations were reached.

The particle size resulted in MMADs between 1.23 and 1.87 μm with GSDs between 2.08 and 2.45. The calculated mass fractions of particles below 3 μm aerodynamic size was greater than 70 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Main group:

- Test group 3 (30 mg/m³): Increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in BAL

- Test group 2 (10 mg/m³), test group 1 (3 mg/m³): No treatment-related adverse effects

Recovery group:

- Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³): No treatment-related adverse effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Species:

rat

Quality of whole database:

According to OECD TG 412, GLP, Klimisch 1

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

The test substance was administered daily as a suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3). 0.5% Sodium carboxymethyl cellulose in deionized water served as vehicle, control animals were dosed daily with the vehicle only. The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Regarding clinical examinations, clinical pathology, pathology, no treatment-related, adverse findings were observed up to a dose level of 1000 mg/kg bw/d.  All 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. Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test the oral administration by gavage to male and female Wistar rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.

 

Inhalation:

In the course of this study, nine week old male Wistar rats (10 rats per concentration group) were nose-only exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) on 5 days for 6 hours per day. Body weight, mortality and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and histopathological changes in the target organs. During the exposure period the target concentrations were reached. The particle size resulted in MMADs between 1.23 and 1.87 μm with GSDs between 2.08 and 2.45. The calculated mass fractions of particles below 3 μm aerodynamic size was greater than 70 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. Inhalation exposure of rats to 30 mg/m³ of the test substance on 5 consecutive days caused increased absolute and relative neutrophil and monocyte counts as well as IL-8/CINC-1 values in bronchoalveolar lavage. The local pulmonary effects observed are considered to appear at lung overload causing concentrations. There was no histological correlate and all effects were reversible within 3 weeks exposure-free recovery period. Based on the findings in lavage fluid, the no observed adverse effect concentration (NOAEC) for local effects was 10 mg/m³ under current study conditions. The systemic NOAEC was above 30 mg/m³ (high concentration group).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.