Registration Dossier

Administrative data

Description of key information

Non-human information
Oral: The repeated dose toxicity of water soluble zinc sulphate and zinc monoglycerolate has been examined in a total of 3 subchronic oral feeding key studies.
The lowest NOAEL was determined to be 31.5 mg/kg bw/day of zinc monoglycerolate which equals a total zinc exposure of approximate 13 mg/kg bw/day.
Inhalation: The repeated dose inhalation toxicity of micro and nano-ZnO has been examined respectively in a subacute (28 days) and subchronic (90 days) inhalation study. The lowest NOAEL was determined to be 0.5 mg/m3 for micro ZnO. For nano-ZnO the NOAEL was determined to be 1.5mg/m3.
Dermal: 28 days dermal toxicity study with nano-ZnO: LOAEL= 75 mg/kg bw/day
Human information
Oral: In studies in which humans were supplemented with zinc (as zinc gluconate), at a LOAEL of 2.5 mg Zn/kg bw/day (150mg/day) decreased ESOD activity and effects are seen as a result of copper imbalance. The NOAEL= 0.83 mg/kg bw/day (50mg/day)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study, key study used in EU risk assessment report for Zinc metal 2004
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
none
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.05% (= male 31.52 mg/kg bw and female 35.78 mg/kg bw)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.2% (= male 127.52 mg/kg bw and female 145.91mg/kg bw)
Basis:
nominal in diet
No. of animals per sex per dose:
20 males
20 females
Details on study design:
Groups of 20 male and 20 female Sprague-Dawley rats were fed zinc monoglycerolate at dietary levels of 0, 0.05 or 0.2% (equal to 0, 31.52 or 127.52 mg/kg for males and 0, 35.78 or 145.91 mg/kg bw for females, respectively) for a period of 13 weeks in a study performed according to OECD 408. Asimilar group was fed 1% (equal to 719 and 805 mg/kg bw/day for males and females, respectively) of zinc monoglycerolate up to day 58 of the study when a deterioration in their clinical condition (poor physical health and reduced food intake) necessitated reducing the dietary level to 0.5% (equal to 632 and 759 mg/kg bw/day for males and females, respectively). However, as no improvement occurred these rats were killed on humane grounds on day 64 of the study.
Observations and examinations performed and frequency:
according to guideline
Sacrifice and pathology:
according to guideline
Statistics:
according to guideline
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Details on results:
The rats fed 1% of test substance for 58 days and then 0.5% to day 64 had were killed before the end of the study due to ill-health developed hypocupremia manifested as a hypochromic microcytic regenerative type anaemia (low haemoglobin and haematocrit, decreased MCV and MCH, and increased MCHC, red blood cell and reticulocyte count). Enlargement of the mesenteric lymph nodes and slight pitting of the surface of the kidneys were noted. Severe pancreatic degeneration and pathological changes in the spleen, kidneys, incisors, eyes and bones were observed. The testes of all males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female the uterus was hypoplastic.

In the main study, a dietary level of 0.2% increases in plasma ALAT, alkaline phosphatase and creatine kinase were observed in males and in plasma creatine kinase in females. Total plasma cholesterol was reduced in both males and females. The changes were statistically significant but small in absolute terms. No changes in haematological parameters were seen at 0.05 and 0.2%. A dose related reduction in the quantity of abdominal fat was noted in male rats at 0.05 and 0.2%. Enlargement of the mesenteric lymph nodes was apparent in 6 out of 20 rats fed 0.2% and in one male fed 0.05%. Microscopic examination showed a reduction in the number of trabeculae in the metaphysis of the tibia of 5 male and 3 female rats fed 0.2%, 4 males and 1 female had a similar reduction in the metaphysis of the femur. Pancreatic cell necrosis was seen in both sexes at 0.2% and a slight, but statistically not significant increase could be noted at 0.05% (3 males and 1 female). This pancreatic cell necrosis was seen also in 1 control male. A reduction in the number of pigmentated macrophages in the red pulp of the spleen was observed in both sexes at 0.2% and a marginal reduction was also seen in males at 0.05%. In the animals given 0.05 and 0.2% no effects were found on the reproductive organs.
Dose descriptor:
NOAEL
Effect level:
31.52 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
NOAEL in this study is 31.52 mg/kg bw (approximately 13.26 mg Zn2+/kg bw)
Executive summary:

Groups of 20 male and 20 female Sprague-Dawley rats were fed zinc monoglycerolate at dietary levels of 0, 0.05 or 0.2% (equal to 0, 31.52 or 127.52 mg/kg for males and 0, 35.78 or 145.91 mg/kg bw for females, respectively) for a period of 13 weeks in a study performed according to OECD 408. A similar group was fed 1% (equal to 719 and 805 mg/kg bw/day for males and females, respectively) of zinc monoglycerolate up to day 58 of the study when a deterioration in their clinical condition (poor physical health and reduced food intake) necessitated reducing the dietary level to 0.5% (equal to 632 and 759 mg/kg bw/day for males and females, respectively). However, as no improvement occurred these rats were killed on humane grounds on day 64 of the study. These rats developed hypocupremia manifested as a hypochromic microcytic regenerative type anaemia (low haemoglobin and haematocrit, decreased MCV and MCH, and increased MCHC, red blood cell and reticulocyte count). Enlargement of the mesenteric lymph nodes and slight pitting of the surface of the kidneys were noted. Severe pancreatic degeneration and pathological changes in the spleen, kidneys, incisors, eyes and bones were observed. The testes of all males showed hypoplasia of the seminiferous tubules to a varying degree and in addition the prostate and seminal vesicles showed hypoplasia. In all but one female the uterus was hypoplastic.

All other rats survived to the end of the 13 weeks treatment. At a dietary level of 0.2% increases in plasma ALAT, alkaline phosphatase and creatine kinase were observed in males and in plasma creatine kinase in females. Total plasma cholesterol was reduced in both males and females. The changes were statistically significant but small in absolute terms. No changes in haematological parameters were seen at 0.05 and 0.2%. A dose related reduction in the quantity of abdominal fat was noted in male rats at 0.05 and 0.2%. Enlargement of the mesenteric lymph nodes was apparent in 6 out of 20 rats fed 0.2% and in one male fed 0.05%. Microscopic examination showed a reduction in the number of trabeculae in the metaphysis of the tibia of 5 male and 3 female rats fed 0.2%, 4 males and 1 female had a similar reduction in the metaphysis of the femur. Pancreatic cell necrosis was seen in both sexes at 0.2% and a slight, but statistically not significant increase could be noted at 0.05% (3 males and 1 female). This pancreatic cell necrosis was seen also in 1 control male. A reduction in the number of pigmentated macrophages in the red pulp of the spleen was observed in both sexes at 0.2% and a marginal reduction was also seen in males at 0.05%. In the animals given 0.05 and 0.2% no effects were found on the reproductive organs.

Since the pancreatic cell necrosis, being without statistical significance at 0.05%, was also apparent in 1 control male and because the reduction in pigmented macrophages in the spleen was only marginal at 0.05% without any haematological changes the dose level of 0.05%, is considered as a NOAEL. This dose level is equal to 31.52 or 35.78 mg zinc monoglycerolate/kg bw for males and females, respectively, so the NOAEL in this study is 31.52 mg/kg bw (»13.26 mg Zn2+/kg bw)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
13.3 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the OECD Guideline with few deviations and in compliance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
,additional endpoints investigated
Deviations:
yes
Remarks:
(only male rats were used)
Principles of method if other than guideline:
Additional endpoints: bronchoalveolar lavage, cell proliferation, electron microscope analysis (non-GLP), toxicokinetics (non-GLP); three doses of the test substance (coated ZnO nanomaterial) were compared to one dose of a reference substance (non-coated microscaled ZnO)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld/Germany
- Age at study initiation: Approx. 8 weeks
- Weight at study initiation: Approx. 230g
- Fasting period before study: No
- Housing: 2 rats per cage, absorbing softwood bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 1 d followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2°C
- Humidity: 55 +/- 15°C
- Air changes (per hr): fully airconditioned
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Particle size: MMAD was checked using a Marple impactor. The MMAD of the aerosol entering the exposure units was < 3.0 μm (GSD: about 1.5).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1L/min
- System of generating particulates/aerosols: Feeding system and high-pressure, high-velocity pressurized air dispersion with computerized control
- Temperature, humidity, pressure in air chamber: 22 +/- 2°C, 55 +/- 15%,
- Air flow rate: 1L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetrically by filter samples
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The target aerosol concentrations of 0.3, 1.5 and 4.5 mg Z-COTE HP1/m3 as well as 4.5 mg microscaled ZnO/m3 were achieved to 103%, 99%, 99%, and 100%, respectively.
Duration of treatment / exposure:
3 months, 5 consecutive days per week, 6 h per day
Frequency of treatment:
5 consecutive days per week
Remarks:
Doses / Concentrations:
0.3, 1.5 and 4.5 mg/m3
Basis:
other: target aerosol concentration of test substance
Remarks:
Doses / Concentrations:
4.5 mg/m3
Basis:
other: target aerosol concentration of reference substance
No. of animals per sex per dose:
65/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the results of the DRF study (Fraunhofer ITEM study no. 02G09005) nominal aerosol concentrations of 0.3, 1.5 and 4.5 mg/m3 were used for the test substance in the low, mid and high dose groups, respectively. For the reference substance an aerosol concentration of 4.5 mg/m3 was used.
- Rationale for animal assignment: The animals were allocated to groups on a body weight basis. The animals were weighed, randomized and grouped by the PROVANTIS system (management of toxicology laboratory data, Instem Computer System Ltd., Walton Industrial Estate, Stone, Staffs, ST 15 OLT, Great Britain, ProvantisTM version 8.2.0.8).
Positive control:
No
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Twice a day and once on weekends during exposure period

BODY WEIGHT: Weekly during exposure period

FOOD CONSUMPTION:
- Food consumption for each dose group determined and mean daily diet consumption calculated as g food/day

HAEMATOLOGY:
- Time schedule for collection of blood: First day after end of exposure period
- Anaesthetic used for blood collection: Halothane
- Animals fasted: 16 h, water ad libitum
- No. of animals: 10 animals per dose group

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: First day after end of exposure period
- Animals fasted: 16 h, water ad libitum
- No. of animals: 10 animals per dose group

URINALYSIS:
- Time schedule for collection of urine: First day after end of exposure period
- Animals fasted: 16 h, water ad libitum

OTHER:
- Bronchoalveolar lavage (1, 8, and 29 d after end of exposure period: cell count, biochemical parameters, cytokines)
- Lung cell proliferation (9 and 29 d after end of exposure period)
- Toxicokinetics according OECD TG 417, chemical Zn analysis in organs, blood, and urine (1 and 29 d after end of exposure period)
- Electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kideny, liver, spleen, and erythrocytes (1, 8, and 29 d after end of exposure period)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
- Bronchoalveolar lavage (1, 8, and 29 d after end of exposure period: cell count, biochemical parameters, cytokines)
- Lung cell proliferation (9 and 29 d after end of exposure period)
- Toxicokinetics according OECD TG 417, chemical Zn analysis in organs, blood, and urine (1 and 29 d after end of exposure period)
- Electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kideny, liver, spleen, and erythrocytes (1, 8, and 29 d after end of exposure period)
Statistics:
Differences between groups were considered statistically significant at p < 0.05. Data were analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups were compared with the means of the control groups using Dunnett's Test. The statistical evaluation of the histopathological findings was done with the two-tailed Fisher Test by Provantis system.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see "Any other information on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see "Any other information on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see "Any other information on results"
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see "Any other information on results"
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see "Any other information on results"
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Due to a technical defect of the clean air supply 15/65 rats of group 3 died during the exposure. One animal died during narcosis performed for the administration of BrdU. 4 animals died during the study or were killed in a moribund condition. 2 of them (group 1 and 5) died due to a severe urogenital infection (group 5) and one due to malignant lymphoma while the cause of death remained unclear for the fourth rat (group 1). Therefore no animal died due to test substance related effects.
Several rats of the treatment group and a few rats of the control group showed very slight red-brown coloured noses and brown-red encrusted eyelids on some days directly after the 6 hour exposure period. These are temporary - probably stress-related - findings often seen in rodent nose-only inhalation studies which disappeared directly after the end of the daily exposure. Generally, the clinical health of all animals was within the normal range seen in rats of this strain and age.

BODY WEIGHT AND WEIGHT GAIN
Group 3 showed statistically significant reduced body weights on Day 28 and 35 compared to control.

FOOD CONSUMPTION
Statistically significant decrease of food consumption as compared to controls was observed on several dates in groups 2 to 5.

HAEMATOLOGY
No test item related findings were observed.

CLINICAL CHEMISTRY
Inorganic phosphate was significantly decreased in group 3, 4, and 5.

URINALYSIS
No significant changes were observed.

ORGAN WEIGHTS
Based on the conclusion of the authors stastically significant lung weights were determined in the animals exposed to the microscale ZnO, no other relevant changes.

GROSS PATHOLOGY
No test item-related findings were observed.

HISTOPATHOLOGY
see "Any other information on results incl. tables"

OTHER FINDINGS
-Bronochoalveolar lavage:
At day 1 after exposure statistically significant increases of polymorphnuclear neutrophils (PMN), lymphocytes, lactic dehydrogenase, beta-glucuronidase and total protein and decreases of macrophages were detected in group 4 and 5. These effects were fully reversible within 8 d except of the PMN increase in group 5 (not significant). The measurement of the reactive oxygen species (ROI) produced by alveolar macrophages showed a maximum activation status of the respiratory burst in alveolar macrophage cultures whithout any difference between nano- and microscaled ZnO. Significantly decreased ROI secretions were observed in the 1.5 and 4.5mg/m3 Z-COTE HP1 treated animals compared to the control, a stronger decrease in the microscaled treatment group. Including Zymosane stimulation significant increases were detected in the 1.5 and 4.5mg/m3 Z-COTE HP1 groups after 1 and 8 d with a normalization after 29 d. CINC-1 (IL-8 analogue in rats) as chemotactic factor for the neutrophilic influx after inflammatory stimuli into the lung was significantly increased in group 5 from day 1 until Day 8 postexposure and had normalized to control level after 29 d.
- Cell proliferation: No indication of an induction of a hyperplastic effect of the test substance or the reference substance was observed.
- Toxicokinetics: On the first day postexposure the Zn content in lungs of animals treated with the Z-COTE HP1 high dose was increased to 180% compared to the control. The deposite mass of the test substance in the 90 d exposure period was approx. 2000 µg/lung and the analytical results demonstrated a practically complete dissolution of the retained test substance. No significantly increased amounts of the test item were detected in any other body compartment demonstrating the rapid elimination.
- Electron microscopy: Electron dense structures were found one and 8 days postexposure in the cytoplasm of different cells and free in the lung lining fluid of animals treated with nanoscaled and microscaled ZnO as well as in animals of the control group. These structures were composed of irregular homogenous to fine granular material which measured only a few nanometers.
Dose descriptor:
NOAEL
Effect level:
1.5 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

CLINICAL SIGNS AND MORTALITY

Due to a technical defect of the clean air supply 15/65 rats of group 3 died during the exposure. One animal died during narcosis performed for the administration of BrdU. 4 animals died during the study or were killed in a moribund condition. 2 of them (group 1 and 5) died due to a severe urogenital infection (group 5) and one due to malignant lymphoma while the cause of death remained unclear for the fourth rat (group 1). Therefore no animal died due to test substance related effects. Several rats of the treatment group and a few rats of the control group showed very slight red-brown coloured noses and brown-red encrusted eyelids on some days directly after the 6 hour exposure period. These are temporary - probably stress-related - findings often seen in rodent nose-only inhalation studies which disappeared directly after the end of the daily exposure. Generally, the clinical health of all animals was within the normal range seen in rats of this strain and age.

BODY WEIGHT AND WEIGHT GAIN

Group 3 showed statistically significant reduced body weights on day 28 and 35 compared to control.

FOOD CONSUMPTION

Statistically sigificant decrease of food consumption as compared to controls was observed on several dates in groups 2 to 5.

HAEMATOLOGY

No test item related findings were observed.

CLINICAL CHEMISTRY

Inorganic phosphate was significantly decreased in group 3, 4, and 5.

URINALYSIS

No significant changes were observed.

ORGAN WEIGHTS

Based on the conclusion of the authors stastically significant lung weights were determined in the animals exposed to the microscale ZnO, no other relevant changes.

GROSS PATHOLOGY

No test item-related findings were observed.

BRONCHOALVEOLAR LAVAGE

At day 1 after exposure statistically significant increases of polymorphnuclear neutrophils (PMN), lymphocytes, lactic dehydrogenase (LDH), beta-glucuronidase and total protein and decreases of macrophages were detected in group 5. In group 4 only LDH was increased. These effects were fully reversible within 8 d except of the PMN increase in group 5 (not significant). The measurement of the reactive oxygen species (ROI) produced by alveolar macrophages showed a maximum activation status of the respiratory burst in alveolar macrophage cultures whithout any difference between nano- and microscaled ZnO. Significantly decreased ROI secretions were observed in the 1.5 and 4.5 mg/m3 Z-COTE HP1 treated animals compared to the control, a stronger decrease in the microscaled treatment group. Including Zymosane stimulation significant increases were detected in the 1.5 and 4.5 mg/m3 Z-COTE HP1 groups after 1 and 8 d with a normalization after 29 d. CINC-1 (IL-8 analogue in rats) as chemotactic factor for the neutrophilic influx after inflammatory stimuli into the lung was significantly increased in group 5 from day 1 until day 8 postexposure and had normalized to control level after 29 d.

CELL PROLIFERATION

No indication of an induction of a hyperplastic effect of the test substance or the reference substance was observed.

TOXICOKINETICS

On the first day postexposure the Zn content in lungs of animals treated with the Z-COTE HP1 high dose was increased to 180% compared to the control. The deposite mass of the test item in the 90 d exposure period was approx. 2000 µg/lung and the analytical results demonstrated a practically complete dissolution of the retained test substance. No significantly increased amounts of the test item were detected in any other body compartment demonstrating the rapid elimination.

ELECTRON MICROSCOPY

Electron dense structures were found one and 8 d postexposure in the cytoplasm of different cells and free in the lung lining fluid of animals treated with nanoscaled and microscaled ZnO as well as in animals of the control group. These structures were composed of irregular homogenous to fine granular material which measured only a few nanometers.

HISTOPATHOLOGY

Animals sacrified 1 d postexposure:

Effect

Group 1 (control)

Group 2

Group 3

Group 4

Group 5

Nasal and Paranasal Cavities

focal degeneration of the olfactory epithelium

 

 

 

 

1/10 slight

(multi)focal hyperplasia of the olfactory epithelium

 

 

 

 

3/10 very slight, 1/10 slight

(multi)focal mucous cell hyperplasia affecting mainly the respiratory epithelium lining the nasal septum

 

1/10 very slight, 1/10 slight

2/10 very slight

1/10 slight

1/10 very slight, 2/10 slight

(multi)focal epithelial hyaline (eosinophilic) droplets

3/10 very slight

3/10 very slight

2/10 very slight

6/10 very slight

6/10 very slight

Lung

(multi)focal accumulation of particle-laden macrophages

 

1/10 very slight

10/10 very slight

6/10 very slight, 4/10 slight

4/10 very slight, 6/10 slight

(multi)focal bronchiolo-alveolar hyperplasia , (bronchiolar type)

 

 

 

4/10 very slight

9/10 very slight

(multi)focal bronchial/bronchiolar

mucous cell hyperplasia

 

1/10 slight

 

2/10 slight

1/10 slight

(multi)focal alveolar granulocyte infiltration

 

 

 

2/10 very slight

5/10 very slight

(multi)focal interstitial mononuclear cell infiltration

1/10 very slight, 1/10 slight

2/10 very slight

3/10 very slight

8/10 very slight, 1/10 slight

6/10 very slight, 4/10 slight

Lung associated lymph nodes

(multi)focal accumulation of particle-laden macrophages

 

4/10 very slight

4/10 very slight

1/10 very slight

2/10 very slight, 6/10 slight

Lymphoid hyperplasia

 

1/10 slight

 

1/10 slight

3/10 slight, 1/10 moderate

Animals sacrified 29 d postexposure:

Effect

Group 1 (control)

Group 2

Group 3

Group 4

Group 5

Nasal and Paranasal Cavities

(multi)focal mucous cell hyperplasia affecting mainly the respiratory epithelium lining the nasal septum

2/10 very slight, 1/10 slight

1/10 slight

1/10 very slight

2/10 very slight

1/10 very slight, 2/10 slight

(multi)focal epithelial hyaline (eosinophilic) droplets

7/10 very slight

4/10 very slight

2/10 very slight

7/10 very slight

4/10 very slight

Lung

(multi)focal accumulation of particle-laden macrophages

 

 

3/10 very slight

 

3/10 very slight, 1/10 slight

(multi)focal bronchiolo-alveolar hyperplasia , (bronchiolar type)

1/10 very slight

1/10 very slight

 

1/10 very slight

2/10 very slight

(multi)focal bronchial/bronchiolar mucous cell hyperplasia

 

 

1/10 slight

1/10 slight

 

(multi)focal interstitial mononuclear cell infiltration

3/10 very slight

 

1/10 very slight

3/10 very slight

2/10 very slight, 2/10 slight

Lung associated lymph nodes

(multi)focal accumulation of particle-laden macrophages

 

1/10 very slight

3/10 very slight

2/10 very slight

5/10 very slight, 2/10 slight

Lymphoid hyperplasia

 

1/10 slight

 

1/10 slight

3/10 slight

At the end of the recovery period all the lesions regarding the nasal and paranasal cavities were diagnosed as fully reversible. The lung effects were reduced in severity or fully reversible at the end of the recovery period. Spontaneous changes like tubular basophilia in the kidney, microgranuloma in the liver, inflammatory prostate lesions and testicular atrophy were found in the ZnO treated animals as well as in control animals in the same extent. In part the incidences of these effects were unusually high for rats of this strain and age. However, these changes were considered to be not test substance-related due to similar incidences in animals of the the control and the treatment groups.

Conclusions:
Under the study condition, the NOAEL for the nanoscaled ZnO was assessed to be 1.5 mg/m3.
Executive summary:

A 90-day repeated dose inhalation toxicity study was conducted to compare the effects of the nanoscaled and microscaled ZnO in rats using nose-only exposure according to the OECD Guideline 413 in compliance with GLP. Additional endpoints (bronchoalveolar lavage, cell proliferation, electron microscopy analysis, toxicokinetics) were included to investigate potentially nano-specific aspects of toxicity.

In this study, male Wistar rats were exposed (nose only) at 0.3, 1.5 and 4.5 mg/m3 with coated nanoscaled ZnO and at 4.5 mg/m3 with non-coated microscaled ZnO. Fresh air treated animals served as concurrent control.

The target aerosol concentrations of 0.3, 1.5 and 4.5 mg nanoscale ZnO/m3 as well as 4.5 mg Microscaled ZnO/m3 were achieved to 103%, 99%, 99% and 100%, respectively. Test substance related findings or losses of animals did not occur. Effects indicating systemic toxicity were not observed. Body weight development did not show any relevant statistically significant changes. Food consumption data show some statistically significant changes, however, these are considered as incidental. Organ weights resulted in statistically significant lung weights in the microscaled ZnO group. Haematology, clinical chemistry and urinalysis data did not show any relevant statistically significant changes as compared to concurrent controls. At 1 d after exposure statistically significant increases of polymorphonuclear neutrophils, lymphocytes, lactic dehydrogenase, ß-glucuronidase and total protein were detected in the high dose group of microscaled ZnO; in the nanoscaled ZnO high dose group only the LDH level was significantly increased. All these effects were reversible and had returned to control levels after 8 d of recovery. Significantly decreased reactive oxygen intermediates (ROI) secretions were observed in the 1.5 and 4.5 mg/m3 nanoscaled ZnO treated animals as compared to the clean air treated control group, a substantially stronger decrease in the 4.5 mg/m3 microscaled ZnO treated group. At Days 8 and 29 of recovery, these effects had more or less normalized (but the microscaled ZnO treated group). Including zymosane stimulation statistically significant increases were detected in the 1.5 and 4.5 mg/m3 nanoscaled ZnO after 1 and 8 d with normalization after 29 d. Cytokine-induced neutrophil chemoattractant-1 (CINC-1) represents the IL-8 analogue in rats inducing as chemotactic factor the neutrophilic influx after inflammatory stimuli into the lung. At Day 1, the CINC-1 concentration showed a statistically significant increase only in the microscaled ZnOgroup as compared to clean air group. This increase persisted until day 8 and had normalized to control levels at Day 29. Within the animals investigated electron dense structures were found in the cytoplasm of different cells and free in the lung lining fluid. Moreover, these structures were found in animals treated with clean air as well as in the nanoscaled ZnO and microscaled ZnO treated group 1 and 8 d after exposure. As these structures were composed of irregular homogenous to fine granular material and measured only few nanometers and, in addition,did not resemble nanoparticles similar to nanoscaled ZnO and was found also in the clean air treated group, some of them in the nanoscaled ZnO treated group might have been nanoparticles which were solved but still led to a higher metal ion concentration at this spot resulting in a higher electron density. During histopathology, a slight focal degeneration of the olfactory epithelium (1 of 10 males of the microscaled ZnO group) was observed. In addition, 4/10 males of this group revealed (multi)focal hyperplasia of the olfactory epithelium. Between 1/10 and 4/10 males of the ZnO exposure groups only (not observed in the control group) showed (multi)focal very slight to slight mucous cell hyperplasia affecting mainly the respiratory epithelial lining of the nasal septum and the ventral nasal meatus in levels 2 to 3 of the nasal cavity sections. Very slight (multi)focal epithelial hyaline (eosinophilic) droplets was markedly increased in the microscaled ZnO group (6/10) while in the other groups (including the control group) the incidences of this finding ranged between 2/10 and 3/10 rats per groups. The occurrence/increased severity of the above findings is considered to be test substance related. At the end of the recovery period all these lesions were diagnosed as fully reversible. In lungs, (multi)focal very slight to slight accumulation of particle-laden macrophages was observed dose-dependently (all very slight) in the nanoscaled ZnO groups as well as in the Microscaled ZnO group, in a single male (very slight) of the high dose group and in 8/10 males of the Microscaled ZnO group. (Multi)focal very slight bronchiolo-alveolar hyperplasia, mainly of the bronchiolar type (=alveolar bronchiolisation) was observed exclusively in 4/10 and 9/10 males of the nanoscaled ZnO high dose and Microscaled ZnO groups. (Multi)focal very slight alveolar granulocyte infiltration and (multi)focal very slight to slight interstitial mononuclear cell infiltration was diagnosed as exposure-related in the nanoscaled ZnO dose and Microscaled ZnO groups. At the end of the recovery period all these lesions were reduced in severity or fully reversible. The unit length labelling index of the terminal bronchiolar epithelium was significantly decreased at 9 and 29 d post-exposure in both high dose groups (at 29 d for microscaled ZnO not statistically significant). Thus, no indication of an induction of a hyperplastic effect of the substances was observed.

Toxicokinetics revealed practically complete dissolution of the retained test substance. Overall, no relevant amounts of increased nanoscaled ZnO were detected in any body compartment demonstrating the rapid elimination.

Under the study condition, the NOAEL for the nanoscaled ZnO was assessed to be 1.5 mg/m3.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1.5 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
yes
Remarks:
(different dose levels, biochemical parameters, and collagen content estimation)
GLP compliance:
not specified
Remarks:
GLP compliance not specified in publication
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
The healthy Sprague-Dawley rats of both sex, aged between 6 and 8 weeks and body weights of 180–220 g were used. Female rats were nulliparous and nonpregnant. The animals were procured from breeding facilities of International Institute of Biotechnology and Toxicology. Animals were housed in polypropylene cages with stainless steel grills and gamma irradiated corn cobs were used as bedding. Bedding material, cages, grills, and water bottles were changed on alternate days. Animals were housed individually sex wise. Animals were acclimated for a minimum period of 5 d in the controlled environment (temperature: 22 ± 3°C; relative humidity: 50 ± 20%; and light: 12-h light/dark cycle) and ad libitum supply of reverse osmosis water and a standard rodent pellet food (supplier: M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India). Food alone was withdrawn over-night prior to the blood collection on Days 0, 28, and 42. One hundred animals were distributed randomly into different groups.
Type of coverage:
open
Vehicle:
water
Details on exposure:
Approximately 10% of the total body surface area of each rat was closely clipped free of hair once in 5 d. The nano ZnO were moistened with distilled water to prepare a paste and the paste thus prepared was evenly applied at dose levels of 75, 180, and 360 mg/kg bw /day to the groups of male and female rats through dermal route to the clipped skin on a 5 d per week basis for 28 consecutive days. Microsize zinc oxide was applied at a limit dose of 2,000 mg/kg bw/day. Control group of animals were similarly treated but with distilled water alone. Restrainer was used to prevent the ingestion of the test
substance from the application sire. At the end of the 6-h exposure period, the residual nano and microsize zinc oxide were removed with cotton soaked in water without altering the integrity of the skin.
Duration of treatment / exposure:
28 d
Frequency of treatment:
6 h per day for 5 d/week
Remarks:
Doses / Concentrations:
0, 75, 180, and 360 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Selection of doses: In order to understand the risk associated with the realistic exposure to consumers, the potential daily uptake quantity of nanomaterial mg/kg bw/day was calculated as follows:
Uder pot= (Ader× n)/bw
Ader= Qprod X Fprod; where Uder pot is the potential daily uptake quantity of nanomaterial mg/kg bw/day; Ader the quantity of the active substance on the skin per application (mg); and n number of application times, minimum of single application was considered; bw is the average body weight of the consumer which is 60 kg bw; Qprod is the quantity product (sunscreen) recommended for an adult is 36,000 mg; Fprod concentration of active substance in the product. As per FDA monograph the maximum allowable concentration of zinc oxide is 2 to 20%. Considering the worst-case scenario, 2% was taken as the concentration of active substance in the product. Fprod = 2%; Ader=2/100×36000 = 720; Uder pot = 720×1/60=12 mg/kg bw/day
Keeping in view the above quantity of nanomaterial uptake for an average adult woman, we have calculated dose for rat using rat conversion factor with 1, 2.5, and 5 times.
Low dose = Uder pot × 1 × 6 = 12 × 1 × 6 = 72 rounded to 75
Intermediate dose = Uder pot × 2.5 × 6=12 × 2.5 × 6 = 180
High dose = Uder pot × 5 × 6 = 12 × 5 × 6 = 360
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (Daily)

DETAILED CLINICAL OBSERVATIONS: Yes (Daily)

DERMAL IRRITATION (if dermal study): Yes (Daily)

BODY WEIGHT: Yes (Weekly)

FOOD CONSUMPTION: Yes (Weekly)

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes (Blood samples were collected from the orbital sinus of the overnight fasted rats on Day 28 from all groups and satellite groups on Day 42)

CLINICAL CHEMISTRY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

COLLAGEN ESTIMATION: At the end of the experimental period (Days 28 and 42), the skin and tail of the animals were collected. The amount of hydroxyproline was estimated and the total collagen content was calculated.
Sacrifice and pathology:
Gross pathology was performed at the end of experimental period (Days 28 and 42). The wet weight of the liver, lungs, kidneys, spleen, brain, and heart were recorded at the end of the experimental period. Histopathology of organs (skin, liver, spleen, kidneys, heart, adrenals, lungs, pancreas, stomach, and representative lymph nodes) was evaluated. Tissues were collected and preserved in 10% buffered formalin. All tissues required for histopathology evaluation were subjected to dehydration procedure and processed in tissue processor, embedded in paraffin wax, and sectioned at 5–8 μm thickness and stained with hematoxylin-eosin stain.
Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in clotting time was observed in all the treatment groups of nano zinc oxide with that of micro size ZnO.
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical biochemistry and hematology: No significant changes in clinical biochemistry parameters were observed in both micro and nano ZnO treated rats. There were no statistically significant changes in the hematologic parameters when compared with control. A statistically significant increase in clotting time was observed in all the treatment groups of nano zinc oxide with that of micro size ZnO.
Collagen content: There was a significant decrease in the collagen content of skin and tail in all the nano-size zinc oxide-treated group of rats compared with the control as well as with the micro-size ZnO treated group. An inverse dose-dependent relationship was observed in the treated groups.
Pathology: No gross pathological lesions were observed in any of the treatment groups. No histopathological lesions were observed in any of the organs observed.
Dose descriptor:
LOAEL
Effect level:
75 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Increase in clotting time and decrease in the collagen content of skin and tail in all the nano-size zinc oxide-treated group of rats. However, these effects were reversible in a period of 14 d.
Critical effects observed:
not specified

Amount of skin collagen, tail collagen, and percentage loss:

Group

Males

Females

Skin (g/100 g tissue)

% Loss

Skin (g/100 g tissue)

% Loss

Skin collagen

G1/Control

46.73 ± 5.80

37.31 ± 8.66

 

G2/Micro size2000 mg/kg bw

41.68 ± 2.21

10.80

34.21 ± 3.56

8.32

 

G3/Nano size 75 mg/kg bw

16.85 ± 2.27

63.94

20.70 ± 1.03

44.53

 

G4/Nano size 180 mg/kg bw

21.41 ± 3.84

54.19

24.43 ± 3.74

34.53

 

G5/Nano size 360 mg/kg bw

34.91 ± 1.72

25.30

29.19 ± 1.53

21.76

 

G6/Micro size satellite 2000 mg/kg bw

40.00 ± 2.31

10.00

38.63 ± 2.83

0.42

 

G7/Nano size satellite 75 mg/kg bw

21.16 ± 1.01

52.38

31.15 ± 5.50

19.69

 

G8/Nano size satellite 180 mg/kg bw

24.86 ± 3.86

44.07

34.16 ± 3.86

11.94

 

G9/Nano size satellite 360 mg/kg bw

39.11 ± 4.47

12.01

36.56 ± 1.53

5.74

 

G10/Control satellite

44.44 ± 5.70

38.79 ± 5.09

 

Group

Males

Females

Tail (g/100 g tissue)

% Loss

Tail (g/100 g tissue)

% Loss

Tail collagen

G1/Control

78.84 ± 12.40

68.70 ± 12.14

G2/Micro size 2000 mg/kg bw

72.58 ± 4.32

7.94

61.72 ± 3.21

10.16

 

G3/ Nano size 75 mg/kg bw

36.64 ± 4.28

53.53

41.95 ± 6.35

38.94

 

G4/ Nano size 180 mg/kg bw

48.13 ± 2.57

38.95

60.55 ± 2.27

11.87

 

G5/ Nano size 360 mg/kg bw

62.66 ± 2.65

20.52

57.30 ± 1.84

16.60

 

G6/ Micro size satellite 2000 mg/kg bw

70.26 ± 2.08

8.63

60.38 ± 2.34

12.51

 

G7/ Nano size satellite 75 mg/kg bw

52.59 ± 5.03

31.61

65.62 ± 4.07

4.93

 

G8/ Nano size satellite 180 mg/kg bw

63.60 ± 4.49

17.29

59.77 ± 5.41

 

13.40

G9/ Nano size satellite 360 mg/kg bw

70.18 ± 5.58

8.73

60.03 ± 2.95

13.02

 

G10/ Control satellite

76.89 ± 10.84

69.02 ± 5.49

 

Conclusions:
Based on decrease in collagen content in all the nano ZnO treated groups, the LOAEL for systemic toxicity was at the lowest tested dose of 75 mg/kg bw/day. However, these effects were reversible in a period of 14 d.
Executive summary:

The study was conducted to determine the repeated dose dermal toxicity of nano ZnO according to the OECD guideline 410 with modifications for dose levels, biochemical parameters and measurement of collagen content.

Sprague-Dawley rats (5/sex/dose) were applied with three different doses (75, 180, and 360 mg/kg bw/day) of nano ZnO (20 nm) at 5 d/week basis for a period of 28 d. Animals were observed for mortality/morbidity, clinical signs of toxicity, weekly body weight, and weekly food consumption during the experimental period. Blood samples were collected from the orbital sinus of the overnight fasted rats on Day 28 from all groups and satellite groups on Day 42. Different groups of rats were killed on Days 28 and 42 for gross and histopathology. Skin and tail from all the groups were collected for collagen estimation.

No significant changes were observed in the clinical chemistry parameters in both micro and nano ZnO treated rats. There were no statistically significant changes in the hematologic parameters compared with the control. A statistically significant increase in clotting time was observed in all treatment groups of nano ZnO compared to the micro-sized ZnO. No gross pathology or histopathological lesions were observed in any of the organs investigated. There was a significant decrease in the collagen content of the skin and the tail in all the nano ZnO treated groups of rats compared to the control, as well as with the micro-sized ZnO treated groups. The loss was higher in the skin than in the tail. There was an inverse dose relationship with the higher doses inducing a lower decrease.

Based on increase in clotting time and decrease in the collagen content in all the nano ZnO treated groups, the LOAEL for systemic toxicity was established at the lowest tested dose of 75 mg/kg bw/day. However, these effects were reversible in a period of 14 d.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
75 mg/kg bw/day
Study duration:
subacute
Species:
rat

Additional information

The biological activities of zinc compounds are determined by their ability to release zinc under the respective exposure conditions. Hence, information on the effects of systemically available zinc allows the repeated dose toxicity assessment across all those zinc compounds covered in this safety report.

Non-human information

The repeated dose toxicity of water soluble zinc sulphate and zinc monoglycerolate has been examined in a total of 3 subchronic oral feeding studies. Due to the different dosing regimens, the lowest NOAEL was determined to be 31.5 mg/kg bw/day of zinc monoglycerolate which equals a total zinc exposure of approximate 13 mg/kg bw/day. The zinc NOAEL derived from the feeding studies with zinc sulphate was determined to be 104 mg Zn/kg bw/day in mice and approximately 53.5 mg/kg bw/day in rats. At higher doses the most important effects in the rats were the development of hypocupremia, and significant changes in the pancreas (i.e., focal acinar degeneration and necrosis) and a decreased number of pigmented macrophages in spleen.

In a subacute inhalation study the toxicity profile of ZnO after inhalation exposure, was performed in Wistar rats nose-only exposed to dynamic atmosphere of ZnO for 6 hours per day on 5 consecutive days per week for 4 weeks (28-day study). The target concentrations were 0.5, 1.5, 3.0 and 4.5 mg/m³.Inhalation exposure of 4.5 mg/m3 ZnO caused alopecia in ear region of female animals and impaired the body weight development in males. In bronchoalveolar lavage fluid, neutrophils and other cytological and biochemical parameters were changes significantly in animals exposed to 1.5 mg/m³ and higher. At 3.0 and 4.5 significantly increased absolute and relative lung weight was found. Histological examination

revealed degeneration/regeneration of the olfactory tract in nasal cavity. In accordance to findings in lavage fluid and the increased lung weight, histology of the lung reveals multifocal alveolar histiocytosis which were associated with single or few inflammatory cells. Based on the above mentioned findings, the No Observed Adverse Effect Concentration (NOAEC) was 0.5 mg/m3 under the current study condition.

In a short term 3-day inhalation study with guinea pigs, a concentration of 2.3 mg ultrafine ZnO/m3(3 hours/day) resulted in changes in neutrophils and activities of lactate dehydrogenase and alkaline phosphatase in the pulmonary fluid. At higher concentrations increased protein concentration, neutrophils, and enzyme activities in lung lavage fluids were seen, together with significant centriacinar inflammation of the pulmonary tissue. Inhalatory doses of 2.7 mg ultrafine ZnO/m3for 5 days 3hours/day did not alter the lung function parameters in guinea pigs, but at 5 and 7 mg ultrafine ZnO/m3exposure according to a similar pattern, a gradual decrease in total lung capacity, vital capacity and reduction of the carbon monoxide diffusing capacity was seen in combination with inflammatory changes and edema. The relevance of the findings in studies with ultra-fine zinc oxide fumes is unclear with respect to commercial grade zinc oxide, as the latter is of much larger particle size and can have different toxicological characteristics.

Zinc oxide nanomaterial:

The above short term studies performed with so-called ultra fine zinc oxide fumes can be interpreted as data on zinc oxide nanomaterial, however their nanomaterial characterisation was poorly defined.

Recent tests performed specifically on nano-ZnO demonstrate:

-In a 5 days Dose Range Finding studyon the repeated dose toxicity of nano-scale zinc oxide and pigmentary zinc oxide by inhalationthat the inhalation of Z-COTE HP1 for 5 days resulted in local inflammations in the lungs of the rats, indicated by changes in several parameters in the bronchoalveolar lavage fluid (BALF) and histological examinations. Secondary to the effect in the lung, activation of the draining lymph nodes was noted. Moreover, minimal to moderate necrosis of the olfactory epithelium was noted. The effects were in a concentration-related manner and were reversible within the recovery period. Only a multifocal increase in alveolar macrophages was still present at the end of the recovery period. Similar effects were also observed in the animals exposed to ZnO powder. At the low concentration of 0.55 mg/m3, increased levels of a few mediators in the BALF and in serum were determined. Moreover minimal multifocal necrosis of the olfactory epithelium of grade was noted in the nasal cavity in one of the six animals. Therefore, a No Observed Adverse Effect Concentration (NOAEC) could not be established in this study. The lowest concentration of 0.55mg/m3 is considered to be the Low Observed Adverse Effect Concentration (LOAEC).

 

-A14-day repeated dose inhalation toxicity study was conducted to establish exposure dose-response relationships of the nanoscaled ZnO (Z-COTE HP1)(with functionalized surface) in rats after subacute exposure and to compare the effects observed with two reference substances, i.e., nanoscaled ZnO (uncoated) and a microscaled ZnO in rats using nose-only exposure according to the OECD Guideline 412. Additional endpoints (bronchoalveolar lavage, electron microscope analysis, toxicokinetics, genotoxicity) were included to investigate potentially nano-specific aspects of toxicity. In this study, male rats were exposed (nose only) atconcentration levels of 0, 0.5, 2, or 8 mg/m³ with nanoscaled ZnO (with functionalized surface) and at 8 mg/m3 with reference test substances. Fresh air treated animals served as concurrent control. No systemic but local effects in the olfactory epithelium and the lung were detected after exposure to nanoscaled ZnO (with functionalized surface) or uncoated ZnO nanoparticles. Increases of polymorphonuclear neutrophils and lactic dehydrogenase, ß-glucuronidase and total protein levels as well as effects oninflammatory markers in the BAL study combined with slight inflammatory response of the lung in histopathology at day 1 post exposure plus slight degeneration of the olfactory epithelium at day 1 were found solely at the high dose level of 8 mg/m³. These effects at the high dose level are considered to be adverse and treatment related. Most of the effects are reversible. Under the study conditions, the NOAEL and LOAEL were established at 2 and 8 mg/m³, respectively.

 

-In a 90-day repeated dose inhalation toxicity study, male Wistar rats were exposed (nose only) at 0.3, 1.5 and 4.5 mg/m3 with coated nanoscaled ZnO (Z-COTE HP1)and at 4.5 mg/m3 with non-coated microscaled ZnO. Fresh air treated animals served as concurrent control. Test substance related findings or losses of animals did not occur. Effects indicating systemic toxicity were not observed. The LOAEL was 4.5 mg/m³ and the NOAEL 1.5 mg/m³. Adverse effects were restricted to the high dose group: very slight bronchiolo-alveolar hyperplasia and increased incidences in very slight to slight infiltration of interstitial mononuclear cells in the lung, increased activity of lactate dehydrogenase in BAL and the increased numbers of lymphocytes in the BAL. The reversibility of all effects has been demonstrated. Effects on the olfactory epithelium were detected only in the reference group exposed to 4.5 mg/m³ microscaled ZnO particles.

In conclusion repeated dose toxicity inhalation studies showed local irritation of nasal cavity and respiratory tract, local lung inflammation and all effects were reversible within 28d after 90 d exposure.

For nano-ZnO, no nano-specific long term toxic effects could be identified. Zn2+ion determines the toxicity of ZnO and read across between various forms of ZnO (micro-scale, nano, coated or not) is fully supported.

  

In the only study available on repeated dose toxicity in the skin, the dose levels tested (75, 180 and 360 mg/kg bw) on Sprague-Dawley rats through dermal route did not show toxicity. On repeated application, nano zinc oxide at low doses caused collegen decrease compared with their high dose and control (LOAEL= 75 mg/kg bw/day). However, these effects were reversible in a period of 14 days. In general no systemic effects were observed.

Human information

Upon supplementing men and women with 150 mg Zn/day (as zinc sulphate capsules), women appeared to be more sensitive than men to the effects of high zinc intake: clinical signs such as headache, nausea and gastric discomfort were more frequent among women a nd women but not men had decreased activities of serum ceruloplasmin and ESOD. In some earlier oral studies in which humans were supplemented with moderately high amounts of zinc (50 mg Zn/day), a reduction in ESOD activity was also observed and again women appeared to be more sensitive to this effect. Hence, a reduction in ESOD was thought to be a sensitive indicator of copper status. However, in more recent and more sophisticated studies using the same dose level, ESOD was only marginally reduced (without a correlation with changes in copper balance), while findings on more specific copper deprivation signs (decreased serum ceruloplasmin and platelet cytochrome c oxidase) indicated that a sub-optimal intake of zinc was more effective than a moderately high intake of zinc in inducing changes associated with a decreased copper status in postmenopausal women. Given this, and the degree of the observed ESOD reduction in comparison to the natural variability in its activity, the zinc-induced decrease in ESOD activity is considered to have marginal biological significance, if any and also because it may not have been caused by an interference with copper metabolism as deep tissue SOD increases as a function of zinc exposure was observed.

Overall, it can be concluded that from studies in which humans were supplemented with zinc (as zinc gluconate), that women are more sensitive to the effects of high zinc intake and that a dose of 50 mg Zn/day is the human NOAEL. This equals a daily exposure of 0.83 mg/kg bw. At the LOAEL of 150 mg Zn/day, clinical signs and indications for disturbance of copper homeostasis have been observed.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The lowest established NOAEL = 13.3 mg Zn/kg bw/day selected out of 3 subchronic oral feeding studies with soluble zinc sulphate and zinc monoglycerolate

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: other; digestive: pancreas

Repeated dose toxicity: dermal - systemic effects (target organ) other: skin

Justification for classification or non-classification

Zinc is essential for human growth and development, neurological functions and immunocompetence. The main clinical manifestations of zinc deficiency are growth retardation, delay in sexual maturation or increased susceptibility to infections (SCF, 2003). Health specialists recommend supplementing the diet with zinc in case human diet is zinc deficient. The maximum allowable daily intake has been established to be 50 mg zinc per day. There is no experimental sufficient evidence for specific target organ toxicity based on the reversibility of the ‘adverse’ effects demonstrated. Hence no classification is required.