Zinc nitrate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-30 to 2015-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to the OECD Guideline and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2013-12-18

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Zinc oxide

EC Number:

215-222-5

EC Name:

Zinc oxide

Cas Number:

1314-13-2

Molecular formula:

OZn

IUPAC Name:

oxozinc

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): ZnO; Zinkweiss HARZSIEGEL Standard - Zinkoxid
- Source of test material: Norzinco GmbH, Harzer Zinkoxide
- Test substance Ni.: 11/0849-1
- Homogeneity: homogenous
- Physical state: Solid/white
- Lot/batch No.: 29666
- Expiration date of the lot/batch: NI
- Date of production: 2009-11-16
- Storage condition of test material: Room temperature, dry storage

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Sulzfeld, Germany)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approx. 7 weeks
- Housing: up to 5 rats per cage (Polysulfon cages). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding. For enrichment wooden gnawing blocks were added.
- Diet: mouse/rat laboratory diet GLP, 10 mm pellets; ad libitum, but not during exposure
- Water: tap water; ad libitum, but not during exposure
- Acclimation period: 3 days

DETAILS OF FOOD AND WATER QUALITY: Food and drinking water analyses showed the food and drinking water used to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15/h ; fully airconditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2012-06-13 To: 2012-07-11

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.8 - <= 2.1 µm

Remarks on MMAD:

MMAD / GSD: please refer to: 'Any other information on materials and methods incl. tables'.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow-past nose-only exposure system, individually exposure of each rat, exhaled air is immediately exhausted
- Method of holding animals in test chamber: Individual acrylic tubes
- Source and rate of air: Pressurized air, 1L/min
- System of generating particulates/aerosols: dust aerosol was generated with the dust generator and compressed air mixed with conditioned dilution air and passed via the cyclonic separator and the dilution tube into the inhalation system.
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 50 ± 20%,
- Air flow rate: 3L/min
- Method of particle size determination: Cascade impactor/ Marple impactor
- Treatment of exhaust air: Disposal in compliance with local, federal and state regulations

TEST ATMOSPHERE
- Brief description of analytical method used: Scattered light photometers (Visguard (Sigrist, Germany) in test group 1 and (RAM1 (Mie, USA); Gravimetrically by filter samples
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
- A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust/aerosol was drawn through the filter. The dust concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling with reference to the sample volume of the inhalation atmosphere.

Duration of treatment / exposure:

28 days

Frequency of treatment:

5 consecutive days per week, 6 h per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.47 mg/m³ air (analytical)

Remarks:

SD: ± 0.20 mg/m³; target concentration: 0.5 mg/m³

Doses / concentrations 2

Dose / conc.:

1.63 mg/m³ air (analytical)

Remarks:

SD: ± 0.65 mg/m³; target concentration: 1.5 mg/m³

Doses / concentrations 3

Dose / conc.:

3.01 mg/m³ air (analytical)

Remarks:

SD: ± 1.19 mg/m³; target concentration: 3.0 mg/m³

Doses / concentrations 4

Dose / conc.:

4.37 mg/m³ air (analytical)

Remarks:

SD: ± 1.31 mg/m³; target concentration: 4.5 mg/m³

No. of animals per sex per dose:

5 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available data, on request of sponsor, concentrations were selected for the study:
4.5 mg/m³: as high concentration causing toxic effects
3.0 mg/m³: as high intermediate concentration
1.5 mg/m³: as mid concentration
0.5 mg/m³: as low concentration and expected NOAEC
- Fasting period before blood sampling for clinical biochemistry: no fasting

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day and once on weekends during exposure period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: At the start of the pre-exposure, at the start of the exposure period and then, as a rule, twice a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Study termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Study termination
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: study termination
- Dose groups that were examined: all
- Number of animals: 5 males per test group
- Parameters checked: Cytology: Total cell count, macrophages, lymphocytes, eosinophils, monocytes, non-classified cells. Protein and enzymes: Total protein, GGT, lactate dehydrogenase, ALP, N-acetyl-β-glucosaminidase (NAG BAL).

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- The animals (main groups) were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.
- Organ weights determined: adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, spleen, testes, thymus, and thyroid glands.

HISTOPATHOLOGY: Yes
- Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings in all gross lesions, nasal cavitiy (4 levels), larynx (3 levels), trachea, lungs (5 lobes), tracheobronchial lymph nodes, mediastinal lymph nodes, adrenal glands, bone marrow (femur), brain, oesophagus, heart, kidneys, liver, ovaries, seminal vesicles, spinal cord (cervical, thoracic, and lumbar cords), spleen, stomach (forestomach and glandular stomach), testes, thyroid glands, thymus, and uterus.
- A correlation between gross lesions and histopathological findings was performed.

Statistics:

- Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means. Differences were considered statistically significant if p ≤ 0.05.
- Blood parameters and BAL: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. Differences were considered statistically significant if p ≤ 0.05.
- Weigth parameters in pathology: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. Differences were considered statistically significant if p ≤ 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the high concentration (4.37 mg/m³), in 4 of the 5 females alopecia was seen, in two of them it was accompanied by injury of the same region, probably caused by scratching. In females, alopecia and injury were observed first on study day 19 in two animals, followed by another two animals on study day 22 and 23. All findings in male and female animals continued until the animals were sacrificed at the end of the study.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight (please refer to: 'Attached background material')::
- At 4.37 mg/m³: Males of the main group showed decreased mean body weights on study day 19 (-10 %, p< 0.05) through to study day 28 (-13 %, p<0.01; terminal body weight).

Body weight changes (please refer to: 'Attached background material')::
- At 4.37 mg/m³: Males of the main group showed a reduced body weight gain from study day 19 to 23 (0.6 g while it was 6.5 g in the control, p<0.05) and from study day 26 to 27 (-10.8 g while it was -1.6 g in the control, p<0.01)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- At 4.37 mg/m³: The absolute and relative lung weights were statistically significantly increased in males (118% and 135%, respectively) and females (127% and 130%, respectively), when compared to the negative control group (100%) (please refer to: 'Attached background material').
- At 3.01 mg/m³: The absolute and relative lung weights were statistically significantly increased in males (120% and 126%, respectively) and females (131% and 134%, respectively), when compared to the negative control group (100%).
- At 1.63 mg/m³: The relative lung weight was statistically significantly increased in females (111%), when compared to the negative control group (100%).

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nasal cavity:
- The inhalation of the test substance led to single small (minimal) to multiple large areas (severe) of degeneration/ regeneration in the olfactory epithelium at the septum, the nasoturbinate and/or ethmoid turbinate (please refer to: 'Attached background material'). This finding was observed in 4 out of 5 males and 3 out of 5 females of test group 2 (1.63 mg/m³) as well as in all males and females of test groups 3 (3.01 mg/m³) and 4 (4.37 mg/m³). The severity increased with concentration. The occurrence of degeneration/ regeneration of the olfactory epithelium in males and females of test groups 2 (1.63 mg/m³) to 4 (4.37 mg/m³) was considered to be a consequence of irritant effects of the test substance and adverse.

Lungs:
- In the lungs, alveolar histiocytosis was observed in all male and female animals of test groups 3 (3.01 mg/m³) and 4 (4.37 mg/m³) (please refer to: 'Attached background material'). In contrast to the spontaneously occurring histiocytosis (one/ few foci of alveolar histiocytosis), in these animals, single or few alveolar macrophages were seen in some or many alveoli and were distributed multifocally in all lobes over the whole lung. The alveolar histiocytosis was associated with single or few inflammatory cells showing the same distribution pattern. In addition, granular material, probably test substance, was apparent in alveolar lumina. These findings were correlated with the increased lung weights in these treatment groups. The occurrence of alveolar histiocytosis and inflammatory infiltrates in animals of test groups 3 (3.01 mg/m³) and 4 (4.37 mg/m³) was considered a response to irritant effects of the test substance and was regarded as adverse.
Because the distribution pattern of alveolar histiocytosis and inflammatory cells in one female (no. 64) of test group 2 (1.63 mg/m³) was comparable to that seen in animals of test groups 3 and 4, the occurrence of these findings was also considered to be treatment-related and adverse.
- Although there was no clear histopathological correlate for the increased mean relative weight of the lungs in females of test group 2 (1.63 mg/m³), the weight increase was regarded to be treatment-related considering the changes found in BAL. The minimal or slight activation of the tracheobronchial lymph nodes (lympho-reticulocellular hyperplasia) in 2 out of 5 males and all females of test group 3 (3.01 mg/m³) and in all males and females of test group 4 (4.37 mg/m³) was considered as reaction to the inflammatory process in the lungs.

Tracheobronchial lymph nodes:
A minimal or slight lympho-reticulocellular hyperplasia was observed in 2 males and all females of test group 3 (3.01 mg/m³), as well as in all males and females of test group 4 (4.37 mg/m³). In males and females of test groups 3 and 4, the lympho-reticulocellular hyperplasia might be seen as response to the inflammatory process observed in the lungs and was therefore regarded to be treatment-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL):
- After 4 weeks of inhalation, in males of the satellite test groups 31 and 41 (3.01 and 4.37 mg/m³) total BAL cell counts (not statistically significant in test group 31) as well as absolute neutrophil, lymphocyte and monocyte cell counts were increased (please refer to: 'Attached background material'). Absolute monocyte and neutrophil counts were not dose-dependently altered between these two test groups. Additionally, in males of the satellite test group 21 (1.63 mg/m³) absolute neutrophil and monocyte cell counts were relevantly higher compared to controls. Absolute macrophage counts were increased only in males of the satellite test group 41 (4.37 mg/m³). Higher non classified cells in the satellite test groups 21 up to 41 (1.63; 3.01, 4.37 mg/m³) were degraded cells which could not be categorized.
- The alterations in the absolute cell counts were also reflected in changes of the relative BAL cell counts: increased relative lymphocyte, neutrophil, monocyte and non-classified cell counts in the satellite test groups 31 and 41 (3.01 and 4.37 mg/m³) and additionally increased relative neutrophil, monocyte and non-classified cell counts in the satellite test group 21 (1.63 mg/m³). Relative monocyte and neutrophil counts were not dose-dependently altered between the satellite test group 31 and 41. Relative macrophages counts were decreased in the satellite test groups 21 up to 41 (1.63, 3.01 and 4.37 mg/m³), but also not dose-dependently between test groups 31 and 41.
- In rats of the satellite test groups 21 up to 41 (1.63; 3.01 and 4.37 mg/m³), total protein content as well as lactate dehydrogenase (not statistically significant in test group 31) and alkaline phosphatase activities in BAL were increased. However, the increases of all three parameters in the satellite test groups 21 (1.63 mg/m³) were below a 2-fold increase of the historical control ranges (total protein: 18-64 mg/L; LDH BAL 0.19-0.50 μkat/L; ALP BAL 0.23-0.87 μkat/L. Therefore, these alteration in test group 21 were regarded as treatment-related, but not adverse.
- N-acetyl-β-glucosaminidase and γ-glutamyltransferase activities in BAL of rats of the satellite test groups 31 and 41 (3.01 and 4.37 mg/m³) were increased. However, the increases were not dose-dependent and the means were below or only about 2-fold higher compared to historical controls (NAG BAL 7-45 nkat/L; GGT BAL 0-41 nkat/L, PART III). Therefore, these alteration in test groups 31 and 41 (3.01 and 4.37 mg/m³) were regarded as treatment-related but not adverse.

Details on results:

MORTALITY:
- No deaths were recorded throughout the study

CLINICAL SIGNS:
- During the pre-exposure period the animals showed no clinical signs and findings different from normal.
- During the exposure period the male and female animals of the control group, low concentration (0.47 mg/m³) and intermediate concentration (3.01 mg/m³) groups showed no clinical signs and findings different from normal. In 2 of the total 10 male animals (5 main and 5 satellite group animals) of the low intermediate group (1.5 mg/m³), alopecia was observed in the ear region starting from study day 20 and 21. In one of them injury in the same region was seen, probably due to scratching. As this finding in males was only observed in test group 2, it is considered incidental due to lack of relation-ship to the exposure concentrations.

BODY WEIGHT AND WEIGHT GAIN
Mean body weight:
- At 0.47 mg/m³: mean body weights decreased on study day 19, (-9 %, p< 0.05), 27 (-10 %, p< 0.05) and 28 (-10 %, p< 0.05). The significantly decreased body weight in test group 1 male animals is considered as incidental because the body weight of the satellite animals exposed to the same concentration of ZnO, as well as those of group 2 and 3 were all comparable to the control.
- The mean body weights of the test substance exposed female animals and male satellite group animals were not statistically significantly different from the control group 0.

Body weight gain:
In male animals of the main groups, following statistically significant changes were observed, when compared with the control.
- Test group 1 (0.47 mg/m³) from study day 5 to 9 (-1.1 g while it was 7.7 g in the control,
p<0.05)
- Test group 2 (1.63 mg/m³) from study day 5 to 9 (-2.4 g while it was 7.7 g in the control,
p<0.05)
- Test group 2 (1.63 mg/m³) from study day 19 to 23 (0.0 g while it was 6.5 g in the control,
p<0.05)
The changes in test group 1 and 2 are considered as incidental due to lack of concentration response relationship.

FOOD CONSUMPTION
No substance-related changes of food consumption were observed during the whole study period.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

ORGAN WEIGHT INCLUDING ORGAN / BODY WEIGHT RATIOS:
- The terminal body weight was significantly decreased in males of test group 4 (4.37 mg/m³) resulting in increased relative brain and testes weights.
- The reduced terminal body weight in males of test group 1 (0.47 mg/m³) resulted in increased relative weights of adrenal glands, brain, and testes. Because there was no concentration-response relationship, the reduced terminal body weight in test group 1 was considered to be incidental.
- The increased relative weights of adrenal glands and testes in males of test group 2 (1.63 mg/m³) were related to the slightly decreased terminal body weight (-6%) in this test group.
- For the increased weights of thyroid glands in females of test groups 2 (1.63 mg/m³), 3 (3.01 mg/m³), and 4 (4.37 mg/m³), a treatment related effect could not be ruled out.

GROSS PATHOLOGY
No test item-related findings were observed.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- A minimal or slight lympho-reticulocellular hyperplasia was observed in 2 males of test group 2 (1.63 mg/m³). Because the 2 males (animal nos. 12 and 15) of test group 2 with minimal lympho-reticulocellular hyperplasia of the tracheobronchial lymph nodes did not show any finding in the lungs, a treatment-related effect seemed rather unlikely.
- For the increased weights of thyroid glands in females of test group 2 (1.63 mg/m³), 3 (3.01 mg/m³) and 4 (4.37 mg/m³), a treatment related effect could not be ruled out, but because there were no histopathological correlates, the weight increase was regarded as nonadverse.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

0.47 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

A 28-day repeated dose inhalation toxicity study was conducted to evaluate the effects of microscaled ZnO in rats using nose-only exposure according to the OECD Guideline 412 in compliance with GLP. In this study, male/female Wistar rats were exposed (nose only) at 0.5, 1.5, 3.0 and 4.5 mg/m³ (analytical concentration: 0.47, 1.63, 3.01, and 4.37 mg/m³) microscaled ZnO. Fresh air treated animals served as concurrent control.
Inhalation exposure of 4.37 mg/m³ ZnO for 28 days (20 exposure days) caused alopecia in ear region of female animals and impaired the body weight development in males. In bronchoalveolar lavage fluid, neutrophils and other cytological and biochemical parameters were changes significantly in animals exposed to 1.63 mg/m³ and higher. At 3.01 and 4.37 mg/m³ significantly increased absolute and relative lung weight was found. Histological examination revealed degeneration/regeneration of the olfactory tract in nasal cavity (level II to IV). In accordance with the findings in lavage fluid and the increased lung weight, histology of the lung reveals multifocal alveolar histiocytosis which were associated with single or few inflammatory cells.

Based on the above-mentioned findings, the No Observed Adverse Effect Concentration (NOAEC) was 0.47 mg/m³ under the current study condition.

The study was conducted according to the OECD Guideline and is considered to be reliable without restriction.