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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: 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:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
TEST ITEM
- Name of test material (as cited in study report): ZnO
- Physical state: Solid/white
- Analytical purity: 99.7%
- Lot/batch No.: 29666
- Expiration date of the lot/batch: NI
- Storage condition of test material: Room temperature, dry storage

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld/Germany
- 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 (e.g. ad libitum): mouse/rat laboratory diet GLP, 10mm pellets, ad libitum, but not during exposure
- Water (e.g. ad libitum): tap water ad libitum, but not during exposure
- Acclimation period: 3 days

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

Administration / exposure

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.
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: Gravimetrically by filter samples
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days, 5 consecutive days per week, 6 h per day
Frequency of treatment:
5 consecutive days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
0.5, 1.5, 3.0 and 4.5 mg/m3
Basis:
other: target aerosol concentration of test substance
No. of animals per sex per dose:
5/dose
Control animals:
yes, concurrent no treatment
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
Positive control:
No

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Twice a day and once on weekends during exposure period

BODY WEIGHT: The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, 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:
- Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

HAEMATOLOGY: yes

CLINICAL CHEMISTRY: yes

OTHER:
- Bronchoalveolar lavage (29 d after end of exposure period: cell count, biochemical parameters, cytokines)
- Electron microscope analysis in nasal cavities, lung, trachea, bronchioles( 29 d after end of exposure period)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
-body weight, body weight change --> Dunnett's test
-blood parameters and BAL--> Kruskal-Wallis test
-weigth parameters --> Kruskal-Wallis test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths were recorded throughout the study
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 (0.5 mg/m³) and intermediate concentration (3.0 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. At the high concentration (4.5 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.

BODY WEIGHT AND WEIGHT GAIN
In male animals of the main groups, following statistically significant changes were observed, when compared with the control.
- Test group 1 (0.5 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 (1.5 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 (1.5 mg/m³) from study day 19 to 23 (0.0 g while it was 6.5 g in the control,
p<0.05)
- Test group 4 (4.5 mg/m³) from study day 19 to 23 (0.6 g while it was 6.5 g in the control,
p<0.05)
- Test group 4 (4.5 mg/m³) from study day 26 to 27 (-10.8 g while it was -1.6 g in the
control, p<0.01)
The changes in test group 1 and 2 are considered as incidental due to lack of concentration response relationship. The decreased body weight change of test group 4 reflex the decreased body weight and is considered as treatment-related.

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 WEIGHTS
increased mean weights of the lungs in females (relative) of test group 2 (1.5 mg/m³) as well as in males and females (absolute and relative) of test groups 3 (3.0 mg/m³) and 4 (4.5 mg/m³) were regarded to be treatment-related.
The terminal body weight was significantly decreased in males of test group 4 (4.5 mg/m³) resulting in increased relative brain and testes weights.
The reduced terminal body weight in males of test group 1 (0.5 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 (1.5 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 (1.5 mg/m³), 3 (3.0 mg/m³), and 4 (4.5 mg/m³), a treatment related effect could not be ruled out

GROSS PATHOLOGY
No test item-related findings were observed.

HISTOPATHOLOGY
In the 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. This finding was observed in 4 out of 5 males and 3 out of 5 females of test group 2 (1.5 mg/m³) as well as in all males and females of test groups 3 (3.5 mg/m³) and 4 (4.5 mg/m³). The severity increased with concentration. The occurrence of degeneration/ regeneration of the olfactory epithelium in males and females of test groups 2 (1.5 mg/m³) to 4 (4.5 mg/m³) was considered to be a consequence of irritant effects of the test substance and adverse.

In the lungs, alveolar histiocytosis was observed in all male and female animals of test groups 3 (3.0 mg/m³) and 4 (4.5 mg/m³). 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 (3.0 mg/m³) and 4 (4.5 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 (1.5 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 (1.5 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 (3.0 mg/m3) and in all males and females of test group 4 (4.5 mg/m3) was considered as reaction to the inflammatory process in the lungs.
For the increased weights of thyroid glands in females of test group 2 (1.5 mg/m³), 3 (3.0 mg/m³) and 4 (4.5 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

Dose descriptor:
NOAEC
Effect level:
0.5 mg/m³ air
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:
Under the study condition, the NOAEC for the ZnO powder test was assessed to be 0.5 mg/m3.
Executive summary:

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 microscaled ZnO. Fresh air treated animals served as concurrent control.

Inhalation exposure of 4.5 mg/m3 ZnO for 28 days (20 exposures) 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 paramters

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 (level II to IV). In

accordance to findings in lavage fluid and the increased lung weight, histology of the lung reveals multifocal alveolar histoctosis 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.