Zinc oxide

Repeated dose toxicity: inhalation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

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

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: nanoform

Details on test material:

TEST ITEM
- Name of test material (as cited in study report): Z-COTE HP1
- Molecular weight (if other than submission substance): 81.38 g/mol
- Physical state: Solid
- Composition of test material, percentage of components: Z-COTE HP1 (98%), coated with triethoxycaprylylsilane (CAS # 2943-75-1; 2%)
- Lot/batch No.: NPL Ref#: ZB250#65
- Expiration date of the lot/batch: June 2014
- Storage condition of test material: Room temperature, dry, exclusion of light
- Other: MMAD determination:

Z-COTE HP1 Z-COTE HP1 Z-COTE HP1
0.3 mg/m3 1.5 mg/m3 4.5 mg/m3

MMAD (µm) GSD MMAD (µm) GSD MMAD (µm) GSD
Mean 0.67 3.56 0.64 3.22 1.17 3.23
STD 0.23 0.75 0.008 0.24 0.35 0.66


REFERENCE ITEM
- Name of test material (as cited in study report): Zinc Oxide 205532, Micron Size Powder
- Molecular weight (if other than submission substance): 81.38 g/mol
- Physical state: Solid
- Composition of test material, percentage of components: Non-coated ZnO
- Lot/batch No.: NPL Ref#: ZrA250#60
- Expiration date of the lot/batch: May 2014
- Storage condition of test material: Room temperature, dry, exclusion of light
- Other: MMAD determination:

Microscaled ZnO
4.5 mg/m3

MMAD (µm) GSD
Mean 1.7 2.4
STD 0.21 0.25

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system 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

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. 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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.3, 1.5 and 4.5 mg/m3
Basis:
other: target aerosol concentration of test substance

Doses / concentrations 2

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1.5 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.

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