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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study, only male rats used

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
,additional endpoints investigated
Deviations:
yes
Remarks:
only male rats 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

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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): 81.38g/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 of the aerosol < 3.0 µm, GSD about 1.5 REFERENCE ITEM- Name of test material (as cited in study report): Zinc Oxide 205532, Micron Size Powder- Molecular weight (if other than submission substance): 81.38g/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 of the aerosol < 3.0 µm, GSD about 1.5

Test animals

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 day followed by 3 weeks of training in nose-only tubes without exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 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 at Frauenhofer ITEM 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 4.5mg microscaled ZnO/m3 were achieved to 103%, 99%, 99%, and 100%, respectively.
Duration of treatment / exposure:
3 months, 5 consecutive days per week, 6 hours per day
Frequency of treatment:
5 consecutive days per week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.3, 1.5 and 4.5mg/m3
Basis:
other: target aerosol concentration of test item
Remarks:
Doses / Concentrations:
4.5mg/m3
Basis:
other: target aerosol concentration of reference item
No. of animals per sex per dose:
65
Control animals:
yes, concurrent no treatment
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
- How many 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
- How many 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 days after end of exposure period: cell count, biochemical parameters, cytokines)
- lung cell proliferation (9 and 29 days after end of exposure period)
- Toxicokinetics according OECD TG 417, chemical Zn analysis in organs, blood, and urine (1 and 29 days after end of exposure period)
- electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kideny, liver, spleen, and erythrocytes (1, 8, and 29 days after end of exposure period)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
- bronchoalveolar lavage (1, 8, and 29 days after end of exposure period: cell count, biochemical parameters, cytokines)
- lung cell proliferation (9 and 29 days after end of exposure period)
- Toxicokinetics according OECD TG 417, chemical Zn analysis in organs, blood, and urine (1 and 29 days after end of exposure period)
- electron microscope analysis in nasal cavities, lung, trachea, larynx, bronchioles, kideny, liver, spleen, and erythrocytes (1, 8, and 29 days 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 days 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 days with a normalization after 29 days. 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 days.
- cell proliferation: No indication of an induction of a hyperplastic effect of the test item or the reference item 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 days exposure period was approx. 2000µg/lung and the analytical results demonstrated a practically complete dissolution of the retained test item. 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: decisive endpoints: LDH in BAL fluid, histopathology: bronchiolo-alveolar hyperplasia and mononuclear cell infiltration

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 days 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 days with a normalization after 29 days.

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 days.

CELL PROLIFERATION

No indication of an induction of a hyperplastic effect of the test item or the reference item 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 days exposure period was approx. 2000µg/lung and the analytical results demonstrated a practically complete dissolution of the retained test item. 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.

HISTOPATHOLOGY

Animals sacrified one day 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 days 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 item-related due to similar incidences in animals of the the control and the treatment groups.

Applicant's summary and conclusion

Executive summary:

A 90-day inhalation study was conducted in rats using nose-only exposure according to OECD TG 413. Additional endpoints (bronchoalveolar lavage, cell proliferation, electron microscopy analysis, toxicokinetics) were included to investigate potentially nano-specific aspects of toxicity. The animals were treated with 0.3, 1.5 and 4.5 mg/m3 Z-COTE HP1 (coated nanoscaled ZnO), respectively, as well as 4.5 mg/m3 non-coated microscaled ZnO. Fresh air treated animals served as concurrent control.

In conclusion coated nanoscale (Z-COTE HP1) and non-coated microscale ZnO caused comparable and reversible histopathological findings restricted to the respiratory tract. The retained material was completely solved and eliminated rapidly since no increased Zn contents were detected in any body compartment after the post-exposure period. The status of the respiratory burst of alveolar macrophages was temporarily increased by treatment with nano- and microscale ZnO and was fully reversible for Z-COTE HP1. Markers for cellular damage and inflammation were reversibly increased to a higher extent by microscaled ZnO than by Z-COTE HP1. Based on the results of the present study the NOAEL for Z-COTE HP1 was assessed to 1.5 mg/m3.