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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:
supporting study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.

Data source

Reference
Reference Type:
publication
Title:
Functional and morphologic changes in the lungs of guinea pigs exposed to freshly generated ultrafine zinc oxide
Author:
Lam HF, Conner MW, Rogers AE, Fitzgerald S & Amdur MO
Year:
1985
Bibliographic source:
Toxicol. Appl. Pharmacol. 78(1):29-38

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Guinea pigs were exposed by nose-only exposure at 3h/d for 6 d to ZnO ultrafine particles and examined at 24, 48 and 72 h postexposure for functional and morphological changes in the lungs.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinc oxide

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass.)
- Weight at study initiation: 260 -300 g
- Housing: Four animals per suspended stainless-steel cage
- Diet : Feed (Agway, Syracuse, N.Y.), ad libitum
- Water: Distilled water, ad libitum
- Acclimation period: Five days


ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C
- Humidity: 50 %

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: Mean particle size expressed as projected area diameter = 0.05 µm, σg 2.0.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rectangular stainless-steel chamber with a trapezoidal plenum consisted of three chambers- inlet, exposure and exhaust chambers.
- Method of holding animals in test chamber: Plexiglass animal holders
- System of generating particulates/aerosols: Zinc turnings were heated to 480 °C in a crucible and the zinc vapours were carried by argon gas flow. The vapours react with oxygen to yield ultrafine particles upon condensation.
- Temperature, humidity, pressure in air chamber: Temperature = 25 - 28 °C, humidity = 43-49 %
- Method of particle size determination: Piezoelectric crystal, TSI model 3030

TEST ATMOSPHERE
- Brief description of analytical method used: ZnO concentrations were determined by total aerosol collection on Millipore GSWP 47-mm filters. Gravimetric analysis was done on a Cahn electrobalance Model 21. Chemical analysis consisted of atomic absorption spectrometry and electron spectroscopy for chemical analysis.



Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Atomic absorption spectrometry and electron spectroscopy
Duration of treatment / exposure:
6 d
Frequency of treatment:
3 h/d
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
4.6 ± 2.7 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
5.3 ± 2.9 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
38 (for 4.6 ± 2.7 mg/m3 exposure),
35 (for 5.3 ± 2.9 mg/m3 exposure)
18 & 35 (control)
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the recommended threshold limit value (TLV).
Positive control:
None

Examinations

Observations and examinations performed and frequency:
BODY WEIGHT: Yes


OTHER: Physiological measurements - Measurements were done at 1, 24, 48, or 72 h after the end of sixth exposure for the treated animals and at 1 and 48 h postexposure for control animals. The following pulmonary function parameters were measured in the anesthesised, tracheostomised animals: ventilation, dynamic compliance, flow resistance, vital capacity, inspiratory capacity, total lung capacity and single breath diffusing capacity for carbon monoxide.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, lungs of exposed animals were examined.
HISTOPATHOLOGY: Yes, lungs of exposed animals were examined for microscopic lesions.
Other examinations:
Epithelial permeability: Permeability of the lower respiratory epithelium to macromolecules was assessed in five animals at 1 and 24 h after exposure. Results were expressed as the concentration of horseradish peroxidase in plasma 30 min after instillation.

Morphologic examination: Five animals were sacrificed at 1, 24, 48, or 72 h after exposure for morphologic examination of the respiratory tract under transmission electron microscope.

DNA synthesis: In epithelial cells of the bronchi and terminal bronchioles, DNA synthesis was studied by calculating the [3H]thymidine labelling index of the cells on autoradiographs. Minimum 1000 nuclei were examined and labelling index was expressed as the no. of labelled nuclei per 1000 nuclei examined.
Statistics:
Statistical analysis was done by analysis of variance and additional comparisons were done by the method of Scheffe.

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN: No significant difference was observed in body weight between treated and control animals.


ORGAN WEIGHTS: Elevated lung weights due to inflammation were observed and were not returned to normal by 72 h.


GROSS PATHOLOGY: The lungs of treated animals appeared normal, but some failed to collapse when the chest was opened.


HISTOPATHOLOGY: NON-NEOPLASTIC: Inflammation of the proximal portion of the alveolar ducts and adjacent alveoli was observed. The lesions were characterised by interstitial thickening, increased pulmonary macrophages and neutrophils in adjacent airspaces and alveolar squamous epithelium was replaced with cuboidal cells (type 2 pneumocytes).

OTHER FINDINGS: Physiological measurements - Vital capacity, functional residual capacity, alveolar volume and single breath diffusing capacity for carbon monoxide were decreased and did not return to normal values by 72 h. Increase in flow resistance, decrease in compliance and total lung capacity were returned to normal values by 72 h.
Epithelial permeability - No significant effect was observed in the epithelial permeability to horseradish peroxidase.
DNA synthesis - [3H]Thymidine labelling of bronchiolar epithelial cell nuclei was increased for 48 h.

Effect levels

Dose descriptor:
conc. level:
Effect level:
ca. 4.6 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: Decreased Vital capacity, functional residual capacity, alveolar volume and single breath diffusing capacity for carbon monoxide and elevated lung weights due to inflammation

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Ventilation and lung mechanics

Time post exposure (h) Control

ZnO

1 and 48 1 24 48 72
No. of animals 18 8 9 9 8
Body weight (g) 288 ± 8 292 ± 8 294 ± 9 294 ± 11 294 ± 13
Respiratory frequency (breaths/min) 67 ± 8 63 ± 8 66 ± 11 65 ± 9 56 ± 5*
Tidal volume (mL) 1.7 ± 0.1 1.8 ± 0.1 1.8 ± 0.2 1.8 ± 0.2 1.8 ± 0.2
Flow resistance (cm H2O/mL/sec) 0.31 ± 0.08 0.50 ± 0.13* 0.41 ± 0.10* 0.30 ± 0.07 0.34 ± 0.09
Compliance (mL/cm H2O) 0.28 ± 0.06 0.20 ± 0.09* 0.20 ± 0.06* 0.22 ± 0.05* 0.25 ± 0.08

Note: Values are mean ± SD. All values were from anesthesised, tracheostomatised spontaneously breathing Guinea pigs.

* p < 0.05.

Table 3: Alveolar duct inflammation in Guinea Pigs exposed six times to 5.3 mg/m3 of ultrafine ZnO

Hours following sixth exposure

Exposure

Control gases ZnO
1 0/5  5/5
24 0/5  4/4
48 0/5  3/5
72 0/5  3/5

Applicant's summary and conclusion

Conclusions:
Under the test conditions, vital capacity, functional residual capacity, alveolar volume and single breath diffusing capacity for carbon monoxide were decreased and did not return to normal values by 72 h.
Executive summary:

A study was conducted in guinea pigs to evaluate the potential of ultrafine zinc oxide particles for changes in the functional and morphological changes in the lungs.

Male Hartley Guinea pigs were used in the study. Exposure was given by nose only for 3 h/d for 6 d. Dose selection was based on the recommended threshold limit value (TLV). 38 animals were exposed to 4.6 ± 2.7 mg/m3 ZnO concentration and evaluated for pulmonary function tests at 1, 24, 48 and 72 h postexposure. 35 animals were exposed to 5.3 ± 2.9 mg/m3 ZnO concentration and evaluated for morphology of respiratory tract, lower respiratory epithelial permeability to macromolecules and DNA synthesis in the epithelial cells of bronchi and terminal bronchioles. Statistical analysis was done by analysis of variance and additional comparisons were done by the method of Scheffe.  

Increase in flow resistance, decrease in compliance and total lung capacity were returned to normal values by 72 h. [3H]Thymidine labelling of bronchiolar epithelial cell nuclei was increased for 48 h. Inflammation of the proximal portion of the alveolar ducts and adjacent alveoli was observed. Elevated lung weights due to inflammation were observed and were not returned to normal by 72 h.  

Under the test conditions, vital capacity, functional residual capacity, alveolar volume and single breath diffusing capacity for carbon monoxide were decreased and did not return to normal values by 72 h.