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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
other: 5 d repeated exposure study
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Pulmonary function of guinea pigs exposed to freshly generated ultrafine zinc oxide with and without spike concentrations
Author:
Lam HF, Chen LC, Ainsworth D, Peoples S and Amdur MO
Year:
1988
Bibliographic source:
Am. Ind. Hyg. Assoc. J. 49(7): 333-341

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Guinea pigs were exposed by nose only 3h/d for 5 d to ZnO ultrafine particles and examined after 1, 2, 3, 4 and 5 d post-exposure for functional changes and extent of damage in the lungs.
GLP compliance:
not specified
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 and ZnO
- Physical state: Ultrafine zinc oxide particles

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: 240-300 g
- Housing: Animals were housed 4/cage
- Diet: Ad libitum, Charles River Guinea Pig Formula
- Water: Ad libitum
- Maintenance: According to guidelines of Division of Comparative Medicine at MIT, which is accredited by the American Association for Accreditation of Laboratory Animal Care (AALAC)
- Others: Observed in quarantine for 5 d for any externally detectable evidence of pulmonary disease before exposure to test material


ENVIRONMENTAL CONDITIONS
- Temperature: 23 °C
- Humidity: 30-50 %


Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
other: no data
Remarks on MMAD:
MMAD / GSD: 0.05 µm/2.0
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Dynamic animal exposure chamber
- System of generating particulates/aerosols: Zinc turnings were heated at 480 °C in a crucible, and the zinc vapours were carried by a flow of argon gas to be contacted by purified air downstream in the furnace. The vapours react with oxygen to yield ultrafine zinc oxide particles upon condensation.
- Method of particle size determination: Test material aerosol was collected on pre-coated carbon grids using Thermosystem Model 3100 electrostatic precipitator and examined (400 to 600 particles) and photographed in a Philips 300 electron microscope
- Particle size distribution: Piezoelectric crystal TSI Model 3030


TEST ATMOSPHERE
- Analytical method used: Test material concentration was determined using Millipore GSWQ 47 mm filters for total aerosol collection and weighing of the filters on a Cahn electrobalance Model 21. Chemical analysis consisted of Atomic absorption spectrometry and electron spectroscopy used for chemical analysis (ESCA)
- Samples taken from breathing zone: Yes




Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Measured by aerosol collection on Millipore GSWQ 47 mm filters, followed by weighing on a Cahn electrobalance Model 21
Duration of treatment / exposure:
3 h/d for 5 d
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Experiment I: 7 mg/m3 (For details see Table 1 under 'Any other information on materials and methods incl. tables')
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
Experiment II: 2.7 mg/m3 (For details see Table 1 under 'Any other information on materials and methods incl. tables')
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
Experiment III: Single high-peak exposure at 34 mg/m3 , and terminated exposure (For details see Table 1 under 'Any other information on materials and methods incl. tables')
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
Experiment IV: Single high-peak exposure: 25 mg/m3 and continuing exposure at: 4-8 mg/m3 (For details see Table 1 under 'Any other information on materials and methods incl. tables')
Basis:
analytical conc.
No. of animals per sex per dose:
5-8 animals in each experiment (For details on number of animals in each experiment - see Table 1 under 'Any other information on materials and methods incl. tables')
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Based on the previous acute exposure study at 8 mg/m3 for 3 h and repeated dose study for 3h/d, 6 d exposure at 5 mg/m3: the following doses were selected: (a) 7 mg/m3 for 5 d : to address the time course of alterations of pulmonary function, (b) 2.7 mg/m3 for 5 d: to determine effects at lower concentration and (c) Two peak experiments (25-34 mg/m3): to address the effects of occasional peak exposure of test material during chronic low-level exposures
Positive control:
None

Examinations

Observations and examinations performed and frequency:
OTHER: PULMONARY FUNCTION MEASUREMENT
- Number of animals: 5-8 animals
- Time schedule for examination: After 1, 2, 3, 4 and 5 exposure periods (3 h)
- Anesthetic used for examination: Yes (ketamine hydrochloride and xylazine - 100 mg/kg 1M and 15 mg/kg 1M, respectively)
- Pulmonary function parameters checked: ventilation, dynamic compliance, flow resistance, vital capacity (VC) and inspiratory capacity (IC), total lung capacity (TLC), Functional residual capacity (FRC) and residual volume (RV), single-breath diffusing capacity for carbon monoxide (CO) ( DLco) and apparent alveolar volume (VA)

Sacrifice and pathology:
None
Other examinations:
LUNG WEIGHT:
- Time schedule for examinations: After the last functional measurement
- Brief description of method: The chest was opened and the lungs were removed after exsanguination of animals. The heart and mediastinal tissues were dissected to measure the wet-lung weight. Dry-lung weight recording were obtained by drying the lungs at 45 °C for 48 h, followed by keeping in desiccator for 24 h.
Statistics:
Analysis of variance (ANOVA) was used to compare the means of variables across all exposure groups with the pooled control data. Significant differences (p ≤ 0.05) among group means were analysed for group differences by applying Dunnett's multiple range test. Correlations between variables were computed using StatView 512+©

Results and discussion

Results of examinations

Clinical signs:
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
ORGAN WEIGHTS: Extent of pulmonary damage was measured by:

- Wet-lung weight/Body weight ratio: (see Figure 6 )
(a) Experiment I (7 mg/m3): Ratio increased about 10 % on Day 2 and 29 % by Day 5
(b) Experiment II (2.7 mg/m3): No significant increase
(c) Experiment III (single high-peak exposure (34 mg/m3) with terminated exposure: Ratio increased 30 %, 49 %, 45 % and 19 % on Days 2-5 respectively
(d) Experiment IV (single high-peak exposure (25 mg/m3) with continued exposure at 4-8 mg/m3: No change on day of peak (Day 1), but increased about 45 %, 50 % -60 % on Days 2 and 3-5 respectively

- Wet-lung weight/Dry-lung weight ratio: (see Figure 6)
(a) Experiment I (7 mg/m3): Increased significantly only on Day 4
(b) Experiment II (2.7 mg/m3): No significant increase
(c) Experiment III (single high-peak exposure (34 mg/m3) with terminated exposure: Ratio increased 8 % on Day 2-4 and then decreased on Day 5
(d) Experiment IV (single high-peak exposure (25 mg/m3) with continued exposure at 4-8 mg/m3: Ratio increased by 10 % on Day 2 and remained constant till Day 5


OTHER FINDINGS: PULMONARY FUNCTION MEASUREMENT

- Tidal volume, respiratory frequency, airway resistance or compliance:
(a) Experiment I (7 mg/m3): No alterations
(c) Experiment III (single high-peak exposure (34 mg/m3) with terminated exposure and (d) Experimnet IV (single high-peak exposure (25 mg/m3)(with continued exposure at 4-8 mg/m3: Increased airway resistance and decreased compliance was observed at both peak exposures (see Figure 5)

- Lung volumes (TLC, VC, FRC and RV): Significant decrease below control was observed
(a) Experiment I (7 mg/m3): VC and TLC decreased -22 % and -18 %; No change in FRC and RV was observed (see Figure 1A)
(b) Experiment II (2.7 mg/m3): No change in lung volume (see Figure 2A)
(c) Experiment III (single high-peak exposure (34 mg/m3) with terminated exposure: TLC decreased -15 %, -22 %, -24 %, -17 %, on Days 2-5 respectively; VC decreased -17 %, -28 %, -25 % and 19 %, on Days 2-5 respectively; No change in FRC and RV was observed (see Figure 3A).
(d) Experiment IV (single high-peak exposure (25 mg/m3) with continued exposure at 4-8 mg/m3: TLC decreased -10 %, -32 %, -34 %, -28 % and -28 % on Days 1-5 respectively; VC also decreased in similar way; FRC decreased -17 %, -27% and -20 % on Days 2-4 respectively. No difference on Day 5; RV reduced -20 %, -30 %, -20 % and -10 % on Days 2-5 respectively (see Figure 4A)

- DLco and VA: Significant decrease below control was observed
(a) Experiment I (7 mg/m3): Only a slight increase was observed on the Day 1 of exposure (see Figure 1B)
(b) Experiment II (2.7 mg/m3): No change in DLco was observed (see Figure 2B)
(c) Experiment III (single high-peak exposure (34 mg/m3) with terminated exposure: VA decreased significantly by -10 % on Day 3; DLco decreased -40 %, -50 % and -25 % on Days 2, 3 and 5 respectively (see Figure 3B).
(d) Experiment IV (single high-peak exposure (25 mg/m3) with continued exposure at 4-8 mg/m3: VA only decreased on Day 3; DLco decreased -60 %, -40 %, -30 %, 25 % and -25 % on Days 1-5 respectively (see Figure 4B)

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
2.7 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: All measured parameters
Dose descriptor:
other: decreased lung volume
Effect level:
7 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: Other: Pulmonary function measurement
Dose descriptor:
other: decrease of Lung volumes and diffusing capacity at peaks occurs rapidly and to a greater extent
Effect level:
25 - 34 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: Pulmonary function measurement
Dose descriptor:
other: Pulmonary damage
Effect level:
7 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: Wet-lung weight/Body weight ratio and Wet-lung weight/Dry-lung weight ratio
Dose descriptor:
other: Increased pulmonary damage at peak concentrations
Effect level:
25 - 34 mg/m³ air (analytical)
Sex:
male
Basis for effect level:
other: Wet-lung weight/Body weight ratio and Wet-lung weight/Dry-lung weight ratio

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Correlation analysis: Significant associations exists between TLC and DLco with the ratio of wet-lung weight to body weight (r = 0.93 and r = 0.70, respectively). A correlation also exists between the change in DLco and that of TLC ( r = 0.73). (see Figure 7). Association of decreased of lung volumes and DLco with the ratio of wet-lung weight to body weight indicates presence of pulmonary oedema.

Remarks on results on extent of pulmonary damage: The increased wet-lung weight to body weight ratios on peak exposures indicate high dose of ZnO given within a short period of time was more effective in inducing lung damage than an equivalent dose given over a longer period.

Remarks on results on Pulmonary function measurement: Nonpeak exposures induced gradual decreases in TLC and VC and diffusing capacity, whereas, in peak exposures, the changes occurred very rapidly.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, Exposures to 2.7 mg/ m3, using the same 3 hr/ day, 5 day time frame, did not alter any parameters measured.
Executive summary:

An inhalation study was conducted to evaluate the low-level exposures together with occasional intense exposures of ultrafine test material particles in guinea pigs.

 

5-8 Male Hartley Guinea pigs in each experimental group were given an exposure to different concentrations of test material, by nose-only for 3 h/d for 5 consecutive days. The animals were then evaluated for pulmonary function tests and extent of pulmonary damage after 1, 2, 3, 4 and 5 d post-exposure.

An exposure at concentration of 7 mg/m3 produced a gradual decrease in total lung capacity and vital capacity over the course of the exposure period. The carbon monoxide (CO) diffusing capacity (DLco) dropped abruptly to 30% below control levels on Day 4. Wet-lung weight/body weight ratios and wet-lung/ dry-lung weight ratios increased, indicating the presence of edema. Exposures to 2.7 mg/ m3, using the same 3 hr/ day, 5 day time frame, did not alter any parameters measured.

In two experiments a single high peak of test material (25-34 mg/m3) occurred with or without continued exposure. In both, lung volumes were decreased abruptly and to a greater extent than when peaks were absent. Continued exposure caused greater decrements in TLC and V C as well as decrements in FR C and RV than were observed when exposure was stopped. Peak exposures reduced DLco to 45%-60% below control. These values rose to 25%-30% below control with or without continued exposure. Increased airway resistance and decreased compliance were also observed at both peak exposures.

 

Under the test conditions, the short peaks occurring during normal low-level exposures can induce rapid pulmonary functional changes and greater extent of pulmonary damage and oedema.