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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: EC Commission Directive 2000/32/EC Annex 4C-B.12
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.99 %

Test animals

Species:
rat
Strain:
other: Sprague Dawley CD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Not reported
- Weight at study initiation: Males: 175-215 g; Females: 163-196 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Sexes separated, in cages
- Diet (e.g. ad libitum): ad libitum, except during inhalation exposure
- Water (e.g. ad libitum): ad libitum, except during inhalation exposure
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 34-56%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
Air
Details on exposure:
TYPE OF INHALATION EXPOSURE: Whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body exposure chambers constructed from stainless steel and glass, with an internal volume of approximately 750 litres.
- Method of holding animals in test chamber: Singly in stainless steel mesh compartments
- Source and rate of air: Not reported
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: The test atmosphere was produced by diluting the gaseous test substance with air. Adjustments were made to the gaseous test substance supply to each chamber during exposures in order to maintain the desired concentrations. The dilutions of gaseous test substance with air were determined during preliminary generation trials.
- Temperature, humidity, pressure in air chamber: 22±1°C, 40-60%, 1-10 mm H20 below ambient pressure
- Air flow rate: 150 L/min
- Air change rate: Approximately 12 changes/hour
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: During exposure, chamber atmosphere was sampled at least 6 times during each exposure. Analysis of test substance in air was done by chemical analysis of samples of chamber air. The nominal concentration of test substance was calculated by recording the amount of test substance delivered to the generation system during the exposure. The usage over the 6 hours exposure was divided by the total airflow through the chamber. Any losses during the generation process were quantified and included in the calculation of the nominal concentration.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
6 hours
Frequency of treatment:
Single exposure
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
4875, 9750, 19500 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
5 male and 5 female at 4875 and 9750 ppm
10 male and 10 female at 0 and 19500 ppm
5 male and 5 female for positive controls
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Not reported
- Route of administration: Single oral gavage
- Doses / concentrations: 20 mg/kg (10 mL/kg dosing volume)

Examinations

Tissues and cell types examined:
PCEs (polychromatic erythrocytes) and NCEs (normochromatic erythrocytes) taken from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest target concentration selected was the estimated maximum exposure level equivalent to 50% of the Lower Explosive Limit (LEL).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Five males and 5 females were sacrificed from the negative control and each of the test substance groups 24 hours after completion of the exposure period and from the positive control group 24 hours after dosing. In addition, 5 male and 5 female animals were sacrificed from the negative control and high level treatment groups 48 hours after completion of the exposure period.

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, the bone marrow of both femurs from each animal was flushed out and pooled in Hanks’ Balanced Salts Solution by aspiration. The resulting cell suspensions were centrifuged at 1000 rpm (150 x g) for 5 minutes and the supernatant discarded. Each resulting cell pelleted was resuspended in 2 mL of filtered foetal calf serum before being sedimented by centrifugation. The supernatant was discarded and the final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing on glass microscope slides. Several smears were prepared from each femur. Due to the presence of mast cell granules in rat bone smears, a modified Feulgen staining method was employed.

METHOD OF ANALYSIS: The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. One smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear. Micronuclei were identified by the following criteria: large enough to discern morphological characteristics; should possess a generally rounded shape with a clearly defined outline; should be deeply stained and similar in colour to the nuclei of other cells (not black); should lie in the same focal plane as the cell; lack internal structure; and there should be no micronucleus-like debris in the area surrounding the cell. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept.
Evaluation criteria:
A positive response was normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (p<0.01); individual and/or group mean values should exceed the laboratory historical control range. A negative result was indicated where the individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (p>0.01) and where these values fall within historical control range. An equivocal response was obtained when the results do not meet the criteria specified for a positive or negative response.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (p<0.01).
Statistics:
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. For incidences of micronucleated immature erythrocytes, exact one-sided P-values were calculated by permutation. Comparison of several dose levels was made with the concurrent control using the Linear by Linear Association test for trend, in a step-down fashion if significance was detected; for individual inter-group comparisons this procedure simplifies to a straightforward permutation test. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores were used (i.e., exact versions of Wilcoxon’s sum of ranks test and Jonckheere’s test for trend).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
No mortality or adverse clinical signs were observed. The test substance did not cause an increase in the number of micronucleated immature (polychromatic erythrocytes) or micronucleated mature (normochromatic) erythrocytes. The positive control caused large, highly significant increases in the frequency of micronucleated immature (polychromatic) erythrocytes.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). It was concluded that the test substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure in this in vivo micronucleus test.
Executive summary:

The potential induction of micronuclei by the test substance in bone marrow cells of the rat was examined. Animals were treated for a single 6-hour period of whole body inhalation exposure to the test substance at nominal dose levels of 4875, 9750, and 19500 ppm, expressed in terms of volume of test substance vapor per unit volume of atmosphere. The test substance and negative control were administered by whole body exposure. The negative control group received clean air only. A positive control group was dosed orally, by gastric intubation, with cyclophosphamide at 20 mg/kg body weight. Bone marrow smears were obtained from 5 male and 5 female animals in the negative control and each of the test substance groups 24 hours after completion of the exposure period and from the positive control group 24 hours after dosing. In addition, bone marrow smears were obtained from 5 male and 5 female animals in the negative control and high level treatment groups 48 hours after completion of the exposure period. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept. 

No statistically significant increase in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with the test substance and killed 24 or 48 hours after a single 6-hour period of whole body inhalation exposure, compared to negative control values. The positive control compound produced large, highly significant increases in the frequency of micronucleated immature erythrocytes and a decrease in the proportion of immature erythrocytes. It is concluded that the test substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure in this in vivo test procedure.