Trifluoro(trifluoromethyl)oxirane

Genetic toxicity: in vivo

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 labeling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

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.

Test guideline 2

Qualifier:

equivalent or similar to guideline

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

Test material information

Constituent 1

Reference substance name:

Trifluoro(trifluoromethyl)oxirane

EC Number:

207-050-4

EC Name:

Trifluoro(trifluoromethyl)oxirane

Cas Number:

428-59-1

Molecular formula:

C3F6O

IUPAC Name:

2,2,3-trifluoro-3-(trifluoromethyl)oxirane

Details on test material:

- Purity: 99.97% Hexafluoropropylene Epoxide (HFPO)

Test animals

Species:

mouse

Strain:

other: Crl:CD1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: male average weight = 33.94, female average weight = 24.2
- Housing: housed one per cage
- Diet : PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Administration / exposure

Route of administration:

inhalation: gas

Vehicle:

- Vehicle(s)/solvent(s) used: air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass (NYU style) with a nominal internal volume of 150 L
- Method of holding animals in test chamber: Individually in stainless steel, wire-mesh modules (one animal/module) and exposed, whole-body, inside the exposure chamber
- System of generating particulates/aerosols: Chamber atmospheres were generated by dilution of the test substance in air. The test substance was metered into the chamber inlets with a Brooks model 0154E mass flow controller. High pressure air carried the resulting atmosphere into the exposure chamber. Chamber concentrations of test substance were controlled by varying the mass controller feed rate to the chamber.
- Temperature, humidity, pressure in air chamber: 21–23°C, 33-46%, and the oxygen concentrations 20.9 – 21.0%. There was 27.0 liters per minute air flow through all the chambers, which provided 11 air changes per hour.
- Treatment of exhaust air: discharged into the fume hood

TEST ATMOSPHERE
- Brief description of analytical method used: Known volumes of chamber atmosphere were continually drawn from the breathing zone of the animals and were directly injected into a gas chromatograph equipped with a pneumatically operated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 35°C on a column. The atmospheric concentration of the test substance was determined from a standard curve derived from gas standards. To ensure that the test substance did not degrade into hexafluoroacetone (HFA) as a result of the atmosphere generation techniques, the exposure chambers were sampled for HFA.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

6 hours/day

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 250 and 500 ppm
Basis:
nominal conc.

No. of animals per sex per dose:

5/sex/concentration

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive Control: cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

At least 20000 RETs were analyzed per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (%RETs) among the total erythrocytes (RETs plus NCEs).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The test substance is slightly toxic in the rat by the inhalation route with a 4-hour ALC of 2000 ppm. The only signs of toxicity observed in rats exposed at 400 ppm in a 2-week inhalation study were inactivity and slightly deepened respiration; pathological examination revealed no test substance-related changes. No effects on body weight or food consumption were observed nor were there any clinical signs of toxicity in rats exposed at 100 or 400 ppm for 18-weeks. Significant changes in organ/body weight ratios (spleen, kidneys and adrenals in males; liver in females) occurred at 400 ppm. Based on these results and a pilot study performed with the test substance the concentrations targeted for the current study were 0, 50, 250 and 500 ppm.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Peripheral blood was sampled at approximately 24 and 48 hours after the completion of the last exposure or dosing for all animals.

METHOD OF ANALYSIS: Blood samples were prepared using the In Vitro MicroFlow Plus® Mouse Micronucleus assay kit (Litron Laboratories, Rochester, New York, U.S.A.). All subsequent steps to prepare the samples for flow cytometric analysis were documented in the study records.

Evaluation criteria:

The test substance was judged negative if the following conditions were met: No statistically significant concentration-related increase in the group mean MN-RETs above the concurrent vehicle control value occurred at any concentration of the test substance; and the MN-RET values of the test substance-treated animals were within reasonable limits of the laboratory historical control range.

The test substance was judged positive if the following conditions were met: The group mean MN-RETs was statistically significantly increased at one or more concentrations of the test substance compared to the concurrent vehicle control values; and an accompanying statistically significant concentration-response increase in MN-RETs was observed.

Statistics:

Micronucleus data were evaluated using scientific judgment taking into account both statistical and biological significance. For each treatment group, the mean and standard deviation for %RETs and %MN-RETs were calculated. For those data that were normally distributed and had equal variance, parametric statistics (e.g., analysis of variance (ANOVA) and Dunnett’s test) were performed using the transformed data. For those data that were normally distributed but have unequal variance, a robust ANOVA and unequal-variance Dunnett test were done. For those data that were not normally distributed, nonparametric statistics (e.g., Kruskal-Wallis and Dunn’s tests) utilizing non-transformed data were performed. The individual animal was considered the experimental unit. All data analyses were one-tailed and conducted at a significance level of 5%.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Since there were no previous inhalation studies in mice with the test substance and the previously reported 4-hour ALC for rats is 2000 ppm , a pilot study was performed to ensure that there was no excessive toxicity at the initially targeted concentration of 1000 ppm. For the pilot study, one male and one female mouse were exposed 6 hours/day for 2 days at a target concentration of 1000 ppm. The mean concentration of the first exposure was 980 ppm, with concentrations ranging from 770 – 1100 ppm; the mean concentration for the second exposure was 1000 ppm, with concentrations ranging from 970 – 1000 ppm. One day after the second exposure, the female mouse demonstrated a 5.5 g loss in body weight and was found dead 2 days following the exposure. Therefore, it was determined that repeated exposure to 1000 ppm would result in excessive toxicity that would confound the results of the micronucleus study and 500 ppm was subsequently targeted from the high concentration exposure group.

Any other information on results incl. tables

In the main study, no test substance-related clinical signs of toxicity were observed in any animal in any concentration group. Hyperactivity was observed in 1 of 5 control males. No abnormalities were detected in the female vehicle or positive control groups. No test substance related mortality or moribundity was observed in any animal in any concentration group in the main study. One of 5 male mice in the 50 ppm, 48-hour time point group was found dead prior to sample collection. This was likely caused by the anesthesia administrated for the blood sampling and not considered test substance related.

Following 3 days of exposure to the test substance, male mice demonstrated overall mean body weight losses of 1.2, 1.4, 0.8, and 3.1 g for the 0, 50, 250, and 500 ppm groups, respectively. Female mice demonstrated overall mean body weight losses of 1.1, 1.2, 0.1, and 1.4 g for the 0, 50, 250, 500 ppm groups, respectively.

No statistically significant increases in the % MN-RET frequency were observed in any evaluated test substance treated group of male or female animals at either time point. There were no statistically significant decreases in the %RETs.

The positive control, administered once by gavage to male and female groups, showed a statistically significant increase in the frequency of MN-RETs as compared to the vehicle control group (p ≤ 0.05) at the 48-hour time point. However, due to cell cycle kinetics, at the 24-hour time point the increase in MN-RETs was not statistically significant in either male or female animals. The positive response at 48 hours provides evidence for appropriate sampling times for capturing aneugenic and clastogenic effects in the test substance treated animals.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
The test substance is negative in the mouse micronucleus assay.

Executive summary:

Four groups of 5 male and 4 groups of 5 female Crl:CD1(ICR) mice were exposed whole body for 6 hours/day for 3 consecutive days to 0 ± 0 (air vehicle control), 50 ± 0.70, 250 ± 2.7, and 500 ± 0.56 ppm H-28640 (mean ± S.E.M) for a total of 3 exposures. A concurrent positive control groups was administered 20 mg/kg of cyclophosphamide as a single dose by oral intubation. Animals were weighed and observed for clinical signs of toxicity on a daily basis during the study. After approximately 24 and 48 hours following exposure blood was collected. A total of 20000 reticulocytes (RETs) were analyzed per blood sample for induction of micronuclei, and toxicity as indicated by the frequency of immature erythrocytes (% reticulocytes, RETs) among the total erythrocytes (RETs plus normochromatic erythrocytes, NCEs). The frequency of micronucleated reticulocytes (%MN-RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test substance.

No test substance-related clinical signs of toxicity were observed in any of the animals at all concentrations of the test substance. Hyperactivity was observed in 1 of 5 control males. No abnormalities were detected in the female vehicle or positive control groups. No mortality or moribundity was observed in any animal in any concentration group in the main study. Following 3 days of exposure to the test substance, male mice demonstrated overall mean body weight losses of 1.2, 1.4, 0.8, and 3.1 g for the 0, 50, 250, and 500 ppm groups, respectively. Female mice demonstrated overall mean body weight losses of 1.1, 1.2, 0.1, and 1.4 g for the 0, 50, 250, 500 ppm groups, respectively.

Exposure of mice to 50, 250 or 500 ppm for 3 days did not result in significant alteration %RET, %MN-NCE or %MN-RET when compared to the air exposed (0 ppm) control group. Cyclophosphamide treatment (positive control) did not induce changes in %RET, %MN-NCE or %MN-RET 24 hours following treatment. At the 48-hour post dosing time point, male mice demonstrated a significant reduction in %RET while both male and female mice demonstrated a significant increase in %MN-RET when compared to control which indicated a positive response that was consistent with the micronucleated PCE historical control data.

Under the conditions for this study, the test substance did not induce statistically significant or biologically relevant increases in micronucleated reticulocytes in animal peripheral blood. No statistically significant or biologically relevant decreases in the %RET were observed in either male or female animals. The result of the study was concluded to be negative and the no-observed effect level is >500 ppm test substance.