Fluoroethylene

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 labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

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 draft guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA Health Effects Testing Guidelines 50 CFR Part 798, Federal Register Vol 50, No. 188, Sept 27, Subpart 798.5395 (In vivo mammalian bone marrow cytogenetic tests: Micronucleus assay), pp. 39446-39447.

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:

Fluoroethylene

EC Number:

200-832-6

EC Name:

Fluoroethylene

Cas Number:

75-02-5

Molecular formula:

C2H3F

IUPAC Name:

fluoroethene

Details on test material:

- Purity: 99.99% (excluding inhibitor)
- Inhibitor, D-Limonene 0.31 wt. %

Test animals

Species:

mouse

Strain:

other: Crl:CD®-l(ICR)BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 8 weeks old
- Weight at study initiation: Males: 31-38 g (mean 34 g); Females: 23-29 g (mean 27 g)
- Fasting period before study: No
- Housing: Individually in standard wire mesh cages, except during exposure
- Diet (e.g. ad libitum): ad libitum, except during exposure
- Water (e.g. ad libitum): ad libitum, except during exposure
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 50±10%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Route of administration:

inhalation: gas

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: An approximately 30-liter cylindrical glass exposure chamber.
- Method of holding animals in test chamber: Whole-body. The mice were placed into wire-mesh baskets (sexes separate; maximum 9/basket) and the baskets were stacked in the exposure chamber.
- Source and rate of air: Conditioned house air
- Method of conditioning air: Where appropriate, pure oxygen gas was added to maintain the atmospheric oxygen content at an acceptable level.
- System of generating test atmospheres: Test atmospheres were generated by metering pure test substance gas from a cylinder (20 psi) with Brooks flowmeters into 3-neck glass mixing flasks. The pure gas was diluted to the desired concentrations by adding air at the flasks (approximately 12-18 L/min). The diluted test substance was added to the tops of the exposure chambers.
- Temperature, humidity, pressure in air chamber: Temperature was 20-24°C, relative humidity was 31-70%, pressure was not reported, and mean oxygen concentration was 20.6-21.0% during exposure
- Air flow rate: Approximately 9 L/min of air for the control chamber.
- Air change rate: Not reported
- Treatment of exhaust air: The chamber exhausts were drawn through dry-ice cold traps, MSA cartridge filters and scrubbers containing ethylene glycol prior to being discharged into the exhaust hoods.

TEST ATMOSPHERE
- Brief description of analytical method used: Atmospheric concentration of the test substance in each exposure chamber was monitored at approximately 30-minute intervals. Samples (0.1 mL) were collected in duplicate with a gas-tight syringe and injected directly into a gas chromatograph equipped with a flame ionization detector. The injection port temperature was 200°C and the detector temperature was 250°C. Nitrogen was used as the carrier gas. Samples were chromatographed isothermally at 60°C on a glass column packed with 10% SE-30 on 80/100 mesh Supercoport®. Atmospheric concentrations of the test substance were determined by comparing the peak heights against standard curves calculated daily by regression. Standards were prepared daily.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours

Frequency of treatment:

once

Post exposure period:

24, 48, or 72 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 50000, 200000, 400000 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 50100, 191000, 388000 ppm
Basis:
other: mean analytical concentration

No. of animals per sex per dose:

15/sex/control, low, and intermediate dose levels; 18/sex/high dose level

Control animals:

yes

Positive control(s):

- Positive Control: Cyclophosphamide
- Justification for choice of positive control(s): Not reported
- Route of administration: ip injection
- Doses / concentrations: 20 mg/kg (5 males and 5 females)

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes (PCEs) and Normochromatic erythrocytes (NCEs) from rat bone marrow taken from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The selection of the maximum exposure level was based on a preliminary rangefinding experiment. In the rangefinding experiment groups of 4 male and 4 female mice were exposed for 6 hours to mean test substance concentrations of 205000, 271000, or 357000 ppm. The mice were observed for clinical signs during and immediately after exposure. Surviving mice were weighed and observed daily for 7-13 days post exposure. During exposure, mice in all test groups were unresponsive to sound. Mice exposed to 205000 and 271000 ppm appeared to be hyperactive at the beginning of exposure, but were resting normally by the end of exposure. Mice exposed to 357000 ppm were staggering and had tremors, red ocular discharge and laboured breathing during exposure. The only clinical sign which persisted after the termination of exposure was the ocular discharge observed at 357000 ppm. No deaths occurred during exposure or during the recovery period. Weight loss was observed 24 hours post exposure in all groups and some mice in all exposure groups continued to have sporadic weight loss during the recovery period. Although this rangefinding study did not identify significantly toxic concentrations, 400000 ppm was selected as the maximal concentration for the micronucleus assay; higher concentrations would have been impractical from both a physical and biological point of view. Lower concentrations of 20000 and 50000 ppm were selected.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Groups of 5 or 6 males and females from the negative control and each treatment group were sacrificed approximately 24, 48 and 72 hours after the final exposure. The positive indicator group, consisting of 5 male and 5 female mice were concurrently treated at the initiation of the inhalation exposures and were sacrificed approximately 24 hours after treatment.

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, the marrow from both femurs of each animal was aspirated and flushed into prewarmed (37°C) foetal bovine serum. The marrow was collected by centrifugation at approximately 1000 rpm for 5 minutes. Most of the supernatant was removed and the cells were resuspended in the remaining serum. An automatic blood smearing instrument was used to make the bone marrow smears. Three slides per animal were prepared and fixed in absolute methanol for 8 minutes. The slides were stained for 2.5 minutes in acridine orange in phosphate buffer, followed by phosphate buffer rinses. Immediately prior to scoring, a coverslip was floated on each slide using phosphate buffer.

METHOD OF ANALYSIS: Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy. Only cells showing good morphology and staining were selected for scoring. PCEs were identified by their characteristic reddish staining; NCEs appeared dark green. One thousand PCEs per animal were scored for the presence of micronuclei. Cellular inclusions that were irregularly shaped or stained, or were not in the focal plane of the cell were considered artifacts and not scored. The unit of scoring was the micronucleated cell. PCEs containing more than one micronucleus were counted as a single micronucleated cell. The number of micronucleated NCEs seen in the optic fields scored for PCEs was also recorded. The ratio of PCEs to NCEs was determined for each animal based on one thousand total erythrocytes.

Evaluation criteria:

Not reported

Statistics:

Treatment effects were examined for each sex independently. Statistical significance was judged at the 5% level. Two statistical methods were used to evaluate the data for the proportions of micronucleated PCEs:
1) A Cochran-Armitage Test for trend across exposure groups. This method assumes that there was not interanimal variability within treatment groups.
2) An analysis of variance (ANOVA) of treatment level and time. Where the F-test for treatment level was significant, pairwise comparisons were made between each treatment group and the concurrent control using Dunnett’s Test. PCE:NCE ratios, mean body weight and weight gain date were also evaluated using this method. The calculations for the ANOVA were done using BMDP programs.

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

yes

Remarks:

Clinical signs and weight loss

Negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Sex:

female

Genotoxicity:

positive

Toxicity:

yes

Remarks:

Clinical signs and weight loss

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY

Clinical findings:
During exposure, mice in all test substance exposure groups had a reduce response to sound. Mice exposed to 191000 ppm were more active than the controls. Soon after the onset of exposure to 388000 ppm, mice were less active than the controls, were grooming excessively and staggering and had red ocular discharge. Later during exposure, all mice were hyperactive and were grooming excessively as indicated by frantic preening, licking and scratching. Twenty-four hours after the initiation of treatment, 1 male mouse in the 191000 ppm group and 1 male mouse in the 388000 ppm group exhibited ruffled fur; this condition was still present on the 388000 male at 48 hours. Additionally, at 48 hours, one male mouse in the 50100 ppm group was hyperactive. No clinical signs were noted at the 72-hour observation period. Significant weight loss was seen in the low dose group males at the 72-hour sacrifice time and in the high dose group males at the 48-hour and 72-hour sacrifice times. Females from the high dose group had significant weight loss at the 24-hour sampling time. No significant weight loss or decreased weight gain was observed in any other test groups.

Micronucleus and PCE:NCE evaluations:
No significant depression of the PCE:NCE ratios was seen in either the test substance exposed males or females at any of the sampling times. Additionally, no statistically significant increases or concentration-related trends in MN-PCEs were seen at the 48 or 72 hour sampling times. At the 24 hour sacrifice time, the females showed a statistically significant concentration-related trend in the proportion of micronucleated PCEs, although no single test concentration gave significant increases above the controls. The males in the low and high exposure groups exhibited increased frequencies of MN-PCEs as compared to the concurrent negative control groups; these increases were not statistically significant.

To further investigate the responses demonstrated at the 24-hour sample time, an additional 1000 PCEs per animal were evaluated for both the males and females. Based on the total of 2000 PCEs scored per animal, statistically significant increases in micronucleated PCEs were seen in the females at the middle and high exposure levels; a concentration-related trend was still evident. Elevated frequencies of MN-PCEs seen in the 24-hour test substance exposed males were again not statistically significant, and no concentration-related trend was present. The CP-treated females showed significant increases in micronucleated PCEs compared to the concurrent air controls. The elevated frequency of MN-PCEs seen in the CP-treated males was not statistically significant (p=0.09), possible due to the relatively high number of MN-PCEs in the concurrent 24-hour negative control group. However, when compared to the pooled negative control values across all sacrifice times, the increase was significant (p=0.004). No significant depression PCE:NCE ratios was seen in the CP-treated groups. See Table 1 for summary data for micronucleated PCEs and PCE:NCE ratios.

Any other information on results incl. tables

Table 1

	MN-PCEs/1000 PCEs^a
Test substance (ppm)	Sacrifice Time
	24 hours	48 hours	72 hours
Males 0	2.8 ± 0.5^b	1.6 ± 0.9	2.0 ±.03
50100	4.5 ± 0.6^b	2.2 ± 0.9	1.8 ± 0.5
191000	3.3 ± 0.7^b	2.4 ± 1.0	1.2 ± 0.4
388000	4.7 ± 0.8^b	1.3 ± 0.3	0.8 ± 0.5
Trend	N.S.	N.S.	N.S.
CP	8.4 ± 2.9^c**
Females 0	1.7 ± 1.1^b	2.0 ± 0.7	2.8 ± 0.4
50100	3.0 ± 0.9^b	1.4 ± 0.7	1.0 ± 0.5
191000	5.5 ± 1.2^b*	2.8 ± 0.4	1.2 ± 0.4
388000	5.6 ± 0.6^b*	1.8 ± 0.7	2.5 ± 0.8
Trend	***	N.S.	N.S.
CP	9.8 ± 1.2^***
	Ratio PCEs:NCEs^a
Test substance (ppm)	Sacrifice Time
	24 hours	48 hours	72 hours
Males 0	0.8 ± 0.1^b	1.2 ± 0.1	1.3 ± 0.2
50100	1.2 ± 0.1^b	1.0 ± 0.2	1.1 ± 0.2
191000	1.0 ± 0.1^b	9 ± 0.1	0.9 ± 0.1
388000	0.8 ± 0.0^b	1.2 ± 0.1	0.9 ± 0.1
CP	1.2 ± 0.1
Females 0	1.2 ± 0.1^b	1.1 ± 0.1	1.5 ± 0.3
50100	0.9 ± 0.1^b	0.9 ± 0.2	1.1 ± 0.2
191000	1.0 ± 0.1^b	1.3 ± 0.2	1.3 ± 0.2
388000	1.0 ± 0.0^b	1.0 ± 0.1	1.2 ± 0.2
CP	1.0 ± 0.1

MN-PCEs = micronucleated polychromatic erythrocytes; NCEs = normochromatic erythrocytes; CP = cyclophosphamide, 20 mg/kg i.p.

^a Means ± S.E. of 5 mice (0, 50100 and 191000 ppm) or 6 mice (388000 ppm) per treatment group at each sacrifice time.

^b Based on scoring 2000 PCEs/animal; for other sacrifice times, 1000 PCEs/animal were scored.

^c Statistically compared to pooled male negative controls (24, 48 and 72 hours).

^* = p < 0.05; ^** = p < 0.01; ^*** = p < 0.001; N.S. = not significant

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: equivocal in male mice and positive in female mice
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). Under the conditions of this assay, the test substance is judged equivocal in male mice and positive in female mice for its ability to induce micronuclei in bone marrow cells of rats.

Executive summary:

The test substance was tested for its ability to induce micronuclei in bone marrow polychromatic erythrocytes of male and female mice. The animals were exposed whole-body to test substance atmospheres of up to 388000 ppm for 6 hours, and bone marrow smears were prepared 24, 48, and 72 hours after the beginning of exposure. No significant depression in the ratio of young, polychromatic erythrocytes to mature, normochromatic erythrocytes was detected in either sex.

 

At the 24-hour sampling time, females exposed to the intermediate and high dose levels, 191000 and 388000 ppm, respectively, showed statistically significant increases in the frequency of micronucleated polychromatic erythrocytes as compared to their concurrent air controls; a significant concentration-related trend was also present. The 24-hour treated males also showed increased frequencies of micronucleated polychromatic erythrocytes; however, these were not statistically significant. On the basis of these findings, the test substance is judged equivocal in male mice and positive in female mice.