Trifluoro(trifluoromethoxy)ethylene

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 19th December 1989 to 29th january 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study in compliance with international recognized guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Trifluoro(trifluoromethoxy)ethylene

EC Number:

214-703-7

EC Name:

Trifluoro(trifluoromethoxy)ethylene

Cas Number:

1187-93-5

Molecular formula:

C3F6O

IUPAC Name:

1,1,2-trifluoro-2-(trifluoromethoxy)ethene

Test material form:

other: gas

Details on test material:

name of the substance as cited in the report: FMVE.
Chemical name: Ethene, trifluoro (trifluoromethoxy)
Chemical formula: C3F6O.
Molecular weight: 166.02.
CAS No.: 1187-93-5.
Purity: 99.6% by GC.

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

All animals in this study were Specific Pathogen Free CD-1 outbred mice of Swiss origin weighing between 22 and 24 grams and approximately 35 days old, on despatch. The animals were obtained from Charles River U.K. Limited, Margate, Kent, England.
On arrival the weight of the animals was checked, the animals were randomly assigned to groups and tail marked.
Each group of 5 mice was kept in a plastic disposable cage with the sexes housed separately and maintained in a controlled environment with 30 changes of air per hour and the thermostat set at 22°C. The room was illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted Biosure LAD 1 rodent diet and tap water, except during inhalation exposure. They were acclimatised for approximately four days, examined daily and weighed prior to dosing.

Administration / exposure

Route of administration:

other: inhalation, whole body exposure

Vehicle:

air atmosphere

Details on exposure:

Gas generation
The gas was fed through stainless steel pipes from the cylinder supplied via a 3-way stainless steel manifold, to the base of stainless steel and glass elutriation columns one of which was fitted before the inlet duct of each chamber. The concentration of gas in air within each chamber was regulated by an in-line flow control needle valve; gas flow was monitored with in-line glass flow meters.
Exposure chambers
The exposure chambers were constructed from stainless steel and glass and were approximately 0.7 m^3 internal volume. The chambers were of square cross-section fitted with a pyramidal base and top.
Diluent inlet air at 0.1 m3/min, monitored continuously using tapered tube flow meters, entered the base of the elutriation columns and mixed with the gas (except Group 1 [Negative control]). The gas/air mixture passed into the chamber via the inlet duct.
The chamber atmosphere was extracted by a single extract fan withdrawing air from the chambers, via a manifold, through an activated charcoal filter. Extract flow was controlled by gate valves in the extract line such that the pressure within each chamber was maintained at 10 mm of water below ambient.
Each chamber was fitted with ports for withdrawal of chamber air samples for analytical purposes. Routinely a port mid-way on the side wall was used.
The mice were held in groups according to sex in compartments of stainless steel wire mesh cages during the exposure.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

Single exposure.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.075 other: % v/v

Remarks:

Basis: nominal conc.
Equivalent to 750 ppmv, i.e., ca. 5000 mg/m3

Doses / concentrations 2

Dose / conc.:

0.15 other: % v/v

Remarks:

Basis: nominal conc.
Eq. to 1500 ppmv, i.e. ca. 10000 mg/m3

Doses / concentrations 3

Dose / conc.:

0.3 other: % v/v

Remarks:

Basis: nominal conc.
Eq. to 3000 ppmv, i.e. ca. 20000 mg/m3

Control animals:

yes

Positive control(s):

Mitomycin C, obtained from BDH Limited, was used as the positive control compound. It was prepared as a solution in sterile 0.9% saline at a concentration of 0.6 mg/mi. It was administered by oral gavage at a standard volume of 20 ml/kg bodyweight. The animals in the positive control group were deprived of diet overnight prior to and for two hours after oral dosing.

Examinations

Tissues and cell types examined:

Bone marrow cells from the femurs.

Details of tissue and slide preparation:

Animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone. A direct bone marrow smear was made onto a slide containing a drop of calf serum. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol (>10 minutes). The smears were air-dried and stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Gurr’s improved R66 Giemsa (BDK) with distilled.water). Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal. A proof to evaluate systemic availability of the test item was not made (even if it is not requested by OECD)
The ratio of polychromatic to normochromatic erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of microriucleated normochromatic erythrocytes was also kept.

Evaluation criteria:

A positive response is normally indicated by a substantial, dose-related and statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group. In borderline cases, e.g. where the response is not dose-related or where individual group mean values do not fall outside our historical control range, further testing may be necessary.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial, dose-related and statistically significant decrease in the ratio of polychromatic to normochromatic erythrocytes. This decrease would normally be evident at both the 48 and 72 hour sampling points, a decrease at the 24 hour time point is not necessarily expected because of the relatively long transition time of erythroid cells (late normoblast - polychromatic erythrocyte (approximately 6 hours) - normochromatic erythrocyte (approximately 30 hours)].

Statistics:

Non-parametric statistical methods, based on rank, are chosen for analysis of results because:
(a) They are suited to analysis of data consisting of discrete/integer values such as the incidence of micronucleated polychromatic erythrocytes.
(b) The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of nonparametric analysis and classical analysis of variance are very similar).
(c) ‘outliers’ are frequently found in the polychromatic erythrocyte to normochromatic erythrocyte ratios for both control and treated animals; non-parametric analysis does not give such values an undue weighting.
For a comparison of an individual treated group with a concurrent control group, Wilcoxon’s sum of ranks test is used. For multiple intergroup comparisons Kruskal-Wallis’ version of this test is used. Spearman’s and Jonckheere’s tests are used to test for evidence of a dose-related trend.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

three animals treated withe the test substance at 0.3% v/v died after exposure and prior to sacrifice

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Signs and mortalities:
Three animals died after exposure to PMVE in the micronucleus test. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.
Micronucleated polychromatic erythrocyte counts (mnp)
PMVE did not cause any statistically significant increases in the number of micronucleated polychromatic erythrocytes at any of the three kill times [P>0.05] using Kruskal-Wallis test, Jonckheeres test for trend and Spearma's correlation test.
Mitomycin C caused large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes [P<0.001] using Wilcoxon’s sum of ranks test.
Micronucleated normochromatic erythrocytes (mnn)
PMVE did not cause any substantial increases in the incidence of micronucleated normochromatic erythrocytes at any of the three kill times.
Ratio of polychromatic to normochromatic erythrocytes (p/n)
Statistically significant dose-related decreases in the p/n ratio were obtained for mice treated with FMVE at the 48 and 72 hour sampling times. Mitomycin C also caused a small, statistically significant decrease in the p/n ratio. These decreases may be indicative of bone marrow cell toxicity/depression.

Any other information on results incl. tables

Summary of results per group

time	treatment	Exposure level (% v/v)	ratio p/n			incidence mnp		Incidence mnn
			mean	statistics P		Mean 0/00	statistics P	Total 0/00
24 h	Vehicle control (air)	-	0.727	KJS>0.05		0.9		0.6
	PMVE	0.075	0.839			0.6		0.5
	PMVE	0.15	0.590			0.4	KJS>0.05	0.4
	PMVE	0.30	0.575			0.6		0.5
	Mitomycin C	12 mg/kg	0.406	<0.05W		31.8	<0.001W	1.4
48 h	Vehicle control (air)	-	1.008		JS<0.01	0.4	KJS > 0.05	0.0
	PMVE	0.075	0.800	K>0.05		0.9		0.4
	PMVE	0.15	0.639	K>0.05		0.6		0.5
	PMVE	0.30	0.433	K<0.01		1.1		0.6
72 h	Vehicle control (air)	-	1.187		JS<0.001	0.6		0.6
	PMVE	0.075	0.545	K<0.01		0.7	KJS >0.05	0.9
	PMVE	0.15	0.482	K=0.001		0.8		0.5
	PMVE	0.30	0.377	K<0.001		1.2		0.6

P: probability value (1-sided)

p/n: polychromatic/normochromatic erythrocytes

mnp: number of micronucleated polychromatic erythrocytes observed

mnn: number of micronucealted normochromatic erythrocytes observed

0/00: number per thousand cells

W: results of statistical analysis using Wilcoxon's sum of rank test

K: results of statistical analysis using Kruskal-Wallis test

J: results of statistical analysis using Jonckheere's test for trend

S: results of statistical analysis using Spearman's correlation test

Applicant's summary and conclusion

Conclusions:

negative
From the results obtained it is concluded that the test substance shows no evidence of mutagenic potential after administration via the inhalation route in this in vivo test procedure.

Executive summary:

In this assessment of the effect of Perfluoro (methyl vinyl ether) (hereafter referred to as PMVE) on the incidence of micronucleated polychromatic erythrocytes in mice, animals were exposed to nominal concentrations of 0.075, 0.15 and 0.3% v/v (5000, 10000 and 20000 mg/m3) of the test gas via the inhalation route for 6 hours. The high dose (20000 mg/m3) is the limit dosage recommended in current EEC guidelines on acute inhalation toxicity studies and it has been considered this an appropriate maximum level of exposure for this test.

In addition, three animals treated with PMVE at 0.3% v/v (20000 mg/m3) died after exposure and prior to sacrifice.

The negative control group were subjected to a similar 6 hour exposure but to air alone. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

At all sampling times (24, 48 or 72 hours after initiation of exposure) and dose levels, mice treated with PMVE showed no significant increase in the frequency of micronucleated polychromatic erythrocytes.

At the 48 hour and 72 hour sampling times, statistically significant dose-related decreases in the ratio of polychromatic to normochromatic were obtained for mice exposed to PMVE (p < 0.01 at the highest test concentration (0.30% v/v atmosphere) at the 48 h sampling time and p < 0.01, p = 0.01, p < 0.001 respectively at the three tested concentrations ( 0.075%, 0.15% and 0.30% v/v atmosphere) at the 72 h sampling time). These decreases are indicative of bone marrow cell toxicity/depression.

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated polychromatic erythrocytes (p < 0.001) together with decreases in the ratio of polychromatic to normochromatic erythrocyte (p < 0.05).

It is concluded from the results obtained that PMVE shows no evidence of mutagenic potential in mice following inhalation exposure in this in vivo test procedure.