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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The genetic toxicity of perfluoromethyl vinyl ether (PMVE) has been assessed in 5 in vitro studies (including 2 bacterial reverse mutation assays, 2 mammalian chromosome aberration tests, and a mammalian gene mutation assay) and an in vivo mouse micronucleus assay. Negative results were reported in all the studies with the exception of one positive result showing evidence of clastogenic activity in the absence of S-9 mix but not in the presence of S-9 mix (C. E. Mason, 1998). However an in vivo test, a mouse micronucleus assay, showed no evidence of mutagenic potential after administration of the substance via the inhalation route.
Link to relevant study records
Reference
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
Study period:
from 19th December 1989 to 29th january 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international recognized guidelines
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals 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.
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.
Remarks:
Doses / Concentrations:
0.075 % v/v
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.15% v/v
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.3% v/v
Basis:
nominal conc.
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.
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.
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.
Conclusions:
Interpretation of results (migrated information): 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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In the key bacterial reverse mutation assay (OECD guideline 471) perfluoromethyl vinyl ether was tested, as a gas, for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and Escherichia coil WP2uvrA (pKM101) at nominal exposure levels ranging from 2.5% to 100% (v/v). The tests were conducted, using the pre-incubation method, on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Perfluoromethyl vinyl ether did not induce mutagenic activity in any of the 6 bacterial strains either in the presence or the absence of S9 mix.

In the supporting bacterial reverse mutation assay (OECD guideline 471) the test substance, PMVE, was tested using Gas-Phase Exposure for mutagenic activity in Salmonella typhimurium tester strains TA98, TA 100, TA 1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The test was performed in two phases, using the desiccator method. The first phase, the preliminary toxicity test, was used to establish the dose-range for the mutagenicity test. The second phase, the mutagenicity test, was used to evaluate the mutagenic potential of the test substance. Untreated controls (air controls) were used as the negative controls.The results of the Bacterial Reverse Mutation Test Using Gas-Phase Exposure indicate that, under the conditions of the study, PMVE did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.

In the key in vitro mammalian chromosome aberration test (OECD 473) the effects on chromosomal structure of exposure to Perfluoromethylvinylether were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S9 mix): without S9 mix cells were exposed continuously for 19 hours, with S-9 mix exposure was limited to three hours and cells were harvested 16 hours later. Lymphocyte cultures were established from whole, human blood, and 48 hours later they were exposed to atmospheres containing Perfluoromethylvinylether in air in sealed bottles.

The tests were performed using a range of concentrations of Perfluoromethylvinylether to cover the appropriate range of toxicity. The tests also incorporated negative (air) and positive (Mitomycin C and cyclophosphamide) control cultures. Statistically significant increases (p<0.01) in the frequency of metaphases with chromosomal aberrations were observed in cultures treated in the absence of S9 mix in both tests. The increases at the highest test concentration (70% v/v atmosphere) were reproducible and exceeded the historical negative control range, both including and excluding gap-type aberrations. Smaller increases were observed at the lower test concentrations. No statistically significant increases were observed in cultures treated in the presence of S-9 mix (p>0.05).It is concluded that Perfluoromethylvinylether, under the conditions of test, showed evidence of clastogenic activity in the absence of S-9 mix.

In the supporting in vitro mammalian chromosome aberration test (equivalent to OECD 473) the test substance, PMVE, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced S9 activation system in order to evaluate the clastogenic potential of the test substance. In the chromosome aberration assay, the cells were treated for 4 and 20 hours in the non- activated test system and for 4 hours in the S9 activated test system, and all cells were harvested at 20 hours after treatment initiation. In the absence of both substantial toxicity (at least 50% reduction in cell growth, relative to the air control) and test substance precipitation in treatment medium at any dose level in any of the treatment groups, the top three dose levels were selected for microscopic analysis for chromosome aberrations in all harvests. The positive and air controls fulfilled the requirements for a valid test. Based on the findings of this study, PMVE was concluded to be negative for the induction of structural and numerical chromosome aberrations in Chinese hamster ovary (CHO) cells in the in the presence and absence of Aroclor-induced rat liver S9.

In the key in vitro mammalian cell mutation assay (OECD 476) Perfluoromethylvinylether was examined for mutagenic potential by measuring its ability to induce mutation in mouse lymphoma (L5178Y) cells that are heterozygous at the thymidine kinase (TK+/-) gene locus.

A preliminary toxicity test was first conducted: cells were exposed to seven Perfluoromethylvinylether test atmospheres ranging from 1.25 to 70% (v/v) in the presence and absence of a rat liver-derived metabolic activating system (S9 mix). After three hours of exposure, the cells were transferred to non-selective medium: suspension growth was monitored, over a 48 hour period, by means of cell counts.

Cultures of L5178Y cells were then exposed to five test atmospheres of Perfluoromethylvinylether in each of two mutation assays. In both assays, the selected test atmospheres were 5, 10, 20, 40 and 70% (v/v) in the absence and presence of S9 mix. Control cultures, exposed to an atmosphere of air, were included in all experiments. Methylmethanesulphonate (MMS), a known direct- acting mutagen, and 20-methylcholanthrene (20-MC), a mutagen which requires metabolic activation to achieve optimal activity, were used as positive controls. All treatments were established in duplicate.

Cultures exposed to Perfluoromethylvinylether showed no real increases in mutant colony numbers or increased mutant frequencies (per 10^6 survivors), compared to the negative controls.Under the same test conditions, MMS (at 10 µ/ml) produced significant increases in the incidence of mutant colonies. 20-MC (at 2.5 µ/ml) significantly increased the incidence of mutant colonies only in the presence of S9 mix.

It is concluded that, under the conditions of the test, Perfluoromethylvinylether showed no evidence of mutagenic activity in the absence or presence of S9 mix.

In the key in vivo micronucleus assay it was assessed the effect of PMVE on the incidence of micronucleated polychromatic erythrocytes in mice. The 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.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. 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 together with decreases in the ratio of polychromatic to normochromatic erythrocytes. It is concluded from the results obtained that PMVE shows no evidence of mutagenic potential in mice following inhalation exposure in the in vivo test procedure.


Justification for selection of genetic toxicity endpoint
An in vivo mouse micronucleous test on the substance has been conducted showing no evidence of mutagenic potential after administration via the inhalation route.

Justification for classification or non-classification

The substance does not meet the criteria for classification and labelling for this endpoint, as set out in Regulation (EC) No. 1272/2008.