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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in compliance with international recognized guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: gas
Details on test material:
name of the substance as cited in the report: ethene, trifluoro(trifluoromethoxy)
Appaerance: colorless gas
the substance was stored at ambient temperature.

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver microsomes (S9)
Test concentrations with justification for top dose:
Toxicity test:
an initial test was carried out (test 1) using S. typhimurium TA 100 in the presence and absence of S9 mix. The following concentrations of the test substance were used:
2.5%, 5%, 7.5%, 10% and 12.5% (v/v)
Mutation tests:
Two indipendent mutation tests were conducted using 6 bacterial strains ( TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A pKM 101).
The exposure levels used in the first experiment (test 2) were:
4%, 8%, 12%, 16%, 20% and 24% (v/v)
The second main experiment (test 3) used:
8%, 16%, 24%, 32%, 40% and 48% (v/v).
A further experiment was conducted due to the lack of toxicity in the first 2 experiments, the concentrations used were:
75% and 100% (v/v).
Duplicates plates were poured for each exposure level, bacterial strain and activation system.

Vehicle / solvent:
air was used as the vehicle control in the study.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
air atmosphere
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinyl chloride
Remarks:
30% in air , it was used with TA 1535 and TA 100 with and without S9 mix
Untreated negative controls:
yes
Remarks:
air atmosphere
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-acetylaminofluorene
Remarks:
1000 µg per plate with TA 1538 and TA 98 with S9 mix.
Untreated negative controls:
yes
Remarks:
air atmosphere
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
other:
Positive control substance:
9-aminoacridine
Remarks:
20 µg of 9-aminoacridine per plate with TA 1537 without S9.
Untreated negative controls:
yes
Remarks:
air atmosphere
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methanesulfonate
Remarks:
100 µg of methanesulfonate per plate with E. coli WP2 uvr A pKM 101
Details on test system and experimental conditions:
Diluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was sterilised by autoclaving. L-histidine and biotin solutions, and L-tryptophan solutions were sterilised by filtration.
For use with S. typhimurium strains, top agar was prepared by adding 5.0 ml of 1.0 mM L-histidine and 1.0 mM biotin solution to 100 ml of dilute agar.
Top agar for use with E. coil was prepared by adding 1.0 ml of 1.35 mM L-tryptophan to 100 ml dilute agar. Each top agar (with additions) was thoroughly mixed prior to use. Agar preparations were kept in a water bath at a temperature not exceeding 45°C.
In the course of testing Ethene, trifluoro (trifluoromethoxy), 2.0 ml of soft agar was added to 9 small plastic, sterile tubes. This was followed by 0.5 ml of S9 mix or 0.1 M phosphate buffer, pH 7.4 and, finally, 0.1 ml of bacteria.
The tube contents, which were continually cooling, were mixed and then poured in minimal medium plates. These plates contained 20 ml of 1.5% Difco Bacto-agar in Vogel-Bonner Medium E (2) with glucose. When the soft agar had set, the plates were inverted and metal spacers inserted under the lids. The plates were then placed in wide- necked, straight-sided flasks of known volume (6.25 litre). The greased lids were replaced and the openings filled with quickfit Dreschel tops (T-junction) through which the gas passed into the jars.
Evaluation criteria:
A significant mutagenic response was recorded if there was:

i) for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98 and for E. coil, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant rnutagenic response was identified.

ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example,
(1) toxicity to the bacteria generally,
(2) specific toxicity to the mutants and
(3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.

iii) a reproducible effect in independent tests.
Statistics:
Not required (revertant colonies were counted)

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the initial test ( Test 1), toxicity to the bacteria was observed at 12.5% in the absence of S9 mix. this effect, however, was not reproduced in any of the other experiments undertaken. In the fourth experiment a reduction in colony numbers was observed at 100% in all the strains in both the presence and absence of S9 mix, although no thinning of the background lawn of microcolonies occured. (See data attached in the field "Attached background material")


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It was concluded that Ethene, trifluoro (trifluoromethoxy) was not mutagenic In S. typhirnurium or E. coli when tested as a gas up to the maximum attainable concentration (100% v/v).
Executive summary:

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.

Ethene, trifluoro (trifluoromethoxy) did not induce mutagenic activity in any of the 6 bacterial strains either in the presence or the absence of S9 mix.

In an initial test, toxicity to the bacteria was observed at 12.5% in the absence of S9 mix. This effect, however, was not reproduced in any of the other experiments undertaken. In the fourth experiment a reduction in colony numbers was observed at 100% in all the strains in both the presence and absence of S9 mix, although no thinning of the background lawn of microcolonies occurred.

It was concluded that Ethene, trifluoro (trifluoromethoxy) was not mutagenic in S. typhimurium or E. coil when tested as a gas up to the maximum attainable concentration (100%).