1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethylene

Genetic toxicity: in vitro

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

Study period:

17-Dec-2015 to 11-Mar-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

EC Number:

615-064-0

Cas Number:

700874-87-9

Molecular formula:

C4F8O2

IUPAC Name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

Test material form:

liquid: volatile

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Sponsor
- lot/batch No.of test material: 150525V10
- Expiration date of the lot/batch: October 2020
- Purity test date: October 2015
- Name of test material (as cited in study report): MOVE3
- Substance type:Clear colourless liquid
- Physical state: Liquid

FORM AS APPLIED IN THE TEST: Liquid

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 94, 469, 938, 1876, 2814, 3752 and 4690 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 938, 1876, 2814, 3752 and 4690 µg/plate
Experiment 2:
Without and with S9-mix: 938, 1876, 2814, 3752 and 4690 µg/plate
Experiment 3:
Without and with S9-mix: 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle:
A solubility test was performed. The test item was soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (pre incubation test)

Inclusion of a pre incubation step of 30 and 60 minutes in the first and second experiment respectively. During the pre incubation phase the temperature has been kept at 20˚C to avoid volatilisation of the test item.

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100, WP2uvrA) or more or a three-fold (TA1535, TA1537, TA98) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that MOVE3 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In this study the mutagenic activity of MOVE 3 was evaluated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

The procedures described in this study were based on the most recent OECD and EC guidelines. The study design has been developed considering the physic-chemical properties of the test item (low boiling liquid).

Since the test item is a low boiling liquid the pre-incubation method has been considered the more appropriate test design for guaranteeing the exposure of the bacteria cells.

 

MOVE3 was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254) with the inclusion of a pre-incubation step for 30 and 60 minutes in the first and second mutation experiment, respectively. An additional experiment was performed at the top dose of 5000 μg/plate with all five tester strains with the inclusion of a pre-incubation step for 60 minutes. During the pre-incubation phase of the experiments the temperature has been kept at 20°C in order to avoid the volatilisation of the test item.

 

In the dose range finding test, the test item was tested up to concentrations of 4690 μg/plate in the absence and presence of 5% (v/v) S9-mix in the strains TA100 and WP2uvrA (pre-incubated by 70 rpm at 20°C for 30 minutes). MOVE3 did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay.

 

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 938 to 4690 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98 (pre-incubated by 70 rpm at 20°C for 30 minutes).

 

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 938 to 4690 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA and with an increased pre-incubation period up to 60 minutes.

 

The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

Since in the mutation assays the top dose of 5000 μg/plate was not reached, an additional mutation assay was performed in the absence and presence of 10% (v/v) S9-mix with the inclusion of a pre-incubation step for 60 minutes in the tester strains TA1535, TA1537, TA98, TA100 and TA102. In this third mutation assay, the top concentration of 5000 μg/plate was tested. The bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed.

 

MOVE3 did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in follow-up experiments.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that MOVE3 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.