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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
Study period:
24 February 2005 to 28 April 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline-conform study under GLP without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane
EC Number:
609-534-4
Cas Number:
382-26-3
Molecular formula:
C5H4F8O
IUPAC Name:
2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Octafluoroisobutyl methyl ether
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: > 86%
- Impurities (identity and concentrations): not reported
- Composition of test material, percentage of components: not reported
- Isomers composition: not reported
- Purity test date: not reported
- Lot/batch No.: 20/01/04-161
- Expiration date of the lot/batch: 31 December 2006
- Stability under test conditions: not reported
- Storage condition of test material: 2°C -8°C

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Experiment I (TA 98, TA 100): 3; 10; 33; 100; 333; 1000; 2500; 5000 microgram/plate
Experiment I (plate incorporation test): 33; 100; 333; 1000; 3500; 5000 microgram/plate
Experiment II (pre-incubation test): 10 ; 33; 100; 333; 1000; 2500; 5000 microgram/plate
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoantracece; 4-nitro-o-phenylenediamina
Evaluation criteria:
A test item is considered as mutagen if a biologically relevant increase in the number of revertants exceeding the treshold of twice (strains TA 98, TA 100 and TA 102) or three times (strains TA 1535 and TA 1537) the colony count of the corrisponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the treshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

ACCEPTABILITY OF THE ASSAY
The Salmonella Typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
Statistics:
Statistical analysis of data was not performed.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxicity was observed at highest tested doses
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Experiment I (plate incorporation test)

Any other information on results incl. tables

In both experiments, reduced background growth was observed with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations (microgram/plate).

 

 

EXPERIMENT I

EXPERIMENT II

Strain

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

5000

5000

2500

1000, 5000

TA 1537

333, 5000

5000

333, 1000

1000 - 5000

TA 98

//

//

1000 - 5000

2500

TA 100

5000

//

1000 - 5000

1000 - 5000

TA 102

2500, 5000

2500, 5000

1000 - 5000

1000 - 5000

 // = no toxic effects observed

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Applicant's summary and conclusion

Conclusions:
2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of 2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane to induce gene mutations according to the plate incorporating test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item eas tested at the following concentrations:

Experiment I (TA 98, TA 100): 3; 10; 33; 100; 333; 1000; 2500; 5000 microgram/plate

Experiment I : 33; 100; 333; 1000; 3500; 5000 microgram/plate

Experiment II : 10 ; 33; 100; 333; 1000; 2500; 5000 microgram/plate

In both experiments, reduced background growth was observed with and without S9 mix in all strains used.

Toxic effect, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.

Therefore, 2-[difluoro(methoxy)methyl]-1,1,1,3,3,3-hexafluoropropane is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.