Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2006 - 14 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
1-(3,3-dimethylcyclohexyl)ethan-1-one
EC Number:
246-799-1
EC Name:
1-(3,3-dimethylcyclohexyl)ethan-1-one
Cas Number:
25304-14-7
Molecular formula:
C10H18O
IUPAC Name:
1-(3,3-dimethylcyclohexyl)ethanone
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5, 6-benzoflavone
Test concentrations with justification for top dose:
- Dose range finding test (first experiment):
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
- Dose range finding test 2 (first experiment:
TA 1535 and TA 100 (without and with S9): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

- Main experiment:
Due to cytotoxicity (in all strains), the following dose levels were used:
TA 100 and TA 1535 (without and with S9): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA 1537, TA 98 and WP2uvrA (without and with S9): 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to and including 50 mg/mL.
Controls
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
Remarks:
(100 µL/plate DMSO)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Dose range finding and main experiment: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance and positive control groups were tested in duplicate and triplicate for the negative control groups in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation
Evaluation criteria:
The test substance was judged positive when the number of revertant colonies increased to twice or more that in the negative control in a concentration-dependent manner and also the reproducibility of the test results was obtained. In all other cases, it was judged negative.
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 313 µg/plate (main test)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 625 µg/plate and above (main test)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 625 µg/plate and above (main test)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 313 µg/plate (main test)
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 625 µg/plate and above (main test)
Vehicle controls validity:
valid
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:
- In TA100 and TA1535 toxicity was observed at 313 µg/plate and above, both in the absence and presence of S9. In TA98, TA1537 and WP2uvrA toxicity was observed at 1250 and 5000 µg/plate, both in the absence and presence of S9

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In all strains toxicity was observed at the higher doses (at >= 313 µg/plate), both in the absence and/or presence of S9

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed equivalent to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated equivalent to OECD 471 guideline and according to GLP principles. The test was performed in two independent preincubation experiments. In the dose range finding test (first experiment) the test substance was tested up to and including 5000 µg/plate in all five strains, in the absence and presence of S9-mix. In the main experiment the test substance was tested in strain TA100 and TA1535 up to and including 313 µg/plate, and in strain TA98, TA1537 and WP2uvrA up to and including 1250 µg/plate in the absence and presence of S9-mix. Toxicity was observed at the highest dose levels tested. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.