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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22-09-2006 to 09-11-2006
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:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
EC Number:
275-156-8
EC Name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
Cas Number:
71048-82-3
Molecular formula:
C13H20O
IUPAC Name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
Test material form:
liquid

Method

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:
phenobarbital and 5,6-benzoflavone induced rats livers: S9
Test concentrations with justification for top dose:
- Dose finding test 1: 0, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate with and without S9 mix
- Dose finding test 2 and main test (without S9): 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1 µg/plate
- Dose finding test 2 and main test (with S9): 0, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-aminoanthracene, 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCI
Details on test system and experimental conditions:
After 0.1 mL of the substance solution, solvent or the positive control solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix and 0.1 mL of the bacterial culture were added to a test tube, the mixture was shaken at 37±0.5°C for 20 minutes. Two milliliters of the soft agar were then added to each tube and the mixture was poured onto a minimal glucose agar plate. The number of revertant colonies was counted after incubation at 37±0.5°C for 48 hours. The precipitation of the test substance was observed macroscopically and the bacterial growth inhibition was observed using a stereomicroscope. For the plates in which the bacterial growth inhibition was observed, the number of colonies was counted with a manual counter, and the other plates were counted by using a colony analyzer (CA-IID, System Science Ltd.).
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:
Any statistical methods were not used.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- Dose finding test 1: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in the groups with and without S9 mix. The bacterial growth inhibition was observed more than 78.1 µg/plate in all test strains without S9 mix and more than 313 µg/plate in all test strains with S9 mix. The precipitation of the substance was not observed at any doses in the groups with and without S9 mix.
- Dose finding test 2: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in the groups with and without S9 mix. The bacterial growth inhibition was observed at 78.1 µg/plate in all test strains without S9 mix and more than 156 µg/plate in TA100, TA1535, TA98 and TA1537 and at 313 µg/plate in WP2uvrA with S9 mix. The precipitation of the substance was not observed at any doses in the groups with and without S9 mix.
- Main test: The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in the groups with and without S9 mix. The bacterial growth inhibition was observed at 78.1 µg/plate in all test strains without S9 mix and more than 156 µg/plate in TA100, TA1535, TA98 and TA1537 and at 313 µg/plate in WP2uvrA with S9 mix. The precipitation of the substance was not observed at any doses in the groups with and without S9 mix.

Applicant's summary and conclusion

Conclusions:
It is concluded that the results obtained with the substance in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in the absence and presence of the S9-mix, indicate that the test substance is not mutagenic under the conditions of this test (OECD TG 471).
Executive summary:

An in vitro mutagenicity test using microorganisms was performed according to a protocol equivalent or similar to OECD Guideline 471, and according to GLP. The ability of the substance to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2uvrA using a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). In the absence of the S9, the highest dose tested was 78.1 µg/plate, while in the presence of S9 mix it was 313 µg/plate based on a dose-range finding test. No precipitation was observed at any dose level with and without S9 mix. Bacterial growth inhibition was only observed at 78.1 µg/plate without S9 mix and at 156 and 313 µg/plate with S9 mix. The numbers of revertant colonies in the substance treatment groups were less than two times that observed in each negative control in all test strains and therefore, the mutagenicity of the substance was judged negative. As such, it is concluded that the substance has no ability to induce mutations under the present test conditions.