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Diss Factsheets

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:
23-05-2007 to 27-06-2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Ministry of Labor, Official Notification, February 8, 1999
Qualifier:
according to guideline
Guideline:
other: "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemical Substances"
Version / remarks:
Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No.2 (2003.11.13) of the Manufacturing Industrie Bureau, MeTI & No. 031121002 of Environmental Health Department, MOE (November 21, 2003)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethylcyclohex-3-ene-1-carbaldehyde
EC Number:
248-742-6
EC Name:
Dimethylcyclohex-3-ene-1-carbaldehyde
Cas Number:
27939-60-2
Molecular formula:
C9H14O
IUPAC Name:
(1R,6R)-3,6-dimethylcyclohex-3-ene-1-carbaldehyde; (1R,6R)-4,6-dimethylcyclohex-3-ene-1-carbaldehyde
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
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
- Dose range finding test 1: TA 100, TA 1535, WP2uvrA, TA 98 and TA 1537 (without and with S9-mix): 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate. The number of relevant colonies in the test substance treatment groups was less than twice compared to the solvent control. The bacterial growth inhibition was observed at 313 µg/plate or more in all test strains with and without S9-mix.

- Dose range finding test 2: TA 100, TA 1535, WP2uvrA, TA 98 and TA 1537 (without and with S9-mix): 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate.
The number of relevant colonies in the test substance treatment groups was less than twice compared to the solvent control. Bacterial growth inhibition was observed for all strains at test substance concentrations of 156 µg/plate or above with S9 mix and 313 µg/plate or above without S9-mix

- Main experiment: Based on the results of dose finding test-1 and 2, a total of 6 doses was employed in all test strains with and without S9-mix. TA 100, TA 1535, WP2uvrA, TA 98 and TA 1537: 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was insoluble in distilled water at 50 mg/mL and dissolved in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable from the facts that there was no change in color nor heat generation at room temperature within 2 hours after preparation.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
100 µL DMSO
True negative controls:
no
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:
- Preincubation method

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

NUMBER OF REPLICATIONS:
Triplicate plates were used for the negative control group and duplicate plates per dose for the test substance treatment groups and the positive control groups.

DETERMINATION OF CYTOTOXICITY
The bacterial growth inhibition was observed by 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. Square correction and miss counting correction were performed in colony analyzer.
Evaluation criteria:
The test substance was judged to be positive when the number of revertant colonies increased to twice or more compared to 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 to be negative.

Results and discussion

Test resultsopen allclose all
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 applicable
Positive controls validity:
valid
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 applicable
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 applicable
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 applicable
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 applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The precipitation of the test substance was not observed at any doses in the groups of with and without S9-mix.
- Other confounding effects: It was confirmed that the test system was free from bacterial contamination, which indicates the test results to be valid.

RANGE-FINDING/SCREENING STUDIES:
- Bacterial growth inhibition was observed for all strains at test substance concentrations of 156 µg/plate or above without S9-mix and 313 µg/plate or above with S9-mix in the dose-range finding studies.

HISTORICAL CONTROL DATA:
- Positive historical control data: The numbers of the revertant colonies in the positive controls were above two times that in the negative controls. The test results showed that the numbers of revertant colonies in the positive controls were within the range of the historical data at the testing facility.
- Negative (solvent/vehicle) historical control data: The test results showed that the numbers of revertant colonies in the negative control was within the range of the historical data at the testing facility.

Applicant's summary and conclusion

Conclusions:
The test substance, under the present test conditions, was negative in the bacterial reverse mutation assay (Ames) according to OECD TG 471.
Executive summary:

The mutagenic activity of the test substance was evaluated in a study equivalent to OECD TG 471 and according to GLP principles. The test was performed according to the pre-incubation method, in the absence and presence of S9-mix. The dose levels were selected based on observed growth inhibition in all strains (156 µg/plate or above without S9 mix and 313 µg/plate or above with S9-mix). Adequate vehicle and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in any of the five tester strains (S. typhimurium TA100, TA1535, TA98, TA1537 and E. coli WP2uvrA), both in the absence and presence of metabolic activation. Based on the results of this study it is concluded that the test substance is not mutagenic in the bacterial reverse mutation assay.