4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 June 2011 - 22 July 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

METI, MHLW and MAFF

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: EPA (TSCA) OPPTS harmonised guidelines

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

EC Number:

238-620-0

EC Name:

4-(1-methoxy-1-methylethyl)-1-methylcyclohexene

Cas Number:

14576-08-0

Molecular formula:

C11H20O

IUPAC Name:

4-(2-methoxypropan-2-yl)-1-methylcyclohex-1-ene

Test material form:

liquid

Method

Target gene:

- S. typhimurium: his-locus
- E. coli: trp-locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/ß-Naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

- Preliminary toxicity test (TA 100, WP2 uvrA, with and without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- Experiment 1 (all strains, with and without S9-mix): 15, 50, 150, 500, 1500, 5000 µg/plate
- Experiment 2 (TA 98, TA 100, TA 1535, TA 1537, with S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate
- Experiment 2 (WP2 uvrA, with S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
- Experiment 2 (all strains, without S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Experiment 1:
METHOD OF APPLICATION: in agar (plate incorporation);
DURATION: Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3

Experiment 2:
METHOD OF APPLICATION: preincubation
DURATION: Preincubation period: 20 min; Exposure duration: 48 hours
NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
The test item was toxic at 5000 μg/plate to the strains of bacteria used (TA100 and WP2uvrA). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns initially noted from 15 and 150 μg/plate in the absence and presence of S9-mix respectively. The sensitivity of the tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on experimental methodology.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and the E.coly assay performed according to OECD guideline 471.

Executive summary:

In a GLP compliant study, in accordance with OECD guideline 471, the test substance was examined for its possible mutagenic activity using a the bacterial reverse mutation test. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 15 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using doses ranging between 0.15 and 5000 μg/plate. The sensitivity of the assay and the efficacy of the S9-mix were validated with negative and positive control plates. In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns initially noted from 15 and 150 μg/plate in the absence and presence of S9-mix respectively. The sensitivity of the tester strains to the toxicity of the test item varied between strain type, exposures with or without S9-mix and experimental methodology. The test item was tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit, depending on experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item was therefore considered to be non-mutagenic under the conditions of the test.