Reaction mass of 1-methyl-4-(1-methylethylidene)cyclohexyl acetate and p-menth-1-en-8-yl acetate

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:

08 August 2019 to 20 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries, 24 November 2000

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1-methyl-4-(propan-2-ylidene)cyclohexyl acetate and 2-(4-methylcyclohex-3-en-1-yl)propan-2-yl acetate

EC Number:

904-693-9

Molecular formula:

C12H20O2

IUPAC Name:

Reaction mass of 1-methyl-4-(propan-2-ylidene)cyclohexyl acetate and 2-(4-methylcyclohex-3-en-1-yl)propan-2-yl acetate

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Moltox
- Inducing agent: Phenobarbital-5,6 and Benzoflavone
- Method of preparation of S9 mix: Matsushima et al., In vitro metabolic activation in mutagenesis testing (F.J. de Serres, ed.), Elsevier, 1976, p85
- Quality controls of S9: enzymatic activity, sterility, and metabolic capability

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Experiment 2: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate (All Salmonella strains)
Experiment 2: 1.5, 5, 15, 50, 150, 500, 1500, 5000 (E.coli strain WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

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:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning

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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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 judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and 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:

Controls:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
- Results for the negative controls (spontaneous mutation rates) are considered to be acceptable.
- The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1:
- Test item treatment caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in both the absence and presence of metabolic activation. Weakened bacterial background lawns were noted in the absence of metabolic activation from 500 μg/plate to all of the Salmonella strains and at 5000 μg/plate to WP2uvrA. In the presence of metabolic activation, weakened bacterial background lawns were noted to all of the tester strains from 500 μg/plate to TA100, TA1535 and TA1537, 1500 μg/plate to TA98 and at 5000 μg/plate to WP2uvrA.
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix). One statistically significant value was noted (TA98 at 50 μg/plate in the presence of metabolic activation (S9-mix)), This value was not identified as statistically significant, as this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

Experiment 2:
- Test item treatment caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in both the absence and presence of metabolic activation. Weakened bacterial background lawns were noted in the absence of metabolic activation from 50 μg/plate to TA100 and 150 μg/plate to TA1537, TA1535, TA98 and WP2uvrA. In the presence of metabolic activation, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate to TA100, 500 μg/plate to TA1535, TA98 and TA1537 and from 1500 μg/plate to WP2uvrA.
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Applicant's summary and conclusion

Conclusions:

The test material was considered non-mutagenic under the conditions of this test.

Executive summary:

The test substance was evaluated in a bacterial reverse mutation assay performed according to OECD 471 and following GLP using Salmonella typhimurium strains TA98, TA100, TA 1535 and TA1537 and Escherichia coli strain WP2 uvrA both in the presence and absence of an exogenous metabolic activation system (S9-mix). The test substance was evaluated using an initial plate incorporation assay and a confirmatory preincubation procedure in triplicate with and without metabolic activation. In the first experiment, test substance concentrations of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate were assessed. In the second experiment, test substance concentrations of 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate (All Salmonella strains), and 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate (WP2uvrA) were assessed. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence and presence of metabolic activation, initially from 500 μg/plate. In the second experiment, the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of metabolic activation, initially from 50 μg/plate. In the presence of metabolic activation, the lowest test item concentration at which toxicity was initially observed was strain dependant and started from 150 μg/plate. No test item precipitate was observed on any of the plates. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. One statistically significant value was noted (TA98 at 50 μg/plate in the presence of metabolic activation (S9-mix)). This value was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. Similarly, 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 (S9-mix) in Experiment 2. Therefore, the test material was considered to be non-mutagenic under the conditions of this test.