Reaction mass of (3R)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one and (3S)-3-methyl-5-[(1R,3R)-2,2,3-trimethylcyclopentyl]pentan-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 18,201 3 to July 25,2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Notification No. 7 of Japanese Ministry of Health, Labour and Welfare, No. 5 of Japanese Ministry of Economy, Trade and Industry, No. 110331009 of Japanese Ministry of the Environment on March 3 1,2011

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: The standard that the Minister of Labor establishes based on the Industrial Safety and Health Law Article 57-3 Paragraph 1

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

NACET00502

IUPAC Name:

NACET00502

Constituent 2

Reference substance name:

3-Methyl-5-(2,2,3-trimethylcyclopentyl)pentan-2-one

Cas Number:

119464-63-0

Molecular formula:

C14H26O

IUPAC Name:

3-Methyl-5-(2,2,3-trimethylcyclopentyl)pentan-2-one

Test material form:

other: light yellow oil

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.610 , 1.22, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance was hardly-soluble in water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: Name: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

RANGE-FINDING STUDY: In the dose-finding study, 226.96 mg (250 µL) of the test substance was weighed and 4.289 ml of dimethylsulfoxide was added and the test substance was dissolved to prepare a 50 mg/ml test solution. In the main study, 352.18 mg (400 µl) of the test substance was weighed and 6.644 ml of dimethylsulfoxide was added and the test substance was dissolved to prepare a 50 mg/mL test solution. In the confirmatory study, 134.45 mg (150 µl) of the test substance was weighed and 2.539 ml of dimethylsulfoxide was added and the test substance was dissolved to prepare a 50 mg/mL test solution. Dimethylsulfoxide was previously dehydrated with molecular sieve (3A). In the dose-finding study, total five lower concentrations were prepared by dilution of the 50 mg/mL solution (highest concentration) with dimethylsulfoxide. In the main and confirmatory studies, total thirteen lower concentrations were prepared.

METHOD OF APPLICATION: in agar.
Soft agar consisting of sodium chloride at 0.5 % (w/v) and Bacto agar at 0.6 % (w/v) was dissolved by heating. A mixture solution of 0.5 mmol/L biotin , 0.5 mmol/L histidine and 0.5 mmol/L tryptophan was added to the soft agar solution in the ratio of 1 : 10.

NUMBER OF CELLS EVALUATED: 1 x 10^9 cells/mL or more

OTHER: Sterility Test
The sterility test was conducted to examine for contamination in the test substance or S9 mix. Mixture of 0.1 mL of the test solution of highest concentration or 0.5 mL of S9 mix and 2 mL of top agar was overlaid on a minimum glucose agar plate. After the top agar solidified, each minimum glucose agar plate was inverted and cultivated for 48 hours at 37°C (FMU-404 1, Fukushima Industries Corporation).

Evaluation criteria:

The test results were judged positive when biologically meaningful increase in the number of revertant colonies, such as dose-related increases with reproducibility were observed.

Statistics:

The number of revertant colonies per plate, the mean values and standard deviation per dose of the test substance, and the negative control were tabulated for each bacterial strain. The number of revertant colonies per plate and the mean values were tabulated for each strain as for the positive control. Dose-response curves of each strain were drawn for the test substance group. Two statistical analyses of Dunnett's multiple comparison method (one-side test) and linear regression method were used in this test. The number of revertant colonies of each bacterial strain at each dose in the main study and confirmatory study was compared with that of the negative control in both the presence and absence of metabolic activation, and statistically significant difference in the number of revertant colonies between those two groups was analyzed first by the multiple comparison method (p<0.05). The dose-reactivity was analyzed by linear regression method (p<0.05) when the statistically significant difference was detected by the multiple comparison method.
Application usage:
Mean and standard deviation; Microsoft EXCEL 2003 for Windows (Version 11 )
Statistical analysis; SAS system for Window (Release 9.1.3)

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was not observed at any dose levels in both the presence and absence of metabolic activation.
- Other confounding effects: No contaminants were found in the sterility test

RANGE-FINDING/SCREENING STUDIES: Six doses including 5000 µg/plate as the highest dose obtained using a common ratio of 4 were tested

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Statistically significant difference in the number of revertant colonies between the test substance group and the negative control was not observed in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. Reproducibility

was observed between the results from the main study and confirmatory study. These results revealed that no biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was observed. The number of revertant colonies in the positive control was twice or more than that of the negative control in all bacterial strains regardless of the presence or absence of metabolic activation. The mean values of colony counts in the negative and positive controls were within the range of background data in both the main study and confirmatory study.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the above results, the test substance was considered to have no potency of mutagenicity under the conditions of this study.

Executive summary:

The inducibility of gene mutation in 3-methyl-5-((1 R)-2,2,3-trimethylcyclopentyl) pentan-2-one was evaluated by the reverse mutation test with a pre-incubation method at 37°C for 20 minutes using five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA. The test was composed of the dose-finding, main and confirmatory studies, and the reproducibility between the results from the main study and confirmatory study was confirmed. All studies were performed in the presence and absence of metabolic activation. As a result, biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was not observed in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. The number of revertant colonies in the positive control was twice or more than that of the negative control in all bacterial strains regardless of the presence or absence of metabolic activation. The mean values of revertant colonies in both the negative and positive controls were within the range of calculated reference value from background data in the main and confirmatory studies. No contaminants were found in the sterility test. These results demonstrated that the test was properly performed. No suspected factor that affected the reliability of the studies was confirmed. Based on the above results, the inducibility of gene mutation in the test substance was determined as negative under the test conditions employed.