Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

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:

18 April 2006 to 19 May 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

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Labor, Notifications No. 77 and 67

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

EC Number:

939-618-9

Cas Number:

25225-08-5

Molecular formula:

C11H20O2

IUPAC Name:

Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

Test material form:

liquid

Method

Target gene:

S. typhimurium TA1535: hisG46 rfa uvrB; S. typhimurium TA1537: hisC3076 rfa uvrB; S. typhimurium TA98: hisD3052 rfa uvrB pKMl01; S. typhimurium TA100: hisG46 rfa uvrB pKM101; E. coli WP2 uvrA: trp

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:

Rat liver S9

Test concentrations with justification for top dose:

Dose-range finding test 1: a total of 6 doses consisting of 5000 μg/plate as the highest dose and 5 lower doses diluted with a geometric progression of 4 were employed.
Dose-range finding test 2 and main test: the highest dose at 78.1 μg/plate without S9 and 313 μg/plate with S9 and each lower 5 doses diluted with a geometric progression of 2.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: the test substance was insoluble in distilled water at 50 mg/mL, but it was soluble in DMSO at 50 mg/mL. The test substance solution of 50 mg/mL prepared with DMSO was considered to be stable as there was no change in colour no heat generation at room temperature within 2 hours after preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (TA98, TA100 and WP2uvrA without S9); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine x 2 HCl (TA1537 without S9); 2-aminoanthracene (all strains with S9)

Remarks:

TA1535 without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS: triplicate plates for the negative control group and duplicate plates per dose for the substance treatment groups and the positive control groups

DETERMINATION OF CYTOTOXICITY:
- Method: bacterial growth inhibition

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:

No statistical methods were used.

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:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In dose-range finding test 1 the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. Precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9 and >313 μg/plate with S9.
In dose-range finding test 2 the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. Precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9, >156 μg/plate in TA100, TA1535, TA98 and TA1537 and >313 μg/plate in WP2uvrA with S9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main test the number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control. Precipitation of the test substance was not observed at any doses. The bacterial growth inhibition was observed at >78.1 μg/plate in all test strains without S9, >156 μg/plate in TA100, TA1535, TA98 and TA1537 and >313 μg/plate in WP2uvrA with S9.

Applicant's summary and conclusion

Conclusions:

The substance gave negative results in an Ames test wih S. typhimurium TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvr A, both with and without metabolic activation (OECD TG 471).

Executive summary:

An in vitro mutagenicity test using bacteria was performed according to OECD TG 471, and in compliance with GLP. The ability of the substance to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2 uvr A 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 two dose-range finding tests. No precipitation was observed at any dose level with and without S9 mix. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9, > 156 μg/plate in TA100, TA1535, TA98 and TA1537 and > 313 μg/plate in WP2uvrA with S9. The numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all test strains, both with and without metabolic activation. Therefore the substance was concluded to give negative results in the Ames test.