Reaction mass of (1R)‐1‐[(1S)‐3,3‐dimethylcyclohexyl]ethyl relacetate and (1R,2R)‐2,6,6‐trimethylcycloheptyl rel‐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:

14 Dec 2007 to 28 Dec 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Labor, Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results

Version / remarks:

Feb 8, 1999

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Environmental Health, Notification No. 1121002

Version / remarks:

Nov 21, 2003

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl rel-acetate

Molecular formula:

C12H22O2

IUPAC Name:

(1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl rel-acetate

Constituent 2

Reference substance name:

(1R,2R)‐2,6,6‐trimethylcycloheptyl rel‐acetate

Molecular formula:

C12H22O2

IUPAC Name:

(1R,2R)‐2,6,6‐trimethylcycloheptyl rel‐acetate

Test material form:

liquid

Method

Target gene:

his (Salmonella typhimurium) and trp (Escherichia coli)

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: Rat liver S9 prepared from the liver of 7 week-old male SD rats treated with a combination of phenobarbital and 5, 6-benzoflavone was used.
- Method of preparation of S9 mix: S9 mix was prepared using Cofactor I® for S9 mix immediately before use. One milliliter of S9 mix consisted of 8 µmol MgCl, 33 µmol KCl, 5 µmol Glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodiumphosphate buffer (pH 7.4) and 0.1 mL of S9.
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9-mix and 0.05 mL S9 in 2.7 mL final incubation mixture
- Quality controls of S9: sterility

Test concentrations with justification for top dose:

Dose Finding Test-1:
- A total of 6 doses consisting of 5,000 µg/plate as the highest dose and 5 lower doses diluted with a geometric progression of 4 were employed.

Dose Finding Test-2:
- Without S9 mix: 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/plate
- With S9 mix: 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/plate

Main test:
- Without S9 mix: 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/plate
- With S9 mix: 313, 156, 78.1, 39.1, 19.5 and 9.77 µg/plate

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent: The test substance was insoluble in distilled water at 50.0 mg/mL but dissolved in DMSO at 50.0 mg/mL. The test substance solution of 50.0 mg/mL prepared with DMSO was considered to be stable from the facts that there was no change in colour nor heat generation at room temperature within 2 hours after preparation. Therefore, DMSO was preferably selected as a solvent.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: AF-2 (2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (TA98, TA100 and WP2uvrA without S9)); ICR-191 (2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine x 2 HCl (TA1537 without S9)); 2-AA (2-aminoanthracene (all strains with S9))

Details on test system and experimental conditions:

PRE-CULTURES OF THE TEST STRAINS
Thirty microliters (for TA100), 5 µL (for WP2uvrA), and 20 µL (for TA98, TA1535 and TA1537) aliquots of frozen stock culture of bacterial strains were respectively inoculated to 11 mL of 2.5 % Nutrient broth No. 2 (in each L tube (volume: 27 mL). The culture was incubated at 37 ± 0.5 °C for 9 hours by shaking at about 50 times/minute in a seesaw type of shaker. The number of viable cells was calculated from O.D. value at 660 nm measured by a spectrophotometry at the end of , incubation to calculate the number of viable cells. It was confirmed that the numbers of viable cells were more than 2.3E9 cells/mL in Salmonella typhimurium and more than 3.8E9 cells/mL in Escherichia coli strain. When the numbers of viable cells were more than 2.7E9 cells/mL in Salmonella typhimurium and more than 4.2xE9 cells/mL in Escherichia coli strain, the O.D. value was measured at 660 nm by a spectrophotometry (the same above) and were adjusted with 2.5 % Nutrient broth No. 2 at 2.3 - 2.7 E9 cells/mL and at 3.8 - 4.2E9 cells/mL, respectively. The culture was used without adjusting when the Salmonella typhimurium strains were in the range of 2.3 - 2.7E9 cells/mL. For the final numbers of prepared viable cells please see "Any other informationon material and methods incl. tables."

PREPARATION METHOD
DMSO was added to the test substance and mixed by a tube mixer to make a 50.0 mg/mL of the test substance solution. The test substance solution was diluted with the same solvent to give each required concentration.

PREPARATION TIME
The test substance solutions were prepared immediately before use, kept under yellow lamps at room temperature and used within 2 hours.

PREPARATION OF POSITIVE CONTROL SUBSTANCES
Preparation method:
NaN3 was dissolved in distilled water. AF-2, ICR-191 and 2AA were dissolved in DMSO
Storage conditions:
Positive control solutions were stored in an ultra-deep freezer below -80°C. The solutions were thawed before use.

METHODS
This study was performed by the pre-incubation method with and without S9 mix. 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. The test code, name of test strain, presence or absence of S9 mix and dose level were noted on each plate.

PROCEDURES
After 0.1 mL of the test substance solution, solvent or the positive control solution, 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.4) or S9 mix and 0.1 mL of the bacterial culture were added to a test tube, the mixture was shaken at 37 ± 0.5 °C for 20 minutes. Two milliliters of the soft agar were then added to each tube and the mixture was poured onto a minimal glucose agar plate. The number of revertant colonies was counted after incubation at 37 ± 0.5 °C for 48 hours.

STERILITY
The highest concentration of the test substance solution (0.1 mL) and S9 mix (0.5 mL) were respectively mixed with 2 mL of the soft agar and were poured onto each minimal glucose agar plate in order to examine bacterial contamination. The plates were incubated at 37 ± 0.5°C for 48 hours.

NEGATIVE CONTROL AND POSITIVE CONTROLS
The solvent used in the tests was employed as a negative control and the following positive controls were used for each bacterial strain. For more information, see "Any other informationon material and methods incl. tables."

OBSERVATION AND COLONY COUNTING
- Observation: The precipitation of the test substance was observed by macroscopically and the bacterial growth inhibition was observed by using a stereomicroscope, at the end of the incubation period.
- Colony Counting: 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 analyser. Square correction and miss counting correction were performed in colony analyser.

Evaluation criteria:

The test substance was judged to be 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 to be negative.

Statistics:

Any statistical methods were not used.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537

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:

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:

DOSE FINDING TEST-1
The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in the groups of treatment with and without S9 mix. The bacterial growth inhibition was observed at 78.1 µg/plate or more in all test strains without S9 mix and at 313 µg/plate or more in all test strains with S9 mix. The precipitation of the test substance was not observed at any doses in the groups of treatment with and without S9 mix.

DOSE FINDING TEST-2
The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in the groups of treatment with and without S9 mix. The bacterial growth inhibition was observed at 39.1 µg/plate or more in TA100, TA1535, TA98 and TA1537 and at 78.1 µg/plate in WP2uvrA in the groups of treatment without S9 mix and at 156 µg/plate or more in TA100, TA1535, TA98 and TA1537 and at 313 µg/plate in WP2uvrA in the groups of treatment with S9 mix. The precipitation of the test substance was not observed at any doses in the groups of treatment with and without S9 mix.

MAIN TEST
The number of revertant colonies in the test substance treatment groups was less than twice that in the solvent control in the groups of treatment with and without S9 mix. The bacterial growth inhibition was observed at 39.1 µg/plate or more in TA100, TA1535, TA98 and TA1537 and at 78.1 µg/plate in WP2uvrA in the groups of treatment without S9 mix and at 156 µg/plate or more in TA100, TA1535, TA98 and TA1537 and at 313 µg/plate in WP2uvrA in the groups of treatment with S9 mix. The precipitation of the test substance was not observed at any doses in the groups of treatment with and without S9 mix.

The test substance was judged to be negative because the number of the revertant colonies in the test substance treatment groups in all test strains was less than twice that in the negative control regardless of the presence or absence of S9 mix. 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 negative control and the positive controls were within the range of the historical data at the testing facility. It was also confirmed that the test system was free from bacterial contamination, which indicates the test results to be valid. From the above results, it was concluded that the test substance had no ability to induce mutations under the present test conditions.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the bacterial reverse mutation assay performed equivalent or similar to OECD TG 471.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test substance conducted equivalent or similar to OECD TG 471 and in compliance with GLP, the ability of the test substance to induce mutations was investigated. For this purpose Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA were used in a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix).

As a result, the mutagenicity of the test substance was found to be negative because 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 with and without S9 mix. Therefore, it is concluded that the test substance has no ability to induce mutations under the present test conditions.