2-tert-pentylcyclohexyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

2-tert-pentylcyclohexyl acetate

EC Number:

267-500-0

EC Name:

2-tert-pentylcyclohexyl acetate

Cas Number:

67874-72-0

Molecular formula:

C13H24O2

IUPAC Name:

2-tert-pentylcyclohexyl acetate

Method

Species / strain

Species / strain / cell type:

other: TA98, TA100, TA1535, TA1537, and WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor­ induced rat liver S-9

Test concentrations with justification for top dose:

Dose range finding test: TA100 and WP2 uvrA: 5.0, 10, 50, 100, 500, 1000, 5000 µg/plate with and without metabolic activation.
Main experiment:
- 1st experiment: Salmonella strains 5, 10, 20, 30 and 50 µg/plate without metabolic activation and 10, 50, 100, 250, 500 µg/plate with metabolic activation. WP2 uvrA: 250, 500, 1000, 2500, and 5000 µg/plate with and without metabolic activation.
- 2nd confirmatory experiment: Salmonella strains: 20, 30, 50, 100, and 500 µg/plate without metabolic activation and 50, 100, 250, 500, and 1000 µg/plate with metabolic activation. WP2 uvrA: 250, 500, 1000, 2500, and 5000 µg/plate with and without metabolic activation.

Vehicle / solvent:

- Solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene

Remarks:

- S9-mix: 2-Nitrofluorene (TA98), Sodium Azide (TA100, TA1535), 9-Aminoacridine (TA1537), Methyl Methanesulfonate (WP2 uvrA). + S-9 mix:2-Aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: Duplicate

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was evaluated based on: Relative cloning efficiencies (RCE); thinning of the background lawn

Evaluation criteria:

- A response was considered to be negative if all of the strains treated with the test article had mean reversion frequencies that were less than twice that of the mean reversion frequencies of the corresponding solvent control plates in TA98 and TA100 and less than three times in TA1535, TA 1537 and WP2 uvrA, and there was no evidence of a concentration-dependent response.
- A response was considered to be positive if either strain TA98 or TA 100 exhibited a mean reversion frequency that was at least double the mean reversion frequency of the corresponding solvent control in at least one concentration, or if either strain TA1535, TA1537 or WP2 uvrA exhibited a three-fold increase in the mean reversion frequency compared to the solvent control in at least one concentration. In addition, the response must be concentration-dependent (i.e.increasing mean reversion frequencies observed over increasing test concentrations). In evaluating the results, consideration was given to the degree of toxicity exhibited by the concentration causing the 2 to 3- fold or greater increase in reversion frequency and the magnitude of the increase in reversion frequency.
- A response was considered equivocal if it did not fulfill the criteria of either a negative or a positive response and/or the Study Director did not consider the response to be either positive or negative.

Results and discussion

Test results

Test results 1

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 2

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 3

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 4

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 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:

SOLUBILITY TEST
The test substance was tested for miscibility in H2O and DMSO. The results indicated the test substance was not miscible in H2O. It was miscible in DMSO at 50 mg/mL and formed a clear solution.

RANGE FINDING TEST
TA100: The Relative Cloning Efficiencies (RCEs) at the concentrations of 5.0 to 5000 µg/plate without activation ranged from 0% to 95%. Significantly decreased RCE values (<50%) were observed at 50 µg/plate and above. Marked thinning of background lawn was also observed at 500 µg/plate and above but the revertant ferquencies were not significantly decreased at any of dose levels. In the presence of the activation system, the RCEs at the concentrations of 5.0 to 5000 µg/plate ranged from 1% to 176%. Significant decreases in RCE (<50%) and the revertant frequency were at test concentrations of 500 µg/plate and above. Noticeable thinning of background lawn was also found at 1000 µg/plate and above. No precipitate was observed at any of the test concentrations.
WP2 uvrA: The RCEs at the concentrations of 5.0 to 5000 µg/plate without activation ranged from 206% to 362%. This may be the result of the low revertant frequency observed in the solvent control. No significant decreases in RCEs were observed at any dose level. A significant decrease revertant frequency and the absence of a background lawn was observed at 5000 µg/plate. In the presence of the activation system, the RCEs at the concentrations of 5.0 to 5000 µg/plate ranged from 77% to 140%. No significant decreases in RCEs (<50%) or effects on the background lawn were found at any dose level. Compared to the solvent control the decreased number of revertant frequency that was observed at some dose levels was not the result of toxicity but rather was due to the number of revertant frequency of the solvent control group being higher than the historical range for solvent controls. No precipitate was observed at any of the test concentrations.

DEFINITVE MUTATION ASSAY
The reversion frequencies for any of the test article-treated plates for any strains were not significantly different than those of their corresponding solvent controls. The background lawns were normal for all concentrations. Based on these results, the Definitive Mutation Assay was negative. Both the solvent and positive controls fulfilled the requirements of a valid test except that the reversion frequency for the TA 98 solvent control was slightly higher than the historical range for solvent controls. This was documented as a protocol deviation. However, the revertant frequencies from all of the test article treated groups were lower than the revertant frequency of the corresponding solvent controls, thereby indicating a negative result and this deviation had no impact on the results or conclusions.

CONFIRMATION MUTATION ASSAY
Without activation, thinning of the background lawn was observed at the highest treatment concentrations, 500 µg/plate for Salmonella typhimurium and 5000 µg/plate for Escherichia coli. This indicated that toxicity was observed at these doses. With activation, the revertant frequency was reduced in TA 98 and TA 100 at 1000 µg/plate, toxicity was also observed. As in the Definitive Assay all strains treated with test article had revertant frequencies that were not significantly greater than those of their corresponding solvent controls. Therefore, the results were negative. The solvent and positive controls for all data presented fulfilled the requirements of the test

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. The test was performed in two independent plate incorporation experiments, both in the absence and presence of S9-mix. The dose levels were selected based on the dose range finding experiment. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) and E. Coli WP2 uvrA), both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic with and without metabolic activation.