Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

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)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 2-hexyl-3-oxocyclopentanecarboxylate

EC Number:

253-379-1

EC Name:

Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Cas Number:

37172-53-5

Molecular formula:

C13H22O3

IUPAC Name:

methyl 2-hexyl-3-oxocyclopentane-1-carboxylate

Test material form:

liquid

Specific details on test material used for the study:

- Test substance storage: At room temperature in the dark
- Description: Clear colourless liquid
- Expiry date: 28 March 2009

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:

Phenobarbital/b-naphthoflavone induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Dose range finding test/Experiment 1:
- TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330, and 5000 μg/plate in the absence and presence of S9-mix
- TA1535, TA1537 and TA98: 10, 33, 100, 333, 1000, and 3330 μg/plate in the absence and presence of S9-mix
Experiment 2:
- TA98 and WP2uvrA: 33, 100, 333, 1000, and 3300 μg/plate in the absence and presence of S9-mix
- TA1535, TA1537 and TA100: 33, 100, 333, 1000, and 2000 μg/plate in the absence and presence of S9-mix

Vehicle / solvent:

Ethanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

- OTHER: In the first experiment 5% (v/v) S9-mix was used and in the second experiment 10% (v/v) S9-mix was used.

Evaluation criteria:

Acceptability of the assay:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean. c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
Data evaluation:
A test substance is considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control. b) The negative response should be reproducible in at least one independently repeated experiment. A test substance is considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment. The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Key result

Species / strain:

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:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING TEST
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards and at 3330 and 5000 µg/plate at the end of the incubation period.
- Toxicity: In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain TA100, reduction of the bacterial background lawn was observed in the absence of S9-mix at concentrations of 333 µg/plate and upwards and in the presence of S9-mix at concentrations of 1000 µg/plate. Microcolonies were observed at concentrations of 3330 µg/plate and upwards.

EXPERIMENT 1
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1000 and 3330 µg/plate and at 3330 µg/plate at the end of the incubation period.
- Toxicity: In the absence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535, TA1537, and TA98. In the presence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535, TA1537 and at a concentration of 3330 µg/plate in tester strain TA98.

EXPERIMENT 2
- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1000 and above and at 3330 µg/plate at the end of the incubation period.
- In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In the absence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535, TA1537, TA100, and TA98. In the presence of S9-mix, toxicity was observed at concentrations of 1000 µg/plate and upwards in tester strains TA1535 and TA1537, at a concentration of 3330 µg/plate in tester strain TA98, and at a concentration 333 µg/plate and upwards in tester strain TA100.