Ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-ene-1-carboxylate

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:

23 February to 23 March 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

rel-(1R,6S)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Molecular formula:

C15H24O3

IUPAC Name:

rel-(1R,6S)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Constituent 2

Reference substance name:

rel-(1R,6R)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Molecular formula:

C15H24O3

IUPAC Name:

rel-(1R,6R)-ethyl 2-methyl-4-oxo-6-pentylcyclohex-2-enecarboxylate

Specific details on test material used for the study:

- Test material name (as stated in the report): Calyxol
- Batch No. 748444534002
- Sanple received on 13 February 2007
- Storage: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver

Test concentrations with justification for top dose:

Salmonella strains: 5, 15, 50, 150, 500, 1500 and 5000 microg/plate (determined in a preliminary toxicity assay)
E.Coli strain WP2uvrA: 50, 150, 500, 1500 and 5000 microg/plate (determined in a preliminary toxicity assay)

Vehicle / solvent:

Dimethyl Sulphoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Controls 2

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Results and discussion

Test results

Key result

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2uvrA-

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1535 in presence of S9 at and above 1500 microg/plate in both range fiding and main tests

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

The test material caused a visible reduction in the growth of the bacterial background lawn of Samonella Typhimurium strains TA100 and TA1537 (absence of S9 only) and TA1535 (presence and absence of S9), initally from 500 to 1500 microg/plate in the absence and presence of S9, respectively. No weakening of the bacterial background lawns were noted for any of the remaining bacterial strains. The sensitivity of the bacterial tester strain to the toxicity of of the test material varied slightly between strain type, exposures with and without S9 and Experiment number. The test material was, therefore, tested up to the maximum recommended dose level of 5000 microg/plate.

No significant increases in the frequency of the revertant colonies were recorded for any of the bacterial strains with any dose of the test material, either with or without matabolic activation.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test, Calyxol does not met the criteria for mutagenic classication according the CLP Regulation (EC) No. 1272/2008.

Executive summary:

Samonella Typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia Coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of rat liver homogenate metabolising system (10% liver S9 in standard co factors). The dosage range for the range finding test was determined in a preliminary toxicity assay and was 5 to 5000 microg/plate and 50 to 5000 microg/plate for the Samonella Typhimurium and Escherichia Coli strain WP2uvrA-, respectively. The experiment was repeated on a separate day using an amended dose range, fresh cultures of the bacterial strains and fresh test material formulations.

Additional dose levels were included for the Samonella Typhimurium strains to allow for the test metarial induced toxicity, ensuring that at least four non toxic doses were archived.

The test material caused a visible reduction in the growth of the bacterial background lawn of Samonella Typhimurium strains TA100 and TA1537 (absence of S9 only) and TA1535 (presence and absence of S9), initally from 500 to 1500 microg/plate in the absence and presence of S9, respectively. No weakening of the bacterial background lawns were noted for any of the remaining bacterial strains. The sensitivity of the bacterial tester strain to the toxicity of of the test material varied slightly between strain type, exposures with and without S9 and Experiment number. The test material was, therefore, tested up to the maximum recommended dose level of 5000 microg/plate.

No significant increases in the frequency of the revertant colonies were recorded for any of the bacterial strains with any dose of the test material, either with or without matabolic activation.

The test material was considered to be non-mutagenic under the conditions of this test.