L-menthyl acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD 471 and GLP, but the plate incorporation method was used for both experiments

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Menthyl acetate

EC Number:

201-911-8

EC Name:

Menthyl acetate

Cas Number:

89-48-5

Molecular formula:

C12H22O2

IUPAC Name:

Menthyl acetate

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

other: TA1535, TA1537, TA102, TA98 and TA100

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9 in standard co-factors

Test concentrations with justification for top dose:

Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment I: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment II: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: not reported

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (TA100, TA1535), 9-Aminoacridine (TA1537), Mitomycin C (TA102), 4-Nitroquinoline-l-oxide (TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: not reported

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the bacterial background lawn, reduction in colony frequency

Evaluation criteria:

The test material may be considered to be positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Species / strain:

other: TA1535, TA1537, TA102, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Visible reduction in the bacterial background lawn in tester strains TA100 and TA1535 with and without S9 (first evidence of a weakened bacterial background lawn at 1500 pg/plate). Tested up to the maximum recommended dose of 5000 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the bacterial background lawn but did cause a reduction in colony frequency at 5000 µg/plate (particularly after the addition of S9-mix).

COMPARISON WITH HISTORICAL CONTROL DATA: Done.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the bacterial background lawn in tester strains TA100 and TA1535 both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 1500 µg/plate. The test material was tested up to the maximum recommended dose of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test material using the plate incorporation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment was determined in a preliminary toxicity assay and ranged from 5 to 5000 µg/plate depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day using a dose range based on results observed in Experiment 1, fresh cultures of the bacterial strainsand fresh test material formulations. The method used conforms with the OECD TG471 and the test was performed under GLP. Extra dose levels were included (where applicable) to allow for the toxicity of the test material and to ensure there were a minimum of four non-toxic doses plated out.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonieswithin the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the bacterial background lawn in testerstrains TA100 and TA1535 both with and without metabolic activation. The first evidence of a weakened bacterial background lawn was observed at 1500 µg/plate. The test material was tested up to the maximum recommended dose of 5000 µg/plate. No test material precipitate was observed an the plates at any of the doses tested in either the presence or absente of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.