Mentha arvensis, ext.

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

Study period:

12-Jul-2010 to 22-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to OECD guideline 471 and under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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:

Mentha arvensis, ext.

EC Number:

290-058-5

EC Name:

Mentha arvensis, ext.

Cas Number:

90063-97-1

Molecular formula:

Not applicable due to UVCB nature of the substance

IUPAC Name:

(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexan-1-ol; (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one

Constituent 2

Reference substance name:

Cornmint oil (menthe arvensis)

IUPAC Name:

Cornmint oil (menthe arvensis)

Details on test material:

- Name of test material (as cited in study report):Cornmint oil (mentha arvensis)
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Composition of test material, percentage of components: Confidential
- Lot/batch No.: Confidential
- Stability under test conditions: Stable
- Test substance handling: Use amber-coloured glassware or wrap container in tin-foil
- Storage condition of test material: In refrigerator (2-8°C) protected from light

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 ug/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 3, 10, 33, 100, 333 and 1000 ug/plate
With S9-mix: 10, 33, 100, 333, 1000 and 2000 ug/plate

Experiment 2:
TA1535, TA1537, TA98 and TA100:
Without S9-mix: 3, 10, 33, 100, 333 and 1000 ug/plate
With S9-mix: 10, 33, 100, 333, 1000 and 2000 ug/plate
WP2uvrA:
Without S9-mix: 10, 33, 100, 333, 1000 and 2000 ug/plate
With S9-mix: 10, 33, 100, 333, 1000 and 3330 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Test compound was stable and soluble in ethanol and ethanol has been accepted and approved by authorities and international guidelines

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: sodium azide (TA1535); 9-aminoacridine (TA1537); 2-nitrofluorene (TA98); methylmethanesulfonate (TA100); 4-nitroquinoline-N-oxide (WP2uvrA). With S9: 2-aminoanthracene (all test strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted

NUMBER OF CELLS EVALUATED: 10E8 per plate

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

OTHER EXAMINATIONS:
- Other: The presence of precipitation of the test compound on the plates was determined

Evaluation criteria:

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 control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of Cornmint oil (mentha arvensis) on the plates was only observed at the start of the incubation period at concentrations of 3330 and 5000 ug/plate. This precipitate manifested as small fatty particles.

RANGE-FINDING/SCREENING STUDIES: In test strain TA100, toxicity was observed at dose levels of 333 and 1000 ug/plate and above in the absence and presence of S9-mix, resp. In test strain WP2uvrA, toxicity was observed at dose levels of 1000 and 3330 ug/plate and above in the absence and presence of S9-mix, resp.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535 and TA1537: without S9: 1000 ug/plate, and with S9: 2000 ug/plate
TA98: without S9: 333 ug/plate and above, and with S9: 2000 ug/plate
TA100: without S9: 333 ug/plate and above, and with S9: 1000 ug/plate and above
WP2uvrA: without S9: 1000 ug/plate and above, and with S9: 1000 ug/plate and above.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Cornmint oil (mentha arvensis) did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Cornmint oil (mentha arvensis) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

Cornmint oil (mentha arvensis) was tested in the Salmonella typhimurium reverse mutation assay with 4 histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and B-naphthoflavone). The study procedures described in this report were based on OECD guideline 471. In the dose range finding test, Cornmint oil (mentha arvensis) was tested up to concentrations of 5000 ug/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. Cornmint oil (mentha arvensis) did not precipitate on the plates at this dose level. Cytotoxicity was observed in both test strains. Results of this dose range finding test were reported as part of the first experiment of the mutation assay. Based on the results of the dose range finding test, Cornmint oil (mentha arvensis) was tested in the first mutation assay up to 1000 and 2000 ug/plate in the absence and presence of 5% (v/v) S9-mix, resp. in test strains TA1535, TA1537 and TA98. In an independent repeat of the assay with additional parameters, Cornmint oil (mentha arvensis) was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in test strains TA1535, TA1537, TA98 and TA100 and up to 2000 and 3330 ug/plate in the absence and presence of 10% (v/v) S9-mix, resp. in test strain WP2uvrA. Appropriate cytotoxicity was observed in all test strains in both experiments. Cornmint oil (mentha arvensis) did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the 4 test strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in test strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Based on the results of this study it is concluded that Cornmint oil (mentha arvensis) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.