L-p-mentha-1(6),8-dien-2-one

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:

11-05-12 to 15-06-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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

Constituent 1

Reference substance name:

L-p-mentha-1(6),8-dien-2-one

EC Number:

229-352-5

EC Name:

L-p-mentha-1(6),8-dien-2-one

Cas Number:

6485-40-1

Molecular formula:

C10H14O

IUPAC Name:

(5R)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one

Test material form:

other: Clear colourless liquid

Details on test material:

- Name of test material (as cited in study report): L-carvone
- Physical state: Clear colourless liquid
- Analytical purity: 99.14%
- Purity test date: 04/19/2012
- Lot/batch No.: 2012041901
- Expiration date of the lot/batch: 04/19/2013
- Stability under test conditions: Assumed stable for 4 hours upon formulation before applying to test system
- Storage condition of test material: Room temperature in the dark

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:

S9 Microsomal fraction from rats induced with Phenobarbitone/Beta-Naphthoflavone prepared in-house (April 2012)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiment 1 (direct plate incorporation): 0, 50, 150, 500, 1500 and 5000 µg/plate

Mutation Test - Experiment 2 (pre-incubation): 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water but was fully miscible in dimethyl
sulphoxide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG: 2 µg/plate WP2uvrA, 3 µg/plate TA100, 5 µg/plateTA1535; 9AA: 80 µg/plate TA1537; 4NQO: 0.2 µg/plate TA98 .

Controls 2

Untreated negative controls:

yes

Remarks:

Untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

2AA: 1 µg/plate TA100, 2 µg/plate TA1535, TA1537, 10 µg/plate WP2uvrA; BP:5 µg/plate for TA98.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Mutation Test - Experiment 1: direct plate incorporation method;
Mutation Test - Experiment 2 : preincubation method;

DURATION
- Preincubation period: 20 minutes (Mutation Test - Experiment 2)

NUMBER OF REPLICATIONS: 3 (Experiments 1 & 2)

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn (Experiments 1 & 2)

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Environmental
Mutagenesis, 1, 87-92.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).

Mahon G A T et al(1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.), Cambridge University PressReport, 26-65.

Results and discussion

Test results

Test results 1

Species / strain:

other: S.typhimurium TA100; E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A test item precipitate (greasy in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.


RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was conducted (see below).

COMPARISON WITH HISTORICAL CONTROL DATA: Combined historical negative, positive and solvent control ranges from the laboratory for 2010 and 2011 were presented (Appendix 2).

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	83	77	76	90	94	104	89	68	92	87	69
+	TA100	116	98	100	123	91	106	118	96	100	97	101
-	WP2uvrA	33	27	37	35	25	36	30	30	24	27	19
+	WP2uvrA	38	30	44	40	37	34	37	27	30	36	12

Numbers refer to revertant colonies.

Study report attachments:

Table 1 Spontaneous Mutation Rates (Concurrent Negative Controls) (41202089)

Tables 2 & 3 Experiment 1 – With & Without Metabolic Activation (41202089)

Tables 4 & 5 Experiment 2 – With & Without Metabolic Activation (41202089)

Appendix 2 History Profile of Vehicle and Positive Control Values (41202089)

Applicant's summary and conclusion

Conclusions:

The test item, L-Carvone, was considered to be non-mutagenic with and without metabolic activation under the conditions of this test.

Executive summary:

In a reverse gene mutation assay in bacteria (41202089), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to L-carvone in DMSO at concentrations of 0, 50, 150, 500, 1500 and 5000 µg/plate (direct plate incorporation; experiment 1) and 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (rat S9). L-carvone was tested up to the limit concentration (5000 µg/plate).

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.