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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): the substance L-carvone (CAS No. 6485-40-1) does not induce mutagenicity using S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvr A in the presence or absence of Phenobarbitone/β-Naphthoflavone-induced rat liver S9 metabolic activation (OECD 471, GLP);

Chromosome aberration (in vitro mammalian cell cytogenicity): the substance L-carvone (CAS No. 6485-40-1) does not induce chromosome aberrations in human lymphocyte cells in the presence or absence of Phenobarbitone/β-Naphthoflavone-induced rat liver S9 metabolic activation (OECD 473, GLP);

Gene mutation (in vitro mammalian cell gene mutation): the substance L-carvone (CAS No. 6485-40-1) does not induce gene mutations in L5178Y TK +/- 3.7.2c mouse lymphoma cells in the presence or absence of Phenobarbitone/β-Naphthoflavone-induced rat liver S9 metabolic activation (OECD 476, GLP).

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

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.

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 .

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.

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

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

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'.

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)

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-05-12 to 07-11-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human (freshly prepared from donors)

Details on mammalian cell type (if applicable):

- Type and identity of media: Eagle's minimal essential medium with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS) (Cell Culture); 9.05 ml MEM, 10% (FBS), 0.1 ml Li-heparin, 0.1 ml phytohaemagglutinin, 0.75 ml heparinised whole blood (Test cultures).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No
- Periodically checked for karyotype stability: No

Metabolic activation:

with and without

Metabolic activation system:

PB/βNF S9 prepared in-house on 15/04/12 and 16/09/12 supplemented with MgCl2 (8mM), KCl (33mM), sodium orthophosphate pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM) . Preliminary Toxicity Test/Experiment 1: 2% S9; Experiment 2: 1% S9

Test concentrations with justification for top dose:

Preliminary test: 0, 5.87, 11.74, 23.47, 46.94, 93.89, 187.78, 375.55, 751.1 and 1502.2 µg/mL
Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

Experiment 1 (4h, without S9): 0, 25, 50, 100, 200, 300 and 400 µg/mL
Experiment 1 (4h, with S9): 0, 50, 100, 200, 400, 600 and 800 µg/mL

Experiment 2:(4h, with S9): 0, 50, 100, 200, 400, 600 and 800 µg/mL
Experiment 2 (24h, without S9): 0, 12.5, 25, 50, 100, 200 and 400 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO;
- Justification for choice of solvent/vehicle: The test item was fully miscible in DMSO at 150.22 mg/mL in an in-house solubility check. Therefore DMSO was used as the solvent vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

mitomycin C

Remarks:

Mitomycin C (MMC) (Sigma, Batch No. 089K0731) in MEM @ 0.4 and 0.2 µg/mL in Experiments 1 and 2 respectively.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) (Acros, Batch No. A0302605) in DMSO was used at 5 µg/mL in both experiments.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4hrs +/- S9, 24hrs - S9
- Fixation time (start of exposure up to fixation or harvest of cells): All culturees were harvested at 24 hrs and fixed


SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Fixed in fresh methanol/glacial acetic acid (3:1 v/v) and 5% Giemsa stain

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
Mitotic Index - 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded.
Scoring of Chromosome Damage - first 100 consecutive well-spread metaphases from each culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as
endoreduplicated.

Evaluation criteria:

The following criteria were used to determine a valid assay:
Negative control:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures will normaly be within the laboratory historical control data range. The level of spontaneous background aberrations may be slightly elevated above the normal range and the experiment still considered to be valid.

Positive control:
All the positive control chemicals must inducte positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case-by-case basis.

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

375.55 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

751.1 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

187.78 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

other: The positive result was considered to be due to a cytotoxic mechanism and to have no biological relevance

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into dimethyl sulphoxide (see pH and osmolality readings below).

- Effects of osmolality: The osmolality did not increase by more than 50 mOsm (see pH and osmolality readings below).

- Precipitation: Precipitate observations were recorded at the beginning and end of the exposure periods from blood-free cultures. In 4 hr groups, greasy/oily precipitate was noted at the end of exposure at and above 375.55 µg/mL; in 24 hr group, greasy/oily precipitate was observed at the end of exposure at 1502.2 µg/mL only

- Other confounding effects: Haemolysis was also observed at and above 375.5 µg/mL in the blood cultures in the absence of S9, whereas in the presence of S9, haemolysis was observed at and above 187.78 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:The current in-house historical aberration ranges from the laboratory for the vehicle and positive control lymphocytes are presented in Appendix 1.

Remarks on result:

other: other: Preliminary (4hr)

Remarks:

Migrated from field 'Test system'.

pH and osmolality readings from preliminary toxicity test:

µg/mL	0	5.87	11.74	23.47	46.94	93.89	187.78	375.55	751.1	1502.2
pH	7.22	7.18	7.21	7.23	7.24	7.23	7.22	7.22	7.21	7.23
mOsm	446	435	-	-	428	443	427	430	437	424

(-) Not determined

Table 1: Mitotic Index - Preliminary Toxicity Test

CONCENTRATION (µg/ml)	4 hr - S9				4 hr + S9	24 hr - S9	S9
	MITOTIC INDEX	% OF CONTROL	MITOTIC INDEX  % OF CONTROL			MITOTIC INDEX	% OF CONTROL
0	4.7	100		3.65	100	2.65	100
5.87	-	-		-	-	-	-
11.74	-	-		-	-	-	-
23.47	-	-		-	-	3.15	119
46.94	-	-		-	-	2.55	96
93.89	-	-		-		1.45	55
187.78	10.4	221		5.05 H	138	0.3	11
375.55	2.30 P H	49		2.85 P H	78	-NM H	-
751.1	5.25 P H	112		1.05 P H	29	-NM H	-
1502.2	- NM P H	-		-NM P H	-	-NM P H	-

- = Not assessed for mitotic index

NM = No metaphases suitable for scoring

P = Greasy/oily precipitate observed at end of exposure period in the blood-free cultures

H = Haemolysis

Study report attachments:

Table 2 Mitotic Index - Experiment 1 (41202090)

Table 3 Mitotic Index - Experiment 2 (41202090)

Tables 4 & 5 Results of Chromosome Aberration Test - Experiment 1 Without & With S9 (41202090)

Table 6 Results of Chromosome Aberration Test - Experiment 2 Without S9 (41202090)

Table 7 Results of Chromosome Aberration Test - Experiment 2 With S9 (41202090)

Appendix I HISTORICAL CONTROL DATA (41202090)

Conclusions:

The test item, L-Carvone, was not considered to be clastogenic to human lymphocytes in vitro following 4 hr exposure in the presence or absence of metabolic activation under the conditions of this test. The test item, L-Carvone, was considered to be clastogenic to human lymphocytes in vitro following 24 hr continuous exposure in the absence of metabolic activation only under the conditions of this test. However this was considered to be due to a cytotoxic mechanism and to have no biological relevance.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration] (41202090), primary lymphocyte cultures were exposed to L-carvone in DMSO at concentrations of 0, 50, 100, 200, 400, 600 and 800 µg/mL (4 hrs, with S9); 0, 25, 50, 100, 200, 300 and 400 µg/mL (4 hrs, without S9) and 0, 12.5, 25, 50, 100, 200 and 400 µg/mL (24 hrs, without S9).

Positive controls induced the appropriate response. In the presence and absence of metabolic activation, there was no evidence of chromosome aberrations induced over background at the 4 hr time point. In the absence of metabolic activation, there was evidence of chromosome aberrations induced over background at the 24 hr time point; however this was due to a cytotoxic mechanism and was considered to have no biological relevance.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 (In vitro mammalian cytogenetics - chromosome aberration).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-07-12 to 18-09-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK+/- locus

Species / strain / cell type:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Routine culture - RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media); Study media - RPMI 1640 with 20% donor horse serum (R20) and without serum (R0).
- Properly maintained: Yes - The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Periodically checked for Mycoplasma contamination: Yes - Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically checked for karyotype stability: No
- Periodically "cleansed" against high spontaneous background: Yes - Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

PB/βNF S9 prepared in-house on 15/04/12 and 01/07/12 supplemented with NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM). Preliminary Toxicity Test/Experiment 1: 2% final concentration S9; Experiment 2:1% final concentration S9.

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 5.87, 11.74, 23.47, 46.94, 93.89, 187.78, 375.55, 751.1, 1502.2 µg/mL
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiment using following criteria:

i) Maximum recommended dose level, 5000 µg/mL or 10mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).

Experiment 1 (4 hrs; with and without S9): 0, 23.25, 46.5, 93, 139.5, 186, 248, 310, 372 µg/mL

Experiment 2 (4 hrs; with S9): 0, 25, 50, 100, 200, 225, 250, 275, 300 µg/mL

Experiment 2 (24 hrs; without S9): 0, 3.13, 6.25, 12.5, 25, 37.5, 50, 75, 100 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item (Harlan Laboratories Ltd. Project No. 41202090; Genetic toxicity in vitro.002). From this solubility check, the test item was fully miscible in DMSO at 150.22 mg/mL. Therefore DMSO was also chosen as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) at 400 μg/mL and 150 μg/mL for Experiment 1 and Experiment 2 respectively.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide (CP) at 2 μg/mL for both experiments

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 hrs and 24 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
Preliminary toxicity test: 1
Mutation test: 2

DETERMINATION OF CYTOTOXICITY
- Method: Relative Total Growth (RTG) values are usually the primary factor to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, % Relative Suspension Growth values may also be taken into account when designating the level of toxicity achieved.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-170 x 10-6 for the TK+/- locus in L5178Y cells in the test laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test item to demonstrate a mutagenic responseit must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by
the Global Evaluation Factor (GEF) of 126 x 10-6 and demonstrates a positive linear trend will be considered positive.

Statistics:

Calculation of Mutation Frequency (MF): MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selectivemedium.

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al, 1989).

Robinson W D et al(1989).Statistical evaluation of bacterial/mammalian fluctuation tests. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing (Kirkland D J Ed.), Cambridge University PressReport part III, pp102-140.

Species / strain:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

187.78 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Metabolic activation:

without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

23.47μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

139.5 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

139.5 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

200 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: L5178Y TK +/- 3.7.2c mouse lymphoma cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

25 μg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no marked change in pH when the test item (in DMSO) was dosed into media and the osmolality did
not increase by more than 50 mOsm for the Chromosome Aberration Test (Harlan Laboratories Ltd. Project No. 41202090; Genetic toxicity in vitro.002 study record; preliminary toxicity test) carried out (see table below).

- Precipitation: Preliminary test - A greasy / oily precipitate of the test item was observed at and above 751.1 µg/ml in all three of the
exposure groups; Mutagenicity tests (Experiments 1 & 2) - Precipitate of the test item was not observed at any of the dose levels.

COMPARISON WITH HISTORICAL CONTROL DATA: The current Historical Vehicle and Positive ControlMutation Frequencies from the test laboratory are presented in Appendix 2.

ADDITIONAL INFORMATION ON CYTOTOXICITY: % RSG are used to determine cytotoxicity in preliminary studies; % RSG and RTG are used to determine cytotoxicity in the Mutagenicity tests - Experiments 1 and 2.

Remarks on result:

other: other: Preliminary (4hr)

Remarks:

Migrated from field 'Test system'.

The pH and osmolality readings from the Chromosome Aberration Test (Harlan Laboratories Ltd. Project No. 41202090; Genetic toxicity in vitro.002 study record; preliminary toxicity test) using the same test item are in the following table:

µg/mL	0	5.87	11.74	23.47	46.94	93.89	187.78	375.55	751.1	1502.2
pH	7.22	7.18	7.21	7.23	7.24	7.23	7.22	7.22	7.21	7.23
mOsm	446	435	-	-	428	443	427	430	437	424

Preliminary Toxicity Test results (MLA assay; 41202091)

Dose (µg/mL)	%RSG (-S9) 4 hrs	%RSG (+S9) 4 hrs	%RSG (-S9)       24 hrs
0	100	100	100
5.87	98	93	90
11.74	107	98	73
23.47	99	88	45
46.94	99	71	18
93.89	72	51	8
187.78	40	34	1
375.55	2	2	0
751.1	1	1	0
1502.2	0	0	0

Study report attachments:

Table 3 Statistical Analysis: Experiment 1 (-S9) 4-Hour Exposure (41202091)

Table 4 Large and Small Colonies Analysis: Experiment 1 (-S9) 4-Hour Exposure (41202091)

Table 6 Statistical Analysis: Experiment 1 (+S9) 4-Hour Exposure (41202091)

Table 7 Large and Small Colonies Analysis: Experiment 1 (+S9) 4-Hour Exposure (41202091)

Table 9 Statistical Analysis: Experiment 2 (-S9) 24-Hour Exposure (41202091)

Table 10 Large and Small Colonies Analysis: Experiment 2 (-S9) 24-Hour Exposure (41202091)

Table 12 Statistical Analysis: Experiment 2 (+S9) 4-Hour Exposure (41202091)

Table 13 Large and Small Colonies Analysis: Experiment 2 (+S9) 4-Hour Exposure (41202091)

Appendix 2 Historical Vehicle and Positive Control Mutation Frequencies (41202091)

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

In a mammalian cell gene mutation assay [TK] (41202091), L5178Y TK +/- 3.7.2c mouse lymphoma cells cultured in vitro were exposed to L-carvone in DMSO at concentrations of 0, 23.25, 46.5, 93, 139.5, 186, 248, 310, 372 µg/mL (4 hrs, with and without S9); 0, 25, 50, 100, 200, 225, 250, 275, 300 µg/mL (4 hrs, with S9) and 0, 3.13, 6.25, 12.5, 25, 37.5, 50, 75, 100 µg/mL (24 hrs, without S9).

L-carvone was tested up to cytotoxic concentrations. The positive controls induced the appropriate response. 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 476 for in vitro mutagenicity (mammalian cell gene mutation assay) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

In a reverse gene mutation assay in bacteria (OECD 471/GLP), 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.

Chromosome aberration (in vitro mammalian cell cytogenicity):

In a mammalian cell cytogenetics assay [Chromosome aberration] (OECD 473/GLP), primary lymphocyte cultures were exposed to L-carvone in DMSO at concentrations of 0, 50, 100, 200, 400, 600 and 800 µg/mL (4 hrs, with S9); 0, 25, 50, 100, 200, 300 and 400 µg/mL (4 hrs, without S9) and 0, 12.5, 25, 50, 100, 200 and 400 µg/mL (24 hrs, without S9). Positive controls induced the appropriate response. In the presence and absence of metabolic activation, there was no evidence of chromosome aberrations induced over background at the 4 hr time point. In the absence of metabolic activation, there was evidence of chromosome aberrations induced over background at the 24 hr time point; however this was due to a cytotoxic mechanism and was considered to have no biological relevance.

Gene mutation (in vitro mammalian cell gene mutation):

In a mammalian cell gene mutation assay [TK] (OECD 476/GLP), L5178Y TK +/- 3.7.2c mouse lymphoma cells cultured in vitro were exposed to L-carvone in DMSO at concentrations of 0, 23.25, 46.5, 93, 139.5, 186, 248, 310, 372 µg/mL (4 hrs, with and without S9); 0, 25, 50, 100, 200, 225, 250, 275, 300 µg/mL (4 hrs, with S9) and 0, 3.13, 6.25, 12.5, 25, 37.5, 50, 75, 100 µg/mL (24 hrs, without S9). L-carvone was tested up to cytotoxic concentrations. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

The results are acceptable for the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance L-carvone (CAS No. 6485-40-1) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.