(S)-2-methyl-5-(1-methylvinyl)cyclohex-2-en-1-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation toxicity study was performed for D- carvone to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. D- carvone did notinduce gene mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed for D-Carvone to evaluate its mutagenic nature

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material: D-Carvone- IUPAC name: (5S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one- Molecular formula: C10H14O- Molecular weight: 150.22 g/mol- Substance type: Organic- Physical state: No data- Purity: 95.4%- Impurities (identity and concentrations): 4.6%

Target gene:

Histidine

Species / strain / cell type:

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

Remarks:

Lab 1 and Lab 2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the S9 preparation of the liver fractions

Test concentrations with justification for top dose:

0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98; -S9), 2-aminoanthracene (all strains, +S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubationDURATION- Preincubation period: 20 mins- Exposure duration: 48 hr- Expression time (cells in growth medium): 48 hr- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No dataSELECTION AGENT (mutation assays): No dataSPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: At least five dose levels of the chemicals were tested, with three plates per dose level.NUMBER OF CELLS EVALUATED: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Other: No dataOTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible;or when the response was of insufficient magnitude to support a determination of mutagenicity

Statistics:

Mean and Standard error of mean

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537, TA98

Remarks:

Lab 1 and Lab 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data - Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: The chemical was initially tested with strain TA100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered. Toxicity was evidenced by one or more of the following phenomena: appearance of his+ pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn. Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility. Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity.COMPARISON WITH HISTORICAL CONTROL DATA: No data

Remarks on result:

other: No mutagenic potential

Table: Mutation data for the test chemical D- carvone

Dose (µg/plate)	TA100
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	89	4.6	121	12.1	133	11.7
3.3	84	7.6	119	9.9	94	14.1
10.0	75	4.0	113	9.6	105	6.7
33.0	89	4.1	107	6.7	108	2.2
100.0	73	7.4	104	6.7	108	2.2
333.0	73	7.4	104	3.0	100	5.0
Positive control	48	9.0	78	8.4	65	11.3

 

Dose (µg/plate)	TA1535
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	4	1.5	6	1.5	8	3.0
3.3	3	0.9	5	1.3	5	0.3
10.0	3	0.7	4	0.9	5	1.2
33.0	3	0.	3	0.7	4	1.5
100.0	2	1.0	5	0.3	3	0.6
333.0	1	0.6	4	0.6	3	2.2
Positive control	122	11.1	41	5.8	76	13.4

 

Dose (µg/plate)	TA1537
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	3	0.7	6	0.6	6	1.3
3.3	3	0.6	5	0.7	5	1.2
10.0	2	0.0	5	1.2	8
33.0	3	1.2	2	0.7	7	1.7
100.0	3	1.0	4	0.7	8	2.7
333.0	3	0.6	6	1.2	6	0.9
Positive control	1041	154.2	261	12.0	211	24.1

 

Dose (µg/plate)	TA98
	NA		10% HLI		10% RLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	14	1.9	25	1.2	21	3.8
3.3	16	2.8	23	5.0	20	3.2
10.0	15	0.3	23	1.2	15	3.2
33.0	11	1.2	25	2.5	22	0.9
100.0	13	0.9	23	2.0	24	1.5
333.0	10	2.1	17	4.0	22	2.0
Positive control	359	21.4	1969	56.9	973	201.3

Conclusions:

D- carvone did not induce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed for D- carvone to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. D- carvone did notinduce gene mutation in theSalmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the target chemical was reviewed to determine the mutagenic nature of D-carvone. The studies are as mentioned below:

Zeiger et al ( Environmental Mutagenesis, 1986) performed gene mutation toxicity study was performed for D- carvone (CAS no 2244 -16 -8) to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system at doses of0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate. DMSO was used at the vehicle. The plates were incubated for 48 hrs after 20 mins preincubation before the evaluation of the revertant colonies could be made. D- carvone did notinduce gene mutation in the Salmonella typhimurium strain TA100, TA1535, TA1537, TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In a study by Stammati et al (Food and chemical toxicology, 1990), Ames assay was performed for S (+)- carvone to evaluate its mutagenic nature. The study was performed as per the standard plate incorporation assay using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system at doses of0, 6.25, 12.5 or 25.0 µL/plate. 85% ethanol was used at the vehicle. Concurrent solvent and positive control chemicals were also included in the study. S (+)- carvone did not induce gene mutation in the Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Ames assay was performed by Stammati et al (Food and Chemical Toxicology, 1999) for S (+)- carvone to evaluate its mutagenic nature. The study was performed as per the standard plate incorporation assay using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system at doses of 0, 6.25, 12.5 or 25.0 µL/plate. 85% ethanol was used at the vehicle. Concurrent solvent and positive control chemicals were also included in the study. S (+)- carvone did notinduce gene mutation in theSalmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Another study was conducted by Rockwell and Raw (Nutrition and Cancer, 1979). Gene mutation toxicity study was performed for carvone to evaluate its mutagenic nature. The study was performed as per the preincubation protocol using Salmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system at doses of 0.05 µl to 100 µL. For each assay, a test sample of 10⁸ bacterial cells, and S9 at a concentration of 800µg protein per plate were incorporated into a tube containing top agar prepared with minimal medium and 0.05 mM histidine and 0.05 mM biotin. The top agar was then poured on a petri dish containing minimal medium supplemented with 20% glucose. After a 48-hour incubation at 37°C, assay plate was counted and the number of spontaneous mutants for either TA98 (40) or TA100 (180) were subtracted from the total number of revertants. Concurrent positive control chemicals were also included in the study. Carvone did not induce gene mutation in theSalmonella typhimurium strain TA100 and TA98 both in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration test was performed (NTP, 1990) to determine the mutagenic nature of d- carvone. The study was performed using Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation system. In the absence of S9 metabolic activation system, cells were incubated in McCoy's 5A medium with the study chemical for 8 hours, colcemid was added, and incubation was continued for 2 hours. For the chromosomal aberration test with S9, cells were treated with the study chemical and S9 for 2 hours, after which the treatment medium was removed and the cells were incubated for 10 hours in fresh medium, with colcemid present for the final 2 hours. For both the treatments, the cells were harvested by mitotic shake-off, fixed, and stained with Giemsa. In cytogenetic tests with CHO cells, d-carvone induced chromosomal aberrations with and without Aroclor 1254-induced male Sprague Dawley rat liver S9. Results were statistically positive in the second trial of the chromosomal aberration test conducted without S9.

In vitro DNA repair assay was performed by Stammati et al (Food and chemical toxicology, 1999) for S(+)-Carvone to evaluate its mutagenic nature. The study was performed using Escherichia coli strain WP2 trpE65 and its isogenic DNA repair-deficient derivative CM871 trpE65, uvrA155, recA56, lexA at dose levels of0, 25, 30, 50, 60 or 80 µmol/plate. The strains were grown overnight in nutrient broth at 37˚C. For the tests, samples of 0.2 ml of either culture were mixed with 2 ml of molten 0.6% agar at 45˚C containing 250 mg L-tryptophan/ml, and the mixtures were poured on Vogel-Bonner agar plates. 12mm diameter filter paper discs impregnated with 25 ml of the solutions of the test compounds at concentrations producing a visible inhibition zone in preliminary tests with WP2, were subsequently placed on the plates. 4-Nitroquinoline-N-oxide was used as a positive control. Each concentration was tested in triplicate. After an overnight incubation at 37˚C the inhibition zones were measured the differential killing between the tests strains being regarded as an indication of the capacity of the test compound to produce DNA damage. It can be seen that carvone had the most marked differential toxicity against the DNA repair-deficient strain CM871. This effect, however, became apparent only at doses that were 10 times higher than with the other plant volatiles tested. At lower doses no antibacterial effects on either of the E. coli strains could be observed. S (+)- carvone did not induce DNA repair in the Escherichia coli strain WP2 trpE65. It however induced gene mutation in the isogenic DNA repair-deficient derivative CM871 trpE65, uvrA155, recA56, lexA.

IIn the same NTP report (1990), Sister chromatid exchange test was performed to determine the mutagenic nature of d- carvone. The study was performed using Chinese hamster ovary cells (CHO) in the presence and absence of S9 metabolic activation system. In the SCE test without S9, CHO cells were incubated for 26 hours with the study chemical in McCoy's 5A medium supplemented with 10% fetal bovine serum, L-glutamine (2 mM), and antibiotics. BrdU was added 2 hours after culture initiation. After 26 hours, the medium containing the study chemical was removed and replaced with fresh medium plus BrdU and colcemid, and incubation was continued for 2 more hours. Cells were then harvested by mitotic shake-off, fixed, and stained with Hoechst 33258 and Giemsa. In the SCE test with S9, cells were incubated with the chemical, serum free medium, and S9 for 2 hours. The medium was then removed and replaced with medium containing BrdU and no study chemical; incubation proceeded for an additional 26 hours, with colcemid present for the final 2 hours. Harvesting and staining were the same as for cells treated without S9.50 second-division metaphase cells were usually scored for frequency of SCEs per cell from each dose.In cytogenetic tests with CHO cells, No slowing of the cell cycle was noted in the CHO cells used for the SCE test. D-carvone induced sister chromatid exchanges with and without Aroclor 1254-induced male Sprague Dawley rat liver S9. Although results were statistically positive in two of the three SCE trials, there was no correlation of dose with response.

Based on the data available for the target chemical, D-carvone does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. The positive result obtained in the chromosome aberration and sister chromatid exchange study however did not show a dose correlated response. taking this into consideration, the test chemical d-carvone is likely to be non-mutagenic.

Justification for classification or non-classification

Based on the data available for the target chemical, D-carvone does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. The positive result obtained in the chromosome aberration and sister chromatid exchange study however did not show a dose correlated response. taking this into consideration, the test chemical d-carvone is likely to be non-mutagenic.