trans-menthone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/mammalian-microsome test was performed on Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98. The test material was exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate. DMSO was used as solvent control. Positive controls were also used for all strain respectively. The test was performed by standard plate method. The toxicity of the substances was tested in the tester strains at 10^-7dilutions. On the basis of the results obtained, the following concentrations of the test substance were selected for testing: 800, 160, 32 or 6.4 µg per plate. Each dosage was tested on 5 parallel plates. The mean number of revertants for n plates at each dose level was calculated. No mutagenic effects were observed in all strain except strain TA1537. Contrary to these results, menthone induced an increased number of revertants in the strain TA1537 (dose levels of 6.4 and 32 µg per plate). But no effects were observed at the dose level of 160 or 800 µg/plate in strain TA1537 without S9. On the basis of obseravations made, the test chemical (CAS no 89-80-5) was considered to be non mutagenic in all strain at highest dose concentration and in the presence of S9 too in all strain. Hence the test material cannot be classified as gene mutant in vitro.

Chromosome aberration:

The test chemical did not induce chromosomal aberration in mammalian cell line used and hence is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

To evaluate the mutagenic potential of Menthone in Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97and TA98.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA1537, TA1535, TA100, TA 97and TA98.

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate.

Test concentrations with justification for top dose:

0, 6.4, 32,160 or 800 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:

2-nitrofluorene

sodium azide

other: For all strains, 2-anthramine with S9 served as a positive control.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar- Cell density at seeding (if applicable): no dataDURATION- Preincubation period: No data- Exposure duration: No data- Expression time (cells in growth medium): No data- Selection time (if incubation with a selection agent): No data- Fixation time (start of exposure up to fixation or harvest of cells): No data SELECTION AGENT (mutation assays): No data SPINDLE INHIBITOR (cytogenetic assays): No dataSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: Each dosage was tested on 5 parallel plates and all the tests were performed on two separate occasions.METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No dataNUMBER OF CELLS EVALUATED: No dataNUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No dataCRITERIA FOR MICRONUCLEUS IDENTIFICATION: No dataDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data- Any supplementary information relevant to cytotoxicity: No dataOTHER EXAMINATIONS:- Determination of polyploidy: No data- Determination of endoreplication: No data- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data- OTHER: No data

Rationale for test conditions:

Not specified.

Evaluation criteria:

The mean number of revertants for n plates at each dose level was calculated. The test was considered to be positive if mean number of revertants for n plates at each dose level was greater than control.

Statistics:

Comparisons of the number of revertants per plate were done as t-tests after a square-root transformation of each number had been performed to give homogeneity of variance. The mean number of revertants for n plates at each dose level was calculated to be the squared value of the mean (y) of the square roots. The standard error of the mean was calculated as 2 y~[' s2/n where s 2 is the pooled variance of all individual square root values

Species / strain:

S. typhimurium, other: strains TA1537, TA1535, TA100, TA 97 and TA98.

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- Definition of acceptable cells for analysis: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: On the basis of the toxicity study, the doses were selected for the main studyCYTOKINESIS BLOCK (if used)- Distribution of mono-, bi- and multi-nucleated cells: No dataNUMBER OF CELLS WITH MICRONUCLEI- Number of cells for each treated and control culture: No data- Indication whether binucleate or mononucleate where appropriate: No dataHISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data:- Negative (solvent/vehicle) historical control data:ADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Remarks on result:

other: No mutagenic effect were observed.

Table: 1

NUMBER OF REVERTANTS AFTER EXPOSURE TO THE TEST CHEMICAL IN THE SALMONELLA/MAMMALIAN-M1CROSOME TEST PRESENTED AS MEAN OF 10 PLATES ± S.E.

 

Dose (µg/plate)	TA1535		TA100		TA1537		TA98
	WITHOUT S9	WITH S9	WITHOUT S9	WITH S9	WITHOUT S9	WITH S9	WITHOUT S9	WITH S9
CONTROL	7 ± 1	8 ± 1	85 ± 4	114 ± 8	7 ± 1	11 ± 1	32 ± 3	33 ± 2
6.4	7 ± 1	10 ± 1	96 ± 4	114 ± 8	13 ±2	11 ± 1	25 ± 2	28 ± 2
32	7 ± 1	9 ± 1	92 ± 4	121 ± 8	12 ± 2	12 ± 1	31 ± 3	27 ± 2
160	5 ± 1	8 ± 1	97 ± 4	123 ± 8	8 ± 1	11 ± 1	28 ± 2	26 ± 2
800	7 ± 1	9 ± 1	59 ± 6	104 ± 7	4 ± 1	11 ± 1	13 ± 3	22 ± 2

Contrary to these results, menthone induced an increased number of revertants in the strain TA1537 without S9 at dose levels of 6.4 and 32 µg per plate (as per table). This increase was statistically significant (p≤0.01). But no effect at highest concentration.

Conclusions:

The test chemical (CAS no. 89-80-5) did not induce gene mutation in Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98 in the presence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/mammalian-microsome test was performed on Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98. The test material was exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate. DMSO was used as solvent control. Positive controls were also used for all strain respectively. The test was performed by standard plate method. The toxicity of the substances was tested in the tester strains at 10^-7 dilutions. On the basis of the results obtained, the following concentrations of the test substance were selected for testing: 800, 160, 32 or 6.4 µg per plate. Each dosage was tested on 5 parallel plates. The mean number of revertants for n plates at each dose level was calculated. No mutagenic effects were observed in all strain except strain TA1537. Contrary to these results, menthone induced an increased number of revertants in the strain TA1537 (dose levels of 6.4 and 32 µg per plate). But no effects were observed at the dose level of 160 or 800 µg/plate in strain TA1537 without S9. On the basis of obseravations made, the test chemical (CAS no 89-80-5) was considered to be non mutagenic in all strain at highest dose concentration and in the presence of S9 too in all strain. Hence the test material cannot be classified as gene mutant in vitro.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

other: Chromosome aberration assay

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: MinimumEssential Medium (MEM; GIBCO) supplemented by 10% calf serum- Properly maintained: yes by 4 day passages - Periodically checked for Mycoplasma contamination: No data available- Periodically checked for karyotype stability: No data available- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

lymphocytes: Human lymphocytes

Remarks:

2

Details on mammalian cell type (if applicable):

- Type and identity of media: RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin- Properly maintained: yes- Periodically checked for Mycoplasma contamination: No data- Periodically checked for karyotype stability: No data- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

1. At three different doses with 0.2 mg/mL being the maximum dose concentration2. 0, 0.1, 1 or 10 mM

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: Ethanol- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Untreated negative controls:

yes

Remarks:

Untreated cells served as negative control

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in mediumDURATION- Preincubation period: No data- Exposure duration: 24 and 48 hrs- Expression time (cells in growth medium): 24 and 48 hrs- 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): Giemsa solution (1.5%, pH 6.8)SPINDLE INHIBITOR (cytogenetic assays): ColcemidSTAIN (for cytogenetic assays): No dataNUMBER OF REPLICATIONS: No dataNUMBER OF CELLS EVALUATED: 100 well spread metaphasesDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No dataOTHER EXAMINATIONS:- Determination of polyploidy: Yes- Determination of endoreplication: No data- Other: No dataOTHER: No data2. METHOD OF APPLICATION: in mediumCells at the start of exposure: 0.5-1.0 x 106DURATION- Preincubation period: No data- Exposure duration: 72 hrs in dark- Expression time (cells in growth medium): 72 hrs in dark- 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: No dataNUMBER OF CELLS EVALUATED: 100 metaphase cells from each donor according to the classification criteria suggested by Savage (1975),DETERMINATION 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:

1/2. No data

Evaluation criteria:

1. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.2. The metaphase cells were observed for chromosomal aberrations

Statistics:

1/2. No data

Species / strain:

S. typhimurium, other: Chinese hamster fibroblast cell line CHL

Remarks:

1

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Species / strain:

lymphocytes: Human lymphocytes

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. 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 maximum dose of each sample was selectedby a preliminary test in which the dose neededfor 50% cell-growth inhibition was estimated using a cell densitometerCOMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data2. 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: 10mM-menthol was chosen as the highest concentrationbecause higher concentrations significantlyaffected the growth of human lymphocytes inphytohaemagglutinin-stimulated culturesCOMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberration in mammalian cell line used and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Data availbale for the test chemicals was reviewed to determine the mutagenic nature of 2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.2 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

In another in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using lymphocytes isolated from the heparinized peripheral blood samples of 12 male and 12 female adult human non-smoking volunteers both with and withoutS9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0, 0.1, 1.0 or 10 mM. About 0.5-1.0 x 10⁶isolated lymphocytes were cultured in RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. Concurrent solvent and positive control chemicals were also included in the study. All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor and tested for statistical significance by the chi-square test. The combined percentage structural aberration rate for males and females in the solvent (DMSO) control was 1.76. Lymphocyte cultures treated with 10mM test chemical alone (10mM), had a rate of 2.11. This difference was statistically insignificant. The presence of S-9 in the culture did not influence the aberration frequency. Cultures grown in the presence of MMC showed a several-fold increase in chromosomal aberration frequency, thus validating the experimental conditions used. Based on these considerations, the test chemical did not induce chromosomal aberrations in human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce chromosomal aberration in mammalian cell line used and hence is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using D. melanogaster strains. The study was performed using D. melanogaster as per the wing somatic mutation and recombination test (SMART). The multiple wing hair strain (mwh), with genetic constitution mwh e/mwh e and the flare (flr³) strain with genetic constitution y w^co/y w^co; flr³se/TM2 Ubx¹³⁰se e were used for the study. Larvae from the cross between flr³virgin females with mwh males were used for testing. The concentration selected for the study was slightly above the crucial dose of each individual compound that causes death to 50% of the tested larvae (LD50: 1.29). Eggs from the Drosophila cross mentioned above were collected during an 8 h period, and 72 (4 h later the larvae were removed from the food. Groups of 50 larvae, after washing with 17% NaCl solution, were transferred to individual Petri dishes (9 cm diameter) containing a Whatman 3 mm paper moistened with Ringer solution and exposed to various amounts of the examined compound. Different amounts of EOs or their components were applied to a small filter paper disk (4 mm). Dishes were kept at 24±1°C and 60% humidity for 18 h. After the exposure period, the larvae were washed with Ringer solution and transferred to new individual vials with food until emergence of adult flies. The trans-heterozygous (mwh/flr3) female flies that emerged from the cross were selected and stored in 70% ethanol-glycerol (1:1). Their wings were mounted in Euparal solution and scored at 400X magnification for the presence of mosaic spots. On the basis of the size, the number, and the type of cells showing malformed wing hairs, different categories of spots were recorded by following the methods and criteria of Graf et al. The test chemical is considered to be potent mutagenic but not recombinogenic inducer based on the observations made using D. melanogaster flr³X mwh crosses.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic and germ cell study: gene mutation

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:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using D. melanogaster strains

GLP compliance:

not specified

Type of assay:

other: Wing somatic mutation and recombination tests (SMART)

Species:

Drosophila melanogaster

Strain:

other: The multiple wing hair strain (mwh), with genetic constitution mwh e/mwh e and the flare (flr3) strain with genetic constitution y wco/y wco; flr3 se/TM2 Ubx130 se e. Larvae from the cross between flr3 virgin females with mwh males were used for testing

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

not specified

Vehicle:

Ringer solution

Details on exposure:

No data

Duration of treatment / exposure:

18 hrs

Frequency of treatment:

No data

Post exposure period:

No data

Remarks:

The concentration selected for the study was slightly above the crucial dose of each individual compound that causes death to 50% of the tested larvae (LD50: 1.29 menthone).

No. of animals per sex per dose:

No data

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Tissues and cell types examined:

Wings

Details of tissue and slide preparation:

Details of tissue and slide preparationCRITERIA FOR DOSE SELECTION: No dataTREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Eggs from the Drosophila cross mentioned above were collected during an 8 h period, and72 (4 h later the larvae were removed from the food. Groups of 50 larvae, after washing with 17% NaCl solution, were transferred to individual Petri dishes (9 cm diameter) containing a Whatman 3 mm paper moistened with Ringer solution and exposed to various amounts of the examined compound. Different amounts of EOs or their components were applied to a small filter paper disk (4 mm). Dishes were kept at 24 ± 1°C and 60% humidity for 18 h. After the exposure period, the larvae were washed with Ringer solution and transferred tonew individual vials with food until emergence of adult fliesDETAILS OF SLIDE PREPARATION: The trans-heterozygous (mwh/flr3) female flies that emerged from the cross were selected and stored in 70% ethanol-glycerol (1:1). Their wings were mounted in Euparal solution and scored at 400X magnification for the presence of mosaic spots. METHOD OF ANALYSIS: On the basis of the size, the number, and the type of cells showing malformed wing hairs, different categories of spots were recorded by following the methods and criteria of Graf et al.OTHER: No data

Evaluation criteria:

No data

Statistics:

For statistical analysis of the genotoxic effects of the tested compounds, the spots were grouped into four categories: (a) small single spots (with one or two affected cells, either mwh or flr3), (b) large single spots (with three or more affected cells, either mwh or flr3), (c) twin spots (consisting of both mwh and flr3 subclones), and (d) total single spots. For the statistical significance of the results, the multiple-decision procedure was used. The procedure is based on the conditional binomial test and the X2 test (K. Pearson’s criterion). Each statistical test was carried out at 5% significance level.

Sex:

female

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Table. Summary of Results Obtained in the Wing Somatic Mutation and Recombination Test (SMART) on D. melanogaster after Treatment with the Essential Oils (EOs) of Menthone

Treatment	Wings analyzed	Spots per wing (no. of spots) diagnosis
		small single spots (1-2 cells) m : 2.0	(large single spots (>2 cells) m : 5.0	twin spots m : 5.0	total spots m : 2
1.3µL	62	1.48 (92) +	0.13 (8) i	0.09 (6) -	1.71 (106) +
control (ringer)	74	0.74 (55)	0.06 (5)	0.08 (6)	0.89 (66)

Conclusions:

The test chemical is considered to be potent mutagenic but not recombinogenic inducer based on the observations made using D. melanogaster flr3 X mwh crosses.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using D. melanogaster strains. The study was performed using D. melanogaster. Themultiple wing hair strain (mwh), with genetic constitution mwh e/mwh e and the flare (flr³) strain with genetic constitution y w^co/y w^co; flr³se/TM2 Ubx¹³⁰se e were used for the study. Larvae from the cross between flr³virgin females with mwh males were used for testing. The concentration selected for the study was slightly above the crucial dose of each individual compound that causes death to 50% of the tested larvae (LD50: 1.29). Eggs from the Drosophila cross mentioned above were collected during an 8 h period, and 72 (4 h later the larvae were removed from the food. Groups of 50 larvae, after washing with 17% NaCl solution, were transferred to individual Petri dishes (9 cm diameter) containing a Whatman 3 mm paper moistened with Ringer solution and exposed to various amounts of the examined compound. Different amounts of EOs or their components were applied to a small filter paper disk (4 mm). Dishes were kept at 24±1°C and 60% humidity for 18 h. After the exposure period, the larvae were washed with Ringer solution and transferred to new individual vials with food until emergence of adult flies. The trans-heterozygous (mwh/flr3) female flies that emerged from the cross were selected and stored in 70% ethanol-glycerol (1:1). Their wings were mounted in Euparal solution and scored at 400X magnification for the presence of mosaic spots. On the basis of the size, the number, and the type of cells showing malformed wing hairs, different categories of spots were recorded by following the methods and criteria of Graf et al. The test chemical is considered to be potent mutagenic but not recombinogenic inducer based on the observations made using D. melanogaster flr³X mwh crosses.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Gene mutation in vitro:

Data available for the target chemical was reviewed to determine the mutagenic nature of the test chemical menthone (CAS no 89 -80 -5). The studies are as mentioned below:

Ames test:

Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/mammalian-microsome test was performed on Salmonella typhimurium strains TA1537, TA1535, TA100, TA 97 and TA98. The test material was exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The S9 mix contained the S9 fraction from Aroclor-1254- induced male Wistar rat liver in amounts which corresponded to 2 mg protein per plate. DMSO was used as solvent control. Positive controls were also used for all strain respectively. The test was performed by standard plate method. The toxicity of the substances was tested in the tester strains at 10^-7dilutions. On the basis of the results obtained, the following concentrations of the test substance were selected for testing: 800, 160, 32 or 6.4 µg per plate. Each dosage was tested on 5 parallel plates. The mean number of revertants for n plates at each dose level was calculated. No mutagenic effects were observed in all strain except strain TA1537. Contrary to these results, menthone induced an increased number of revertants in the strain TA1537 (dose levels of 6.4 and 32 µg per plate). But no effects were observed at the dose level of 160 or 800 µg/plate in strain TA1537 without S9. On the basis of obseravations made, the test chemical (CAS no 89-80-5) was considered to be non mutagenic in all strain at highest dose concentration and in the presence of S9 too in all strain. Hence the test material cannot be classified as gene mutant in vitro.

In another study, Genetic toxicity in vitro study for the test chemical was assessed for its possible mutagenic potential. For this purpose Salmonella/ mammalian-microsome test was performed on Salmonella typhimurium strains TA97. The test material was dissolved in DMSO and exposed to the bacterial strain at the concentration of 0, 6.4, 32, 160 or 800 µg/plate in the presence and absence of S9. The test was performed by standard plate method. No mutagenic effects were observed in the presence of S9. Positive mutagenic effects were observed in the absence of S9. The highly sensitive strain TA97 showed mutagenic effect in the absence of metabolic activation. While no mutagenic effect were observed when the same strain exposed to the test material in the presence of metabolic activation Therefore the test chemical (CAS no 89-80-5) was considered to be non mutagenic in strain TA 97. Hence the test material cannot be classified as a gene mutant in vitro.

Chromosome aberration:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.2 mg/mL being the maximum concentration for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence is not likely to classify as a gene mutant in vitro.

In another in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using lymphocytes isolated from the heparinized peripheral blood samples of 12 male and 12 female adult human non-smoking volunteers both with and withoutS9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose levels of 0, 0, 0.1, 1.0 or 10 mM. About 0.5-1.0 x 10⁶isolated lymphocytes were cultured in RPM1 1640 supplemented with 2 mM+glutamine (BDH), 100 U penicillin/ml, 100 pg streptomycin/ml, 10% foetal calf serum and 1% phytohaemagglutinin. Concurrent solvent and positive control chemicals were also included in the study. All cultures were incubated in the dark at 37°C for 72 hr. Following 1 hr of exposure of the cells to colchicine the slides were prepared. Chromosomal aberrations were scored in 100 metaphase cells from each donor and tested for statistical significance by the chi-square test. The combined percentage structural aberration rate for males and females in the solvent (DMSO) control was 1.76. Lymphocyte cultures treated with 10mM test chemical alone (10mM), had a rate of 2.11. This difference was statistically insignificant. The presence of S-9 in the culture did not influence the aberration frequency. Cultures grown in the presence of MMC showed a several-fold increase in chromosomal aberration frequency, thus validating the experimental conditions used. Based on these considerations, the test chemical did not induce chromosomal aberrations in human lymphocytes in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using D. melanogaster strains. The study was performed using D. melanogaster as per the wing somatic mutation and recombination test (SMART). The multiple wing hair strain (mwh), with genetic constitution mwh e/mwh e and the flare (flr³) strain with genetic constitution y w^co/y w^co; flr³se/TM2 Ubx¹³⁰se e were used for the study. Larvae from the cross between flr³virgin females with mwh males were used for testing. The concentration selected for the study was slightly above the crucial dose of each individual compound that causes death to 50% of the tested larvae (LD50: 1.29). Eggs from the Drosophila cross mentioned above were collected during an 8 h period, and 72 (4 h later the larvae were removed from the food. Groups of 50 larvae, after washing with 17% NaCl solution, were transferred to individual Petri dishes (9 cm diameter) containing a Whatman 3 mm paper moistened with Ringer solution and exposed to various amounts of the examined compound. Different amounts of EOs or their components were applied to a small filter paper disk (4 mm). Dishes were kept at 24±1°C and 60% humidity for 18 h. After the exposure period, the larvae were washed with Ringer solution and transferred to new individual vials with food until emergence of adult flies. The trans-heterozygous (mwh/flr3) female flies that emerged from the cross were selected and stored in 70% ethanol-glycerol (1:1). Their wings were mounted in Euparal solution and scored at 400X magnification for the presence of mosaic spots. On the basis of the size, the number, and the type of cells showing malformed wing hairs, different categories of spots were recorded by following the methods and criteria of Graf et al. The test chemical is considered to be potent mutagenic but not recombinogenic inducer based on the observations made using D. melanogaster flr³X mwh crosses.

However further testing is necessary to prove the mutagenic nature of the test chemical in vivo.

Based on the observations made, the test chemical Menthone (CAS no 89 -80 -5) did not induce gene mutation in bacterial strains and chromosomal aberration in mammalian cell line used iin the presence and absence of S9 metabolic activation system and and hence it is not likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the observations made, Menthone (CAS no 89 -80 -5) does not exhibit gene mutation in vitro. Hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. However, further testing is necessary to prove the mutagenic nature of the test chemical in vivo.