(+)-neomenthol

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Andersen and Jensen

Year:

1984

Bibliographic source:

Mutation Research

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As mention below

Principles of method if other than guideline:

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

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

trans-menthone

EC Number:

201-941-1

EC Name:

trans-menthone

Cas Number:

89-80-5

Molecular formula:

C10H18O

IUPAC Name:

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

Details on test material:

- Name of test material (IUPAC name): (2R,5S)-5-methyl-2-(propan-2-yl)cyclohexan-1-one- Common name: Menthone - Molecular formula: C10H18O- Molecular weight: 154.2512 g/mol- Smiles notation: C1([C@@H](CC[C@@H](C1)C)C(C)C)=O- InChl: 1S/C10H18O/c1-7(2)9-5-4-8(3)6-10(9)11/h7-9H,4-6H2,1-3H3- Substance type: Organic- Physical state: Liquid

Method

Target gene:

Histidine

Species / strain

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

Controls

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

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

The test chemical 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, test chemical 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 observations made, the test chemical 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.