(+)-menthol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Duplicates not systematically performed. Limited documentation

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Chromosomal aberrations and sister chromatid exchange tests in Chinese Ovary Cells in Vitro. IV. Results with 15 chemicals

Author:

JL Ivett, BM Brown, C Rodgers, BE Anderson, MA Resnick and E Zeiger

Year:

1989

Bibliographic source:

Environmental and molecular mutagenesis 14:165-187

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

yes

Remarks:

No systematic duplicates, limited documentation

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

D-menthol

EC Number:

239-388-3

EC Name:

D-menthol

Cas Number:

15356-70-4

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Details on test material:

No further details provided

Method

Target gene:

None

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

No other details provided

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver fraction S9 Aroclor 1254 induced

Test concentrations with justification for top dose:

SISTER CHROMATID EXCHANGE:
First trial without S9 :
0; 5; 16.7 and 50 μg/ml
Second trial without S9:
0; 2.5; 5; 10 and 25 μg/ml
First trial with S9:
0; 16.7, 50 and 167 μg/ml

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C (without metabolic activation trials) and cyclophosphamide (with metabolic activation trials)

Details on test system and experimental conditions:

METABOLIC ACTIVATION SYSTEM
Chemicals were tested in each assay with and without exogenous metabolic activation. The activation mixture consisted of 15μl/ml S9, 1.5 mg/mI NADP, and 2.7 mg/ml isocitric acid in serum-free med ium. S9 was obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats.

DOSE SELECTION
Chemicals were tested up to 5 mg/mI or as limited by solubilily and/or toxicity. Solubility tests were conducted to determine dose range and choice of solvent. The pH of the test chemical solution diluted in the test culture media was measured and was found to be in the range of 7.0-7.5 for all chemicals. In the initial SCE assay, ten doses were tested at half-log increments. The highest three doses that contained sufficient M2 cells were analyzed for SCE. When a positive response was detected at any of the dose levels, a confirmatory trial was required; this generally included doses within a range of about one-half log above and below the positive response.

CONTROLS
Solvent and positive controls were run concurrently with each trial. The solvent conlrol included the same concentration of the solvent as did the test doses. Mitomyciim C (MMC) was used in trials without metabolic activation, and cyclophosphamide (CP) was used with activation, for the positive controls.
In the SCE assay two concentrations of the positive control were tested. The high dose was scored uncoded, and only five cells were examined. The lower dose was scored coded and was used as a control for the sensitivity of the assay at low levels (20—50%) of SCE induction.

SCE ASSAY
In the SCE trial without metabolic activation, cells were exposed to the test chemical for approximately 25 hr; for the trials with metabolic activation the exposure was for 2 hr. For both testing conditions, 10 μM bromodeoxyuridine (BrdUrd) was added 2 hr after dosing. The cells were continuously exposed to BrdUrd up until the time of harvest, with 0.1 μg/ml Colcemid present for the last 2-2.5 hr of incubation. Under standard conditions the total incubation time was 27,5—28 hr. In the cultures without activation the cells were washed to remove the test chemical prior to Colcemid addition (25 hr). In the cultures with metabolic activation the cells were washed to remove the test chemical and the metabolic activation components 2 hr after the initial exposure.
At the time of harvest, visual observations of culture viability were made by estimating the relative monolayer confluence and the availability of mitotic cells in the monolayer. Mitotic cells were obtained by briskly shaking the flask and decanting and centrifuging the cell suspension. The supernatant medium from the cells was returned to the appropriate flasks and the flasks were reincubated to allow for a later harvest if necessaty. The mitotic cells were treated with 0.075 M KCI and fixed in 3:1 methanol:glacial acetic acid. In order to evaluate cell cycle kinetics, slides from the highest doses were stained with Hoechst 3325 (0.5 μg/ml) and examined by fluorescence microscopy. For scoring SCE, slices were coded and stained according to the methods described by Galloway et at, [1985; 1987a]. Fifty cells were scored per dose in the initial trial, and, generally 25 were scored in the repeat trials. In those instances where the number of SCE/cell analyzed for a particular dose consistently exceeded the mean value of the high-dose positive control, only five to ten cells were scored.
If chemical treatment resulted in a delay of the cell cycle progression, as determined by an insufficient number or lack of second-generation mitotic cells (M2), a later harvest was done in an attempt to collect a sufficient number of M2 cells for scoring. In these cases the reincuhated cells were harvested after an additional 6-8 hr incubation. Colcemid and BrdUrd were present throughout the additional incubation period.

Evaluation criteria:

In the SCE assay an increase of 20% or greater increase in SCE per chromosome over the solvent control was considered significant.
Trials with two or more significant doses were considered positive (+), and trials with one significant dose and a significant trend were judged as having weak evidence of a Positive response (+ W).
Trials with a significant response at one dose and no significant trend, and trials with no significant responses but having a significant trend were considered equivocal (?)

Statistics:

SISTER CHROMATID EXCHANGE: none

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

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:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

SCE with and without metabolic activation was found to be negative according to evaluaton criteria.