Cyclohexanemethanol, 4-(1-methylethyl)-

Genetic toxicity: in vitro

Administrative data

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study in compliance with guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Acceptable to the Japanese New Chemical Substance Law (METI)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

[(1r,4r)-4-(propan-2-yl)cyclohexyl]methanol; [(1s,4s)-4-(propan-2-yl)cyclohexyl]methanol

EC Number:

939-719-8

Cas Number:

5502-75-0

Molecular formula:

C10H20O

IUPAC Name:

[(1r,4r)-4-(propan-2-yl)cyclohexyl]methanol; [(1s,4s)-4-(propan-2-yl)cyclohexyl]methanol

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): Mayol [Description: clear colourless liquid. Chemical name: Reaction mass of Cis-4-(isopropyl)cyclohexanemethanol and Trans-4-(isopropyl)cyclohexanemethanol]
- Physical state: liquid
- Analytical purity: 99.8% (Sum of 2 isomers)
- Lot/batch No.: 1000877823
- Expiration date of the lot/batch: 16 August 2013
- Storage condition of test material: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at 37ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction prepared from livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

EXPERIMENT 1:
without S9 (μg/ml): 0, 12.5, 25, 50, 100, 150 and 200
with S9 (2%) (μg/ml): 0, 12.5, 25, 50, 100, 150 and 200
EXPERIMENT 2:
without S9 (μg/ml): 0, 3.13, 6.25, 12.5, 25, 50 and 100
with S9 (1%) (μg/ml): 0, 12.5, 25, 50, 100, 150 and 200

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DSMO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: cyclophosphamide (+S9)

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hours (± S9) in Experiment 1, 4 hours (+S9) and 24 hours (-S9) in Experiment 2
- Expression time (cells in growth medium): 20 hours (± S9) in Experiment 1, 20 hours (+S9) in Experiment 2

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 consecutive well-spread metaphases from each culture were counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, i.e., 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).

Evaluation criteria:

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 were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: >100 μg/ml (-S9) and >150 μg/ml (+S9); Experiment 2: >50 μg/ml (-S9) and >100 μg/ml (+S9).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item showed marked evidence of toxicity in all three exposure groups. Haemolysis was seen in the blood cultures at the end of the exposure period at and above 24.42 μg/ml in the 4(20)-hour exposure group in the absence of S9, and at and above 48.84 μg/ml in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group.

Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 97.69 μg/ml in the 4(20)-hour treatment in the absence of metabolic activation (S9) and in the 24-hour continuous exposure group in the absence of S9. The maximum dose with metaphases present in the 4(20)-hour exposure in the presence of S9 was 195.38 μg/ml.

EXPERIMENT 1:
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test item dose level of 150 μg/ml in both the presence and absence of metabolic activation (S9). The toxicity curve was relatively sharp with no observed metaphases present at the higher dose level of 200 μg/ml in either exposure group. The ‘A’ replicate of the 150 μg/ml dose level in the presence of S9 was lost due to a technical error during the harvesting process and therefore extra scoring was performed on the ‘B’ culture to compensate. This is acceptable under the test guidelines.

The results of the mitotic indices (MI) from the cultures after their respective treatments show 69% and 31% growth inhibition was achieved at 150 μg/ml μg/ml in the absence and presence of S9 respectively.

No precipitate of the test item was observed at the end of the treatment period in either exposure group. Haemolysis was observed at the end of the exposure period in both exposure groups at and above 50 μg/ml.

Thus, the selection of maximum dose level selected for metaphase analysis was 150 μg/ml for both exposure groups and was the highest dose level with metaphases suitable for scoring in both exposure groups.

EXPERIMENT 2:
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test item dose level of 100 μg/ml in both the presence and absence of S9.

The results of the mitotic indices (MI) from the cultures after their respective treatments show a dose related increase in toxicity in the 24-hour exposure group and 36% and 65% mitotic inhibition was achieved at 50 and 100 μg/ml respectively. In the presence of S9 the toxicity curve was very sharp with no toxicity being demonstrated up to the test item dose level of 100 μg/ml and no observed metaphases present at 150 μg/ml. The increase in toxicity in the 4(20)-hour exposure group in the presence of S9 when compared to the same Experiment 1 exposure group is considered to be due to the reduction in the S9 concentration and its protective effects.

No precipitate of the test item was observed at the end of the treatment period in either exposure group. Haemolysis was seen at the end of exposure at and above 50 μg/ml in both exposure groups.

Thus, the maximum dose level selected for metaphase analysis was the highest dose level with metaphases suitable for scoring and was 100 μg/ml for both exposure groups. Although optimum toxicity was not achieved in the 4(20)-hour exposure group in the presence of S9, due to the relative steepness of the toxicity curve, the test item was considered to have been adequately tested.

Any other information on results incl. tables

Experiment 1

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to the test item dose level of 150 µg/ml in both the presence and absence of metabolic activation (S9). 

The results of the mitotic indices (MI) from the cultures after their respective treatments show 69% and 31% growth inhibition was achieved at 150 µg/ml in the absence and presence of S9, respectively. 

Thus, the selection of maximum dose level selected for metaphase analysis was 150 µg/ml for both exposure groups and was the highest dose level with metaphases suitable for scoring in both exposure groups. 

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

 

Experiment 2

The results of the mitotic indices (MI) from the cultures after their respective treatments show a dose related increase in toxicity in the 24-hour exposure group and 36% and 65% mitotic inhibition was achieved at 50 and 100 µg/ml respectively. 

Thus, the maximum dose level selected for metaphase analysis was the highest dose level with metaphases suitable for scoring and was 100 µg/ml for both exposure groups. 

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.

The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. Therefore the substance was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The chromosome aberration test for the substance was conducted according to OECD Guideline No. 473 and under GLP conditions. The test substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments at test concentrations of either 100 or 200 µg/mL, depending on the test. Consequently, the substance was considered to be non-clastogenic to human lymphocytes in vitro.