2-isopropyl-5-methyl-cyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (Klimisch 1) HPRT test: negative (Klimisch 1) MNT test: negative (Klimisch 1) Supporting study Ames: positive (Klimisch 3)

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Gl study

Qualifier:

according to guideline

Guideline:

other: OECD 487

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

primary culture, other: human blood lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

see attached document: Test concentrations MNT test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 40h
- Exposure duration: 4h ( with and without S9 mix) or 20 h ( with S9 mix) (for more details see attached document: Test concentrations MNT test)
- Expression time (cells in growth medium): 16h after 4h exposure, none after 20h exposure
- Selection time (if incubation with a selection agent): 20 h with Cytochalasin B
- Fixation time (start of exposure up to fixation or harvest of cells): harvest 40h after start exposure time


SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4ug/ml)
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2 parallel cultures

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: % cytostasis

OTHER EXAMINATIONS:
- Determination of cytogenetic damage

Evaluation criteria:

A test item can be classified as non-mutagenic - non-clasogenic and non-aneugenic if:
 the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
 no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic clastogenic or aneugenic if:
 the number of micronucleated cells is not in the range of the historical laboratory control data and
 either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Species / strain:

primary culture, other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility:
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Phase separation was observed microscopically in Experiment I in the absence and presence of S9 mix and in Experiment IIA in the absence of S9 mix at 771.5 µg/mL and above at the end of treatment. In Experiment IIB in the presence of S9 mix phase separation was observed microscopically at 600.0 µg/mL and above at the end of treatment.

Remarks on result:

other: other: primary culture

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item 620103 Menthone / Iso Menthone Racemic did not induce micronuclei in human lymphocytes in vitro when tested up to cytotoxic or the highest evaluable concentrations.

Executive summary:

The test item 620103 Menthone / Iso Menthone Racemic, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Three independent experiments were performed.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 1543.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

In Experiment I and IIB in the absence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration (74.2 and 60.5 % cytostasis). In Experiment I and IIB in the presence of S9 mix and in Experiment IIA in the absence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. However, in Experiment IIB cytostasis was 44.1 % at the highest evaluable concentration.

In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.10 – 1.00 % micronucleated cells) were slightly above the range of the solvent control values (0.15 – 0.50 % micronucleated cells) and within the range of the laboratory historical control data. In Experiment I in the absence of S9 mix one single statistically significant increase in the number of micronucleated cells (1.00 %) was observed at the highest evaluated concentration (1543.0 µg/mL). Since this value was clearly in the range of the laboratory historical solvent control data (0.15 – 1.40 % micronucleated cells), this observation is regarded as biologically irrelevant.

Either Demecolcin (125.0 or 150.0 ng/mL), MMC (2.0 µg/mL) or CPA (12.5 or 17.5 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three high quality (K1) genetic toxicity in vitro assays were conducted with the substance covering different modes of action of genetic toxicity. The substance did not produce significant genetic toxicity in a reliable bacterial reverse mutation assay with S. typhimurium and E. coli, in a in vitro micronucleus assay with human lymphocytes and in an in vitro Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT). In a supporting study (Ames test) the test substance showed signs of mutagenic potential. However, since this study is of low quality (K3) it is not considered to be relevant for the overall assessment.

It is therefore concluded that the substance is not genotoxic. Further testing on the genetic toxicity of reaction mass of isomenthone and trans-menthone is not required.

Justification for selection of genetic toxicity endpoint
GLP and guideline study performed in mammalian cells to detect the activity of clastogenic and aneugenic chemicals.

Justification for classification or non-classification

Based on the above stated assessments on genotoxicity the test substance is not considered to be genotoxic and does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.