Methylcyclohexane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. Refer to endpoint summary Genetic toxicity and IUCLID Section 13 for reporting and justification of the analogue approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane

EC Number:

203-806-2

EC Name:

Cyclohexane

Cas Number:

110-82-7

IUPAC Name:

cyclohexane

Details on test material:

- Name of test material (as cited in study report): Cyclohexane
- Molecular formula: C6H12
- Molecular weight: 84.16
- Smiles notation: C1CCCCC1
- InChl: 1S/C6H12/c1-2-4-6-5-3-1/h1-6H2
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: assumed 100%
- Solubility: 50 mg/mL in dimethylsulfoxide

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Fischer’s medium F10p
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes, by treatment with THMG prior to use in experiments

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

8, 12, 17, 24, 34, 50, 70 and 100 µg/mL with and without metabolic activation

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

620 µg/mL without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

1 µg/mL with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time: 12 ± 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 ± 2 days

SELECTION AGENT: 2 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: triplicates

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

Evaluation criteria:

A test substance was considered positive if a dose-related response was obtained in which the mutation frequencies at two or more test concentrations (in the absence of severe toxicity) were at least two to three-fold higher than the mutation frequency of the solvent control.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 100 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: At 8, 24 and 70 µg/mL, the test substance induced a 2.1-, 2.5 and 2.0-fold increase in mutant frequency, respectively. However, there was no evidence for a dose-related response. Therefore, the criteria for a positive result were not met.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In a preliminary toxicity test, cells treated with 100 µg/mL of the test substance exhibited 65% growth inhibition in the presence of metabolic activation. Therefore, 100 µg/mL was the maximum concentration selected for the mutagenicity test. Cytotoxicity was confirmed in the mutagenicity test, in which 63.6% growth inhibition was observed in the presence of S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results of the Mouse Lymphoma Forward Mutation Assay.

Test item	Concentration (µg/mL)	Total survival (%)	Mutation frequency per 1E5 cells	Fold increase
Without S9 mix
Medium	-	70.0	2.7	0.7
DMSO	1 µl/mL	100.0	3.7	1.0
Test substance	8	107.3	3.3	0.9
	12	142.4	1.8	0.5
	17	94.4	2.6	0.7
	24	107.3	2	0.5
	34	167.5	3.1	0.8
	50	93.4	3.8	1.0
	70	162.0	3.6	1.0
	100	142.9	2.9	0.8
EMS	620	43.8	56.7	15.3
With S9 mix
Medium	-	TE	TE	TE
DMSO	1 µl/mL	100.0	2.8	1.0
Test substance	8	57.6	5.8	2.1
	12	119.9	4.5	1.6
	17	31.3	3.6	1.3
	24	105.6	6.9	2.5
	34	108.0	2.8	1.0
	50	75.2	4.7	1.7
	70	87.5	5.6	2.0
	100	36.4	3.8	1.4
MCA	1	96.0	7.1	2.5

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Cyclohexane was tested for its ability to induce forward mutations in a Mouse Lymphoma Assay conducted following a protocol equivalent to OECD guideline 476. Mouse lymphoma L5178Y cells were incubated with the test material at 8, 12, 17, 24, 34, 50, 70 and 100 µg/mL for 4 h in the presence and absence of a metabolic activation system (S9 mix). The maximum concentration of 100 µg/mL was selected on a preliminary toxicity test (65% growth inhibition in the presence of metabolic activation). Untreated, solvent (DMSO) and positive controls (ethylmethanesulphonate, -S9 mix; 3-methylcolanthrene, +S9 mix) were included in the test.
No cytotoxicity and no increase in mutant frequency was observed at any concentration without metabolic activation. In the presence of S9 mix, cytotoxic effects were observed at the highest concentration and a 2- to 2.5-fold increase in mutant frequency was noted at 8, 24 and 70 µg/mL. However, the increases in forward mutations were not dose-related and therefore the criteria for a positive result were not met. Therefore, the test was considered negative in the limits of the range tested.