Cyclohexane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 5 - July 31 1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, conducted to follow regulatory guidelines of the day and main current guideline points, fully adequate for assessment.

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

1982

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Principles of method if other than guideline:

The procedure used was based on that reported by Clive and Spector (1975). L5178Y cells were exposed to the test chemical for 4 h in the presence and absence of rat S9 fraction and expression of the induced TK-/- phenotype determined

GLP compliance:

yes

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

Molecular formula:

C6H12

IUPAC Name:

cyclohexane

Details on test material:

Certified cyclohexane.
Clear colourless liquid
Lot 701739 Class 1B
99 Mol % pure

Method

Target gene:

Thymidine kinase (TK) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The cells were maintained in Fischer's mouse leukaemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10% by volume).
Periodically checked for Mycoplasma contamination.
Periodically checked for karyotype stability (by exposure to methotrexate).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 (Aroclor 1254-induced Fischer 344 male rat liver)

Test concentrations with justification for top dose:

313, 625, 1250, 2500, 3000, 4000, 5000, 6000, 7000 and 8000 nL/mL

Vehicle / solvent:

Deionised water

Controls

Untreated negative controls:

other: Untreated control

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

Positive controls:

yes

Remarks:

N-dimethylnitrosamine in the presence of S9 fraction

Remarks:

Ethylmethane sulfonate (EMS) for nonactivation tests

Details on test system and experimental conditions:

Cultures were exposed to the test chemical for four hours at the preselected doses, in the presence and absence of rat liver S9 metabolic activation. They were washed and placed in growth medium for two or three days to allow recovery, growth and expression of the induced TK-/- phenotype. Cell counts were determined daily and appropriate dilutions made to allow optimal growth rates. At the end of the expression period, 3 x 10E6 cells for each selected dose were seeded in soft agar plates with selection medium and resistant (mutant) colonies are counted after 10 days incubation. To determine the actual number of cells capable of forming colonies, a portion of the cell suspension is also cloned in normal medium (nonselective).

Evaluation criteria:

The test substance was evaluated for its ability to induce mutation in the L5178Y TK+/- mouse lymphoma cell line, as assessed by colony growth in the presence of 5-bromo-2’-deoxyuridine (BrdU) or 5-trifluorothymidine (TFT).
The ratio of resistant colonies to total viable cell number was the mutant frequency.

Statistics:

The mutant frequency was calculated as the ratio of mutant colonies to viable colonies times 10-4.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A separate, preliminary cytotoxicity test was not done. Dose selection was an integral part of the mutation assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Unactivated	Mutant frequency	% relative growth	Activated	Mutant frequency	% relative growth	Activated	Mutant frequency	% relative growth
Solvent control	13.2	100.0	Solvent control	12.6	100.0	Solvent control	17.1	100.0
Solvent control	22.4	100.0	Solvent control	16.8	100.0	Solvent control	21.7	100.0
Untreated control	12.1	136.8	Untreated control	15.9	91.3	Untreated control	23.6	85.3
EMS (0.5 uL/mL)	672.8	11.3	DMN (0.3 uL/mL)	518.4	4.7	DMN (0.3 uL/mL)	222.4	7.1
Cyclohexane (nL/mL)			Cyclohexane (nL/mL)			Cyclohexane (nL/mL)
313	16.4	40.9	313	34.1	60.2	3000	25.2	69.5
625	14.8	31.0	625	36.1	46.4	4000	20.2	56.0
1250	24.7	41.7	1250	29.1	64.5	5000	26.2	23.0
2500	21.5	52.1	2500	36.6	62.1	6000	37.2	30.4
5000	26.7	59.0	5000	40.2	51.6	7000	39.6	27.8
						8000	40.2	26.6

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Cyclohexane did not induce repeatable increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells.

Executive summary:

Cyclohexane did not induce repeatable increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells.

Without activation, concentrations up to 5000 nL/mL were moderately toxic without being detectably mutagenic, and 10000 nL/mL was completely lethal.

With activation, treatments up to 8000 nL/mL were moderately toxic and did not consistently induce significant increases in the background mutant frequency; 9000 – 10000 nL/mL was lethal.