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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Cyclohexane has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown negative results for mutagenicity both in vitro and in vivo. It is concluded that the available data indicates that cyclohexane has no significant genotoxicity.
Link to relevant study records
Reference
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.
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
Target gene:
Thymidine kinase (TK) locus
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
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.
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

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

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.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In vitro data

The key studies are considered to be a bacterial mutation assay (Mortelmans et al, 1986) and a mammalian cell mutation assay (Litton Bionetics, 1982; HLA, 1982h).  These are two recognised core assay types for investigating mutation in vitro.

 

Cyclohexane was tested in a pre-incubation modification of a standard Ames test (Mortelmans et al., 1986). Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) was treated with cyclohexane both with and without metabolic activation. A range of doses was used up to 10,000 μg/plate where toxicity allowed.

 

Cyclohexane was tested in a mouse lymphoma mutation assay at doses ranging from 313 nL/mL to 10,000 nL/mL (250 μg/mL to 7,800 μg/mL) (Litton Bionetics, 1982) both with and without metabolic activation. Toxicity was observed under both sets of conditions, although this was not always dose-related. There was no evidence of forward mutation at any dose in the absence of added metabolic activation, but in its presence there were very slight increases in mutant frequency that were not confirmed in another test. Overall, this test can be considered as negative both with and without metabolic activation. 

 

Cyclohexane was not mutagenic in either test system.

 

There are additional reports of Salmonella and mouse lymphoma mutation data that support the above conclusion. There are also reports of the following further assay types on cyclohexane: sister chromatid exchange in mouse lymphoma cells, unscheduled DNA synthesis in human lymphocytes, a DNA cell binding assay in E coli.  The data from these assays all support the above conclusion that cyclohexane is not mutagenic in vitro (RAR, 2004).

 

In vivo data

The key study is considered to be a cytogenetic study in the rat (Litton Bionetics, 1982). This is a recognised core assay type for investigating mutation in vivo.

 

Cyclohexane was studied in a rodent bone marrow cytogenetic assay in Sprague Dawley rats exposed by inhalation to atmospheres of 0, 97, 307 and 1,042 ppm for 6 hours per day for 5 days (350-1,050–3,650 mg/m3).  Samples of bone marrow cells were taken for cytogenetic analysis 6 hours after completion of the final dose. No significant increases in chromosomal aberrations were found. A small increase in numerical aberrations was recorded in low and medium dose females, however the authors of the report concluded that increases were not of biological importance. Cyclohexane was not mutagenic in this assay. 

 

There is also a further report of a Drosophila sex linked recessive lethal assay that gave a negative result (RAR, 2004).

 

Human information

There is no information indicating any adverse effects of cyclohexane.

 



Justification for selection of genetic toxicity endpoint
Available data indicate that the cyclohexane has no significant genotoxic activity in bacterial and mammalian systems in vitro, or in an in vivo chromosomal aberration assay.

Justification for classification or non-classification

It has shown negative results for mutagenicity both in vitro and in vivo. It is concluded that the available data indicates that no classification under CLP is warranted.