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Toxicological information

Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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.