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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:
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:
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

Reference
Name:
Unnamed
Type:
Constituent
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
Controlsopen allclose all
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
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.