Cyclohexanethiol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexanethiol

EC Number:

216-378-7

EC Name:

Cyclohexanethiol

Cas Number:

1569-69-3

Molecular formula:

C6H12S

IUPAC Name:

cyclohexanethiol

Test material form:

other: liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Expt 1: 3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate; Expt 2: 3, 10, 33, 100, 333, 1000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: none given in study report.
- Since the test substance is reactive to oxygen, the test substance was flushed with nitrogen after weighing, and the test substance concentrations were prepared within 0.5 hour and used in the experiment within 1.5 hours.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 (5 μg) without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA1537 (15 μg) and T98 (10 μg) without metabolic activation

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 100 (650 μg) without metabolic activation

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

WP2uvrA (10 μg) without metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine and tryptophan deficient agar.

NUMBER OF REPLICATIONS: Each concentration was tested in triplicate and the experiment was repeated. The dose range-finding experiment was reported as a part of the first experiment of the mutation assay.

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn; number and size of revertant colonies

OTHER:
METABOLIC ACTIVATION
Before use, all S9 batches were characterised with the mutagens Benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA98 at concentrations of 5 μg/plate and 1 μg/plate, respectively.
S9 mix contained 5% (v/v) S9-fraction), and the following per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml or 5.0 ml Milli-Q water (first or second experiment respectively); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution.

Evaluation criteria:

A substance is considered positive if a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control; b) The increase in the number of revertants is reproducible.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

333 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation observed

RANGE-FINDING/SCREENING STUDIES: results reported as experiment 1

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Background lawn condition and existence of microcolonies were observed at 333 and 1000 μg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2 Experiment 1 (pre-incubation). Mean number of revertant colonies/3 replicate plates (± S.D.)

Dose (μg/plate)	TA1535	TA1537	TA98	TA100	WP2uvrA
Without metabolic activation
positive control	721 ± 65	608 ± 87	826 ± 14	927 ± 19	498 ± 27
solvent control	10 ± 4	4 ± 1	20 ± 1	103 ± 2	34 ± 11
3	7 ± 2	4 ± 2	12 ± 1	109 ± 12	51 ± 25
10	5 ± 2	1 ± 1	19 ± 6	108 ± 14	35 ± 4
33	10 ± 4	3 ± 1	19 ± 3	97 ± 13	49 ± 12
100	7 ± 4	3 ± 1	8 ± 1	117 ± 8	42 ± 4
333	4 ± 2 m	3 ± 1	5 ± 7 m	101	32 ± 0
1000	MC e	MC e	2 ± 3 e	MC e	MC e
3330	-	-	-	MC e	MC e
With metabolic activation^1
positive control	310 ± 14	373 ± 30	845 ± 56	1041 ± 38	362 ± 14
solvent control	4 ± 0	2 ± 2	52 ± 8	101 ± 8	36 ± 5
3	8 ± 4	4 ± 0	41 ± 5	99 ± 8	37 ± 6
10	7 ± 3	4 ± 0	39 ± 9	96 ± 2	44 ± 8
33	8 ± 2	4 ± 2	44 ± 3	99 ± 11	41 ± 5
100	8 ± 3	3 ± 1	44 ± 4	111 ± 3	28 ± 5
333	5 ± 1	4 ± 1	MC e	84 ± 8	41 ± 8
1000	MC e	MC e	MC e	MC e	MC e
3330	-	-	-	MC e	MC e
5000	-	-	-	MC e	MC e

 

Table 3 Experiment 2 (pre-incubation). Mean number of revertant colonies/3 replicate plates (± S.D.)

Dose (μg/plate)	TA1535	TA1537	TA98	TA100	WP2uvrA
Without metabolic activation
positive control	775 ± 12	214 ± 25	826 ± 14	752 ± 87	697 ± 41
solvent control*	6 ± 3	7 ± 3	15 ± 1	136 ± 6	26 ± 3
3	13 ± 5	5 ± 3	16 ± 2	122 ± 16	27 ± 3
10	9 ± 3	3 ± 3	14 ± 4	125 ± 16	28 ± 3
33	11 ± 1	4 ± 2	11 ± 2	123 ± 7	30 ± 3
100	7 ± 2	3 ± 0	14 ± 5	115 ± 18	24 ± 7
333	5 ± 2 m	2 ± 3 s	10 ± 2 m	71 ± 12 m	23 ± 3
1000	12 ± 3 m	2 ± 1 e	MC e	MC e	7 ± 4 m
With metabolic activation^2
positive control	191 ± 20	268 ± 55	272 ± 17	631 ± 121	146 ± 24
solvent control*	5 ± 1	3 ± 1	20 ± 4	75 ± 20	32 ± 3
3	9 ± 4	3 ± 1	14 ± 3	109 ± 2	36 ± 3
10	9 ± 1	3 ± 2	19 ± 5	97 ± 7	34 ± 5
33	10 ± 3	4 ± 2	20 ± 8	95 ± 17	33 ± 5
100	8 ± 1	2 ± 0	19 ± 5	92 ± 11	36 ± 2
333	10 ± 3 s	4 ± 1	22 ± 5	81 ± 19 s	32 ± 9
1000	MC ± m	MC e	MC e	MC e	MC m

Solvent control: 0.1 ml dimethyl sulfoxide

1 The S9-mix contained 5% (v/v) S9 fraction

2 The S9-mix contained 10% (v/v) S9 fraction

m Bacterial background lawn moderately reduced

e Bacterial background lawn extremely reduced

MC Microcolonies

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Cyclohexylmercaptan has been tested for mutagenicity in a reliable study conducted according to OECD 471 and in compliance with GLP. No increase in the number of revertant colonies was observed when the substance was tested up to cytotoxic concentrations in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537 and Escherichia coli WP2 uvrA, in the presence and absence of metabolic activation. The study was conducted using the pre-incubation method, and was repeated to confirm the negative result. Appropriate positive and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.