N-cyclohexylbenzothiazole-2-sulfenamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

2-AA was used as the sole indicator of the efficicacy of the S9-mix

Principles of method if other than guideline:

N-cyclohexylbenzothiazole-2-sulphenamide was tested in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclohexylbenzothiazole-2-sulfenamide

EC Number:

202-411-2

EC Name:

N-cyclohexylbenzothiazole-2-sulfenamide

Cas Number:

95-33-0

Molecular formula:

C13H16N2S2

IUPAC Name:

N-(1,3-benzothiazol-2-ylsulfanyl)cyclohexanamine

Test material form:

solid: granular

Remarks:

greyish white

Details on test material:

Purity: 98.8 %

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not reported
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no stability issue reported
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no reactivity reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): diluted in DMSO

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Kikkoman Corporation, lot number RAA-333, manufactured on 8 September 1995) prepared by enzymatic induction in 7-week-old male Sprague-Dawley rats co-administered phenobarbital (PB) and 5,6-benzoflavone (BF).
- method of preparation of S9 mix: for 1 mL:
S9 0.1 mL
NADH 4 μmol
Magnesium chloride 8 μmol
NADPH 4 μmol
Potassium chloride 33 μmol
Sodium-phosphate buffer (pH 7.4). 100μmol
Glucose-6-phosphate 5 μmol
- concentration or volume of S9 mix and S9 in the final culture medium: not reported
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

Preliminary cytotoxicity test (+/-S9): 0, 50, 150, 500, 1500 and 5000 µg/plate.

Reverse mutation test 1:
TA100, TA1535, TA98 & E.coli WP2 (+/-S9): 0, 156, 313, 625, 1250, 2500 and 5000 µg/plate. Up to limit concentration.
TA 1537 (-S9): 0, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/plate. Up to cytotoxic concentration.
TA 1537 (+S9): 0, 15.6, 31.3, 62;5, 125, 250, 500, 1000 and 2000 µg/plate. Up to cytotoxic concentration.

Reverse mutation test 2:
TA100, TA1535, TA98 & E.coli WP2 (+/-S9): 0, 156, 313, 625, 1250, 2500 and 5000 µg/plate. Up to limit concentration.
TA 1537 (-S9): 0, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/plate. Up to cytotoxic concentration.
TA 1537 (+S9): 0, 15.6, 31.3, 62;5, 125, 250, 500, 1000 and 2000 µg/plate. Up to cytotoxic concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acylamide 

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1 range-finder + 2 main experiments

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium (pre-incubation method)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

The test substance was deemed to be mutagenic (positive) in this test system if 1 or more of the 5 test bacteria strains, either or without S9 mix, had an average number of mutant colonies on test substance plates that was twice or more the number of the solvent control, and the increase was reproducible or dose-dependent. However, it was considered a negative result if the lower number compared to the solvent control value did not demonstrate a dose-dependent increase in the number of mutant colonies in the event only 1 of the 2 main tests at a specific dose was observed with average number of colonies at more than double the solvent control value.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 100 µg/plate without S9; from 500 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 µg/plate with S9. Tested up to recommended limit concentration without S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: None reported

RANGE-FINDING/SCREENING STUDIES:
The results were that in tests without S9 mix, antibacterial activity was observed on TA1537 from 150 μg/plate. In tests with S9 mix antibacterial activity was observed from 500 μg/plate or more for TA1537 and from 1500μg/plate or more for TA100.
Therefore, the highest dose in this study was set at 5000 μg/plate for testing both with and without S9 mix (testing TA100 with S9 mix at 2000 μg/plate, testing TA1537 without S9 mix at 400 μg/plate, and with S9 mix at 1000 μg/plate)


STUDY RESULTS
The results showed no increase in the number of mutant colonies that was more than twice the solvent control value in any strain of the bacteria tested.
- Concurrent vehicle negative and positive control data: an increase in the number of mutant colonies was observed in all test bacteria in the positive control group, and the number of mutant colonies measured with the solvent control group was within the range of historical control values, confirming the effectiveness of this test system.
- Signs of toxicity: cf. result tables
- Individual plate counts: cf result tables
- Mean number of revertant colonies per plate and standard deviation: cf. result tables

HISTORICAL CONTROL DATA
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: not reported

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

In a reverse gene mutation assay in bacteria conducted according to OECD TG 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA were exposed to the test item diluted in DMSO at a concentration up to 5000 µg/plate in the presence and absence of mammalian metabolic activation under the liquid pre-incubation assay.

The positive controls induced the appropriate response in the corresponding strains.

Toxicity effects varied in a strain-specific manner; at high doses from 200 µg/plate upward precipitations were observed.

Under the test conditions, there was no evidence of induced mutant colonies over background. (MHWJ 1997).