N-tert-butylbenzothiazole-2-sulphenamide

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

Remarks:

GLP and guideline study, basic data given (abstract and tables)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 1987

Deviations:

not specified

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Principles of method if other than guideline:

N-tert-butyl-2-benzothiazolesulfenamide 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-tert-butylbenzothiazole-2-sulphenamide

EC Number:

202-409-1

EC Name:

N-tert-butylbenzothiazole-2-sulphenamide

Cas Number:

95-31-8

Molecular formula:

C11H14N2S2

IUPAC Name:

N-(1,3-benzothiazol-2-ylsulfanyl)-2-methylpropan-2-amine

Details on test material:

purity: 96 %

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was sealed and refrigerated until it was being used.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Ouchi Shinko Chemicals Ltd. conducted a chemical analysis of the test substance after testing was completed, and found that the purity was 95.4%.
- 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 / cell type:

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

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). PB and BF doses were PB 30 mg/kg on Day 1, PB 60 mg/kg on Day 2, 60 mg/kg plus BF 80 mg/kg on Day 3, and PB 60 mg/kg on Day 4. All was dosed via intraperitoneal administration. Rat dissection and preparation of S9 was performed on Day 5.
- 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 (-S9): tested up to cytotoxicity limit:
TA100: 0, 39.1, 78.1, 156, 313, 625, 1250 & 2500 µg/plate;
TA 1535: 0, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate;
TA 98: 0, 15.6, 31.3, 62.5, 125, 250, and 500 µg/plate;
TA 1537: 0, 1.56, 3.13, 6.25, 12.5, 25, 50 & 100 µg/plate;
WP2 uvrA: 0, 313, 625, 1250, 2500 and 5000 µg/plate;


Reverse mutation test 1 (+S9):
TA100 / TA 1535 / TA 98 & WP2 uvrA: 0, 313, 625, 1250, 2500 & 5000 µg/plate (cytotoxicity limit);
TA 1537: 0, 62.5, 125, 250, 500, 1000 & 2000 µg/plate (recommended limit concentration).


Reverse mutation test 2 (-S9): tested up to cytotoxicity limit:
TA100: 0, 39.1, 78.1, 156, 313, 625, 1250 & 2500 µg/plate;
TA 1535 / TA 98: 0, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/plate;
TA 1537: 0, 1.56, 3.13, 6.25, 12.5, 25, 50 & 100 µg/plate;
WP2 uvrA: 0, 313, 625, 1250, 2500 and 5000 µg/plate;

Reverse mutation test 1 (+S9):
TA100 / TA 1535 / TA 98 & WP2 uvrA: 0, 313, 625, 1250, 2500 & 5000 µg/plate (cytotoxicity limit);
TA 1537: 0, 62.5, 125, 250, 500, 1000 & 2000 µg/plate (recommended limit concentration).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not reported
- Justification for percentage of solvent in the final culture medium: not reported

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: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in: Preincubation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Rationale for test conditions:

The substance was tested up to cytotoxicity limit or recommended limit concentration.

Evaluation criteria:

The test substance was deemed to be mutagenic (positive) in this test system if 1 or more types of 5 test bacteria, either with 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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 2500 µg/plate -S9. Tested up to limit concentration +S9

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 250 µg/plate (Exp. 1) and from 62.5 µg/plate (Exp.2) -S9. Tested up to limit concentration +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:

cytotoxicity

Remarks:

From 62.5 µg/plate (Exp. 1) and from 125 µg/plate (Exp.2) -S9. Tested up to limit concentration +S9

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

From 25 µg/plate (Exp. 1) and from 50 µg/plate (Exp.2) -S9. From 1000 µg/plate (Exp. 1) and from 2000 µg/plate (Exp.2) +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
- Precipitation and time of the determination: Precipitation was observed on the surface of agar plates at concentration ≥ 150 µg/plate -S9 and ≥ 500 µg/plate +S9.
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:
The results indicated an antibacterial effect was on TA1537 in the tests without S9 mix at all doses and TA1535 at doses of 500 μg/plate and above, and in tests using S9 mix on TA1537 at doses of 1500 μg/plate and above.
Therefore, the highest dose in this study was set at 5000 μg/plate for testing both with and without S9 mix (TA1535 without S9 mix test: 1000 μg/plate, TA1537 without S9 mix at 100 μg/plate and with S9 mix at 2000 μg/plate). However, since results for TA100 and TA98 from Test 1 without S9 mix showed strong antibacterial activity, and the dose without antibacterial activity did not reach 4 doses, Test 1 was repeated with the maximum dose lowered to a maximum dose of 2500 μg/plate for TA100 and 1000 μg/plate for TA98 testing without S9 mix. The non-antibacterial dose was also insufficient for TA98 in this test, so the maximum dose was lowered to 500 μg/plate, and Test 1 was repeated, with this data being used as the Test 1 data. In Test 2 of TA1535 without S9 mix, the non-antibacterial dose did not reach 4 doses, and therefore Test 2 was repeated, lowering the maximum dose to 500 µg/plate, with the resulting data being used as Test 2 data.

STUDY RESULTS
Tests, both with and without S9 mix, were conducted twice by setting 5 to 8 doses at a common ratio of 2 based on the above maximum doses (Tables 2 and 3). As a result, in Test 2 of TA1537 without S9 mix there was an increase in the number of mutant colonies observed, which was more than double the solvent control value at doses from 1.56 to 12.5 μg/plate. However, the solvent control group value was less than 10 and no dose-dependency was observed. Furthermore, Test 1 did not show an increase in the number of mutant colonies that was 2 times or more the control value. Also, there was no increase in the number of mutant colonies at values of twice or more the solvent control value observed when testing TA1537 and the other bacteria with S9 mix.
In order to investigate the factors causing the large differences in doses with antibacterial effect between tests, TA1535 was tested twice and TA98 was tested 4 times in tests of S. typhimurium without S9 mix.
It was determined from the results that in the tests of these bacteria without S9 mix, the test substances exhibit weak antibacterial activity close to detection limits over a wide range of concentrations, and slight variations in test conditions resulted in large differences in the doses that were deemed to be "antibacterial".
- Concurrent vehicle negative and positive control data: In all tests conducted on BBTSA, 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 assay system.
- Signs of toxicity: Precipitates derived from the test substance were observed at all doses+/-S9.
- Individual plate counts: cf. Results tables
- Mean number of revertant colonies per plate and standard deviation: cf. Results 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 or up to cytotoxicity limit in the presence and absence of mammalian metabolic activation under the standard Ames plate incorporation assay.

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

 

Precipitation was observed on the surface of agar plates at concentration ≥ 150 µg/plate -S9 and ≥ 500 µg/plate +S9.

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