N-tert-butylbenzothiazole-2-sulphenamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- In vitro gene mutation study in bacteria: not mutagenic up to limit or cytotoxic concentrations in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA (MHJW, 1997, Key, Rel.2).

- In vitro cytogenicity study in mammalian cells: positive with S9 in CHL/IU cells (MHWJ, 1997, Key, Rel.2). Cf. Genetic toxicity in vivo.

 - In vitro gene mutation study in mammalian cells: inconsistent dataset. A new study is planned.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

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)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 1987

Deviations:

not specified

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

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

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

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

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

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.

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

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

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

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

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

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)

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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

Remarks:

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

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 1987

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: hermetically packaged, refrigerated
- 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
- 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): none

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Chinese hamster-derived CHL/IU (hereinafter referred to as CHL) cells obtained from Research Resource Bank (JCRB)
- Suitability of cells: This line of CHL cell is commonly used because it is generally highly sensitive for detecting chemical substances
- Normal cell cycle time (negative control): not reported
- Absence of Mycoplasma contamination: not reported
- Number of passages if applicable: Less than 10 successive generations were used for testing after thawing (the parent strain had gone through 4 passages when it was obtained in February 1988, and was 12 generations at the time of reporting).
- Methods for maintenance in cell culture: not reported
- Cell cycle length, doubling time or proliferation index : not reported
- Modal number of chromosomes: not reported
- Periodically checked for karyotype stability: no data
- Periodically ‘cleansed’ of spontaneous mutants: no data

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Cells cultured in a CO2 incubator (5% CO2 37°C) using Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum (Cansera International, lot number: 2605420).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: purchased from Kikkoman Ltd. (Lot number: RAA-333, manufactured in September 1995)
- method of preparation of S9 mix : 5% S9, 0.83 mM G-6-P, 0.67 mM β-NADP+, 0.83 Mm magnesium chloride 5.5 mM potassium chloride、0.67 mM HEPES
- concentration or volume of S9 mix and S9 in the final culture medium: 5%
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

-S9 (continuous treatment): 0, 0.015, 0.03, 0.06 mg/ml;
-S9 (short-term treatment): 0, 0.05, 0.1, 0.2 mg/ml;
+S9 (short-term treatment): 0, 0.1, 0.2, 0.4 mg/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used :0.5% carboxymethylcellulose sodium solution (CMC Na aqueous solution, Nacalai Tesque Inc., lot number: M9G8053)
- Justification for choice of solvent/vehicle: not dissolved in DMSO at 1M, so an aqueous solution of CMC Na was used
- Justification for percentage of solvent in the final culture medium: not reported

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: 3

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Continuous treatment (24 and 48 h) without S9
Short term treatment (6 h) with and without S9(18 h sampling time)
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: colcemid, 0.1 µg/mL, 2 hours before the of incubation
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
After incubation, the medium was removed and the cells were liberated with 0.02% EDTA in phosphate-buffered saline (free of Ca2+ and Mg2+), collected in a 10 ml centrifuge tube, and centrifuged (1000 to 1200 rpm for 5 minutes). After discarding the supernatant, 3 ml of 0.075 M potassium chloride aqueous solution was added to the precipitated cells, and hypotonic treatment was performed for 30 minutes. After hypotonic treatment, 6 ml of fixative solution (methanol: water acetic acid = 3:1 v/v) was added and centrifuged. Then the supernatant was removed, fresh fixative was added and the mixture was recentrifuged. The fixative solution was changed several times before the cells were suspended in a small volume of fixative solution, then a small amount of this suspension was dropped onto a slide glass (with the test system ID number, code number, and slide number noted on the frosted part in advance), and left to air dry. 6 slides were prepared from each dish. After staining the slides with 3% Giemsa solution (diluted with 1/15 M phosphate buffer, pH 6.8), the slides were rinsed with distilled water and air-dried. Slides were stored in a slide case in order of their code numbers, clearly labeled with the creation date and study protocol number.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 200 metaphase cells per group were also analyzed for structural abnormalities and 800 metaphase cells per group were analyzed for polyploidy.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Based on the measurements of cell proliferation rates (Tables 1 and 2) and the mitotic index, the highest concentrations possible for chromosome analysis were 0.060 mg/ml with continuous treatment, 0.40 mg/ml for short treatment with S9 mix and 0.20 mg/ml for short treatment without S9 mix, so 3 concentration groups, including these concentrations, were targeted. Since these were the highest possible concentrations for chromosomal analysis, 3 concentration groups including these concentrations were subjected to observation. Chromosome analysis was performed in line with the classification method of the Japanese Environmental Mutagen Society, Mammalian Tests (MMS) Study Group 1). Well-spread metaphase images in which chromosomes were not dispersed were observed. The number of observed cells, the type and number of chromosome-type and chromatid-type structural abnormalities, and the number of polyploid cells for each group were noted on the record sheets. The position of abnormal cells on the slide was indicated by the position of the stage of the microscope and recorded on a recording sheet. 4 slides per dish were analyzed by 4 observers, who did not know the treatment conditions of individual slides.
- Determination of polyploidy: yes
- Determination of endoreplication: no


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Growth inhibition (cell confluency representing cytotoxicity was measured with monocellater and was indicated in % of solvent control (100%))

Rationale for test conditions:

Concentrations were selected based on cell growth inhibition test. In this test, the concentrations ranged from 0.0094 to 0.30 mg/mL for continuous treatment and 0.075 to 2.4 mg/mL (10 mM) for short treatment.
Three concentration groups were decided at the highest targeted concentrations in advance of chromosome analysis, based on the results from measuring cell proliferation rates and mitotic index, from the highest concentrations with a relative growth rate of 20% or more and a mitotic index of 0.5% or more in both dishes.

Evaluation criteria:

The ultimate determinations were based on statistical and biological evaluations.

Statistics:

A significant difference test was performed on the medium background data (Appendix 2) and the test substance-treated groups using Fisher's exact method) with a familywise significance level of 5%. If there was a significant difference with the direct probability method, dose dependence was confirmed by the Cochran-Armitage test for trend) (p < 0.05).

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: none reported

CELL GROWTH INHIBITION TEST
The results of cell growth inhibition testing showed both continuous and short treatment suppressed CHL/IU cell proliferation in a dose-dependent manner. The 50% growth inhibition was at 0.03 mg/ml under continuous treatment, 0.2 mg/ml for short treatment with S9 mix and 0.1 mg/ml without.
For this reason, in the chromosomal aberration test, in all treatment series, the maximum treatment concentration was twice the 50% growth inhibitory concentration, and each concentration was set at a common ratio of 2. (continuous treatment: 0.0038, 0.0075, 0.015, 0.030, 0.060mg/ml, short treatment with S9 mix: 0.025, 0.050, 0.10, 0.20, 0.40 mg/ml, short treatment without S9 mix: 0.013, 0.025, 0.050, 0.10, 0.20 mg/ml). The concentrations for 48-hour continuous treatment were set to the same concentration as the 24-hour treatment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: appropriate responses were obtained in both vehicle negative and positive control groups.
- Results from cytotoxicity measurements: cf Results tables attached. Cytotoxicity of the test substance was indicated by a clear reduction of cell confluency at 0.03 (62.5 %) and 0.06 mg/ml (38 %) (24 hour treatment) and at 0.06 mg/ml (60.5%) (48 hour treatment) without metabolic activation. In the short-term treatment (6 hours) without metabolic activation a clear reduction in cell confluency was noted in all treatment groups (65% to 36.5 % compared to control). In the short-term treatment with metabolic activation (6 hours) no decrease in cell confluency was noted in any of the treatment groups (increase up to 222 %)
- Genotoxicity results: Cf. Result tables attached.
No genotoxic effects of the test substance were noted without metabolic activation.
Whereas in the short-term treatment with metabolic activation a statistically significant increase of polyploidy and total number of cells with aberrations (TAG) were noted at the highest concentrations (0.2 and 0.4 mg/ml) when compared to the historical control data (no historical control data given).
However, the increase in polyploidy is even low (1.13% and 1.63 %) and the authors stated that the increase is not biologically relevant. Moreover, according to recommendation given in the current OECD guideline 473 (in vitro chromosomal aberration assay) this test system is not designed to measure numerical aberrations and should not routinely be used for that purpose.
The increase in aberrant cells including gaps is significant different (p<0.05) from historical control data (5.0% and 6.0% vs. 2.0% solvent control). The number of cells with aberrations without gaps (TA) is also different from control at the highest concentrations (3.5% and 5.0 % vs. 0.5 % solvent control); however, the authors indicated no statistically significant differences for TA of the highest dose groups compared to the historical control data.


HISTORICAL CONTROL DATA: not available

Conclusions:

Interpretation of results: positive with metabolic activation

Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration) conducted according to OECD TG 473 and in compliance with GLP, cultures of Chinese hamster lung (CHL/IU) cells were exposed to the test material diluted in 0.5% CMC in the presence or absence of exogenous metabolism under the following conditions :

-S9 (continuous treatment): 0, 0.015, 0.03, 0.06 mg/ml;

-S9 (short-term treatment): 0, 0.05, 0.1, 0.2 mg/ml;

+S9 (short-term treatment): 0, 0.1, 0.2, 0.4 mg/ml.

The top concentration was limited by toxicity or was up to recommended limit concentrations.

 

Negative (solvent) and positive control cultures were included in the assays to monitor performance.

Following exposure to the test agent, colcemid was added to the cultures to arrest cell division at metaphase and slides were prepared.

The positive control mutagens induced cytogenetic damage as expected.

 

Test item-induced chromosomal structural aberrations were significantly increased in the presence of S9 mix with short treatment (0.20 and 0.40 mg/ml, p<0.05). There was also a significant increase in polyploid cells with short treatment with or without S9 mix (short treatment with S9 mix: 0.20 and 40 mg/ml, short treatment without S9 mix: 0.10 and 0.20 mg/ml (p<0.05). However, the frequency of polyploid cell induction was low (with S9 mix 1.13 to 1.63%, and 1.13 to 1.50% without), and were judged to be negative. 

N-tert-butylbenzothiazole-2-sulphenamide was positive with metabolic activation in this in vitro mammalian chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Genetic toxicity in vivo

Description of key information

- In vivo mammalian erythrocyte micronucleus test: not clastogenic and not aneugenic in mice via i.p. administration up to 2000 mg/kg bw (Durward, 2002, Key, Rel.1).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

Groups, each of seven mice, were dosed once via the intraperitoneal route with the test substance at 500, 100 or 2000 mg/kg bw. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg bw was killed after 48 hours.
Immediately following termination (i.e. 24 or 48 hours after dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil

Duration of treatment / exposure:

24 and 48 h

Frequency of treatment:

once

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 males/ dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

Immediately following termination (i.e. 24 or 48 hours after dosing), both femurs were were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grünwald/Giemsa, allowed to air-dry and cover slipped using mounting medium.
Stained bone marrow smears were coded and examined blind using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE- pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test substance groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 -hours kill times when compared to the corresponding control group.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.

Statistics:

All data were statistically analyzed using appropriate methods as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analyzed using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Range-finding study:

In animals dosed with test material, at maximum recommended dose level of 2000 mg/kg bw, via the oral and intraperitoneal route no premature deaths were recorded. One animal dosed via the intraperitoneal route demonstrated one clinical sign, diuresis.

The test material showed no marked differences in its toxicity to male or female mice; it was therefore considered from the authors to be acceptable to use males only for the main study. The intraperitoneal route was selected for use in the main study in an attempt to maximize exposure of the animals to the test substance. The maximum recommended dose (MRD) of the test substance, 2000 mg/kg bw, was selected for use in the main study, with 1000 and 500 mg/kg bw as the lower dose levels.

Micronucleus study:

Mortality: none during the study

Clinical signs: Clinical signs were observed in animals dosed with test material at and above 500 mg/kg and included as follows: hunched posture, pilo-erection, lethargy and ptosis. The animals appeared to be more sensitive to the test substance in the main-study than observed in the preliminary range-finding study.

Effect on PCE/NCE ratio: a statistically significant decrease in the PCE/NCE ratio was seen in the 48 -hour 2000 mg/kg and 24 -hour 500 mg/kg test material groups when compared to their concurrent vehicle control groups. The PCE/NCE ratios for the 1000 and 2000 mg/kg 24 -hour test material dose groups were also lower than their concurrent vehicle control. These, accompanied by the observation of clinical signs, were taken to indicate that systemic absorption and adequate exposure of the bone marrow had been achieved.

Genotoxic effects: Negative.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

The positive control group showed a market increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Summary of group mean data

Treatment group	Number of PCE with micronuclei per 2000 PCE		PCE/NCE ratio
	Group mean	SD	Group mean	SD
Vehicle control, 48 h sampling time	2.0	1.2	0.88	0.21
Vehicle control, 24 h sampling time	2.6	2.3	0.85	0.26
Positive control, 24 h sampling time	34.00**	18.3	1.56	0.63
2000 mg/kg, 48 h sampling time	1.4	1.3	0.59*	0.25
2000 mg/kg, 24 h sampling time	2.4	2.6	0.71	0.38
1000 mg/kg, 24 h sampling time	2.1	2.3	0.62	0.32
500 mg/kg, 24 h sampling time	1.6	1.4	0.53*	0.27

* p< 0.05

** p< 0.001

Conclusions:

Interpretation of results: negative

Executive summary:

Groups, each of seven mice, were dosed once via the intraperitoneal route with the test substance at 500, 1000 or 2000 mg/kg bw. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg bw was killed after 48 hours.

Immediately following termination (i.e. 24 or 48 hours after dosing), both femurs were were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6.1/1 : Summary of key genotoxicity results:

 

Test n°	Test Guideline / Reliability	Focus	Strains  / cells tested	Metabolic activation	Test concentration	Statement
1 (MHJW, 1997)	Ames Test (OECD 471, GLP) Key, rel.2	Gene mutation	S. thyphimurium TA 1535, TA 1537, TA 98, TA 100, E. coli WP2 uvrA	-S9 +S9	Tested up to cytotoxicity limit or up to limit concentration in DMSO	- S9: not mutagenic + S9: not mutagenic
2 (MHJW, 1997)	CAT (in vitro) (OECD 473, GLP) K, rel.2	Chromosomal aberration	Chinese Hamster lung CHL/IU	-S9 +S9	Tested up to cytotoxicity limit or up to limit concentrationin 0.5% CMC Na	- S9 (24/48 hrs): negative - S9 (6 hrs): negative + S9 (6 hrs): positive
3 (Durward, 2002)	MNT (in vivo) (OECD 474, GLP, K, rel.1)	Chromosomal aberration	CD-1 mouse	Not relevant	500, 1000 or 2000 mg/kg bw in Arachis oil. Single i.p. treatment.	Negative

In vitro data

The mutagenic potential in bacteria of the test substance TBBS was evaluated in a GLP and OECD guideline study.

Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were used. Treatment by the pre-incubation method was done for doses up to 5000 µg/ plate or even lower if toxicity was indicated. The tester strains were evaluated with and without metabolic activation. In this study no biologically relevant and dose dependent increases in revertants was observed in any of the tester strains evaluated with and without metabolic activation (MHWJ 1997).

In addition, in earlier bacterial mutation assays the test substance also indicated a non-mutagenic potential (Monsanto 1976, Hinderer 1983).

The test substance was evaluated in an in vitro chromosome assay (TG 473) with CHL-cells (MHWJ 1997). The cells were treated in a short-term treatment with and without metabolic activation and in continue treatments for 24 and 48 hours without metabolic activation. Cytotoxicity was noted in short- and long-term treatments without metabolic activation, indicated by a reduction of the cell confluence. No cytotoxicity of the test substance was noted in the presence of metabolic activation up to the highest concentration evaluated (0.4 mg/ml). No biologically relevant increase in aberrant cells or polyploidy was noted without metabolic activation. Whereas in the short-term treatment with metabolic activation a statistically significant increase of polyploidy and total number of cells with aberrations (TAG) were noted at the highest concentrations (0.2 and 0.4 mg/ml) when compared to the historical control data. However, the increase in polyploidy was even low (1.13% and 1.63 %) and the authors stated that the increase is not biologically relevant. The increase in aberrant cells including gaps was significant different (p<0.05) from historical control data (5.0% and 6.0% vs. 2.0% concurrent solvent control). The number of cells with aberrations without gaps (TA) was also different from the solvent control at the highest concentrations (3.5% and 5.0 % vs. 0.5 % concurrent solvent control). Thus, based on the findings of this study, the test substance reveals a weak but statistically significant mutagenic effect in presence of metabolic activation.

No increases in aberrant cells were detected in an earlier in vitro chromosome assay (Hinderer 1983). However, this study reveals limitations concerning the test design which limited the validity of the study.

A mutagenic response in presence of metabolic activation was also found in several mouse lymphoma forward mutation studies mainly in the presence of metabolic activation (Monsanto Co.1979, Monsanto 1982, 1982b, Hinderer 1983). Whereas, in a limited documented HGPRT assay with CHO cells (only secondary literature) no mutagenicity was indicated (ECB IUCLID 2000). Based on the result inconsistency, a new in vitro study is planned. 

 

In vivo data

In an in vivo micronucleus assay (OECD TG 474) with CD-1 male mice no genotoxic effects of the test substance TBBS was observed. Male CD-1 mice were treated once ip. with 0, 500, 1000 or 2000 mg/kg test substance. No mortality occurred in any of the treated animals. Clinical signs like pilo-erection, lethargy and ptosis were noted in animals of the 500 mg/kg treatment group and higher. A decrease of the PCE/NCE ratio was noted in all treated animals compared to the concurrent vehicle control. Moreover, the decrease in the PCE/NCE ratio was statistically significant decrease in the 48-hour 2000 mg/kg (0.59) and 24-hour 500 mg/kg (0.53) groups when compared to the concurrent vehicle controls (0.88, 0.85). The indication of clinical signs revealed systemic availability and the decrease in the PCE/NCE ratio indicated that the target tissue was reached by the test substance, and thus reveals the validity of the test system. No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes (PCE) were noted in any of the treated animals compared to the concurrent vehicle control. Whereas, the positive control group showed a market increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the test system to the known mutagenic activity of cyclophosphamide under the conditions of the test (Durward 2002).

In conclusion, the test substance TBBS was negative in an in vivo micronucleus assay with CD-1 mice.

Justification for classification or non-classification

Further testing is on-going to conclude on the mutagenicity potential of TBBS.