N-cyclohexylbenzothiazole-2-sulfenamide

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.1).

 

- In vitro gene mutation study in mammalian cells: negative in L5178Y cells up to cytotoxic concentrations (Monsanto, 1979, Key, Rel.2).

 

- In vitro cytogenicity study in mammalian cells: weak positive / ambiguous with S9 in CHL at non cytotoxic concentrations (MHWJ, 1997, Key, Rel.2).

 

Link to relevant study records

Reference

Reference 1

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

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

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

Species / strain / cell type:

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

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

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: 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.

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

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

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

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

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

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).

Reference 2

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 with acceptable restrictions

Remarks:

GLP and guideline study, but with restriction concerning cytotoxicity determination and data documentation (no historical control data documented)

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

Principles of method if other than guideline:

Genotoxicity of N-cyclohexyl-2-benzothiazolesulfenamide was studied by chromosomal aberration in cultured hamster lung (CHL/IU) cells.

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: 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
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: 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 cells obtained from Research Resource Bank (JCRB)
- Suitability of cells: not reported
- 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 within after thawing (the number of passages of the parent strain was 4 generations when it was obtained in February 1988, and is currently 12 generations).
- Methods for maintenance in cell culture: no data
- 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:
Eagle MEM medium (Nissui Pharmaceutical Co., Ltd.) containing 10% fetal bovine serum (Cansera International, lot number: 2605420 and Filtron, lot number: 55301), and cultured in a carbon dioxide incubator (5% CO2, 37°C)

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, RAA-338, manufactured in December 1995, and RAA-363, manufactured in May 1997) and stored at -80°C in an ultra-low temperature bath until time for use. Prepared from the liver of 7-week-old male Sprague-Dawley rats administered phenobarbital and 5,6-benzoflavone
- 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 (24 h exposure)
0, 10, 20, 40, 80, 160 µg/ml; evaluated: 0, 10, 30, 40 µg/ml
-S9 (48 h exposure)
0, 10, 20, 40, 80, 160 µg/ml, evaluated:0, 10, 20, 40 µg/ml
-S9 (6 h exposure, experiment)
0, 21, 41, 83, 170, 330 µg/ml; evaluated: 0, 21, 41 µg/ml

+S9 (6 h exposure, experiment I)
0, 41, 81, 160, 330 µg/ml; evaluated: 0, 41,81 µg/ml
+S9 (6 h exposure, experiment II)
0, 20, 41, 81, 160, 330, 650 µg/ml; evaluated: 0, 20, 41, 81 µg/ml

Top dose selected based on mitotic index.

Vehicle / solvent:

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

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 cultures per concentration: duplicate
- 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)
- Harvest time after the end of treatment (sampling/recovery times): 24 or 48 hrs
- 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 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. MI max was set at the highest observed concentration based on the mitotic index prior to chromosome analysis.
- 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): 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.
- Determination of polyploidy: yes
- Determination of endoreplication: no

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative metaphase frequency to the solvent control was calculated (Exp.1), cell confluency (Exp.2).

Rationale for test conditions:

The highest concentrations were limited by cytotoxicity.

Evaluation criteria:

The final determinations were made by referring to the results of additional testing and examining reproducibility.

Statistics:

A significant difference test was performed on the solvent background data 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:

ambiguous

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

RANGE-FINDING/SCREENING STUDIES (if applicable):

STUDY RESULTS
- Concurrent vehicle negative and positive control data: appropriate response were obtained in both vehicle negative and positive control groups.
- Results from cytotoxicity measurements: cf. attached Result tables. Cytotoxicity noted from 0.080 mg/mL (continuous treatment without S9 mix, 0.083 mg/mL ( short-term treatment without S9 mix) and 0.16 mg/mL (short-term treatment with S9 mix).
- Genotoxicity results:
Chromosome analysis showed no increase in chromosomal structural abnormalities in any of the treatment groups under continuous treatment. Chromosomal structural aberrations increased significantly with short treatment (4.5%, p<0.05) only at a concentration of 0.041 mg/mL with S9 mix, but no dose dependence was observed. The number of polyploid cells only significantly increased in the short treatment group with S9 mix (0.081mg/mL, 1.88%, p<0.05), and a significant difference was also observed in the trend test (p<0.05). However, polyploid cells did not appear often, and a clear determination could not be made, so it was therefore decided to conduct a confirmatory test only for short treatment with S9 mix. In the confirmatory testing, chromosomal structural abnormalities (including gaps) increased significantly (3.5%, p<0.05) in short treatment with S9 mix at a concentration of 0.081 mg/mL, but the trend test results indicated there was no dose dependence. Due to toxicity, 0.081 mg/mL did not allow a defined number of analyses for polyploid cells. At 0.41 mg/mL, there was a significant increase in polyploid cells (1.13%, p <0.05), but the trend test showed no concentration dependence. Therefore, it was determined from the additional testing of short treatment with S9 mix that both chromosomal structural aberration and polyploidy cell induction were negative.

HISTORICAL CONTROL DATA: not available

Cytotoxicity:
80 µg/ml (24 - 48 h treatment -S9)
83 µg/ml (6 h treatment -S9)
160 µg/ml (6 h Treatment +S9).

After 6 h treatment + S9 there were 4.5 % structural chromosomal  aberrations (incl. gaps) induced at 41 µg/ml and 1.88 % polyploidy at 81 µg/ml.

However a trend test showed no dose dependency for the structural chromosomal aberrations. In a confirmation test, 3.5 % structural chromosomal aberrations (incl.  gaps) were induced at 81 µg/ml and 1.13 % polyploidy at 41 µg/ml.

However a trend test showed no dose dependency for the structural chromosomal aberrations or the polyploidy.

Experiment I and II with metabolic activation, 6 h treatment

Group	concentration µg/ml	TAG (%)	TA (%)	Polyploid (%)
experiment I
Solvent (DMSO)	0	1 (0.5)	1 (0.5)	0.38
CBS	41	9* (4.5)	7 (3.5)	0.88
CBS	81	5 (2.5)	4 (2.0)	1.88*
experiment II
Solvent (DMSO)	0	2 (1.0)	2 (1.0)	0.88
CBS	20	4 (2.0)	4 (2.0)	0.38
CBS	41	2 (1.0)	2 (1.0)	1.13*
CBS	81	7* (3.5)	7 (3.5)	0.79

TAG: total no. of cells with aberrations

TA: total no. of cells with aberrations except gap

*:Significant different from histroical solvent control (p<0.05) by fisher's exact test using a Bonferroni's correction for multiple comparisions.

The statistically significant increase in aberrant cells and polyploidy after treatment with CBS with metabolic activation showed, when trend tested no dose dependency. In addition, according to recommendation given in the current OECD guideline (TG 473), the in vitro chromosome aberration assay is not appropriate method for the detection of polyploidy.

Conclusions:

Interpretation of results: ambiguous with metabolic activation, negative without metabolic activation.

Executive summary:

The test substance CBS was weak positive in an in vitro chromosomal aberration using CHL cells (MHWJ 1997). In the absence of metabolic activation (S-9 mix) no induction of structural chromosomal aberrations was found for doses ranging from 10 to 41 µg/ml; no strong cytotoxicity was induced, but higher doses led to total cytotoxicity; various treatment and sampling times used (24/24 h; 48/48 h and 6/18 h). In the presence of S-9 mix weakly increased frequencies of structural chromosome aberration were found in two experiments after 6 hour treatment and 18 hours sampling. Again, no strong cytotoxicity was induced but higher doses led to total cytotoxicity. The main results were as followed: experiment I: 41 µg/ml, 81 µg/ml results in 3.5 % and 2.0% aberrant cells (excluded gaps); experiment II: 41 mg/ml, 81 µg/ml results in 1.0% and 3.5 % aberrant cells (excluded gaps). Since all solvent controls ranged from 0.5 to 1.0 aberrant cells (excluded gaps), this is interpreted as weak evidence for a clastogenic potential.

However, statistically evaluation revealed no dose dependency for structural chromosomal aberration. In addition, cytotoxicity was indicated in the first experiment as relative metaphase frequency to the solvent control and in the second experiment as cell confluency. No dose dependency in cytotoxicity was seen up to 0.081 mg/ml test substance, whereas higher concentrations could not be evaluated because of very high cytotoxicity. Thus, no dose-dependency in genotoxicity and cytotoxicity was noted in the relevant concentration range. The increase in aberrant cells (3.5 %) is rather weak. In addition, the background frequency was low in both experiments (0.5% and 1%) compared to data from literature (XX). In addition, the slight increase in aberrant cells was stated to be significant different from historical solvent control data, but no historical control data are documented. Based on the rather weak increase in aberrant cells, the lack of a dose response relationship in genotoxicity and cytotoxicity and the low background frequency noted, the findings was assumed to be ambiguous.

Reference 3

Endpoint:

in vitro gene mutation 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 with acceptable restrictions

Remarks:

comparable to guideline study with acceptable restriction (e.g. positive controls not consistent with recent guideline, no differentiation on colony-size; selective agent was BrdU instead of TFT)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

positive controls not consistent with recent guideline, no differentiation on colony-size, BrdU used as selection agent

Principles of method if other than guideline:

Santocure BO-78-221 (N-cyclohexyl-2-benzothiazolesulfenamide) was tested for mutagenicity in a MLA at the TK locus in L5178Y mouse lymphoma cells with or without S9 metabolic activation according to the protocol of Clive, D., Mutation Res., 31, 17-29, 1975).

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not reported
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: will slowly decompose with heat and water as per the TIDS.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not assessed
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium:
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not assessed

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: none

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: derived from the Fischer L5178Y line of Dr. Donald Clive
- Suitability of cells: standard cell line
- Normal cell cycle time (negative control): not reported
- Absence of Mycoplasma contamination: yes, stock are periodically checked by culturing methods.
- Number of passages if applicable: not reported
- 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: not reported
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Fischer's mouse leukaemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10% by volume). Cloning medium consists of the preceding growth medium with the addition of agar to a final concentration of 0.35% to achieve a semisolid state. Selection medium is cloning medium containing 50 or 100 µg/mL of BrDU.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: from Aroclor 1254 induced Fisher 344 male rats liver.
- method of preparation of S9 mix: the final concentration of the activated system components in the cell suspension is: 2.4 mg NADP/mL; 4.5 mg isocitric acid/mL and 50 µL S9/mL.
- concentration or volume of S9 mix and S9 in the final culture medium: 50 µL S9/m
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

Test 1:
-S9: 0.156; 0.625; 2.5; 5 and 10 µg/mL.
+S9: 0.313; 1.25; 5; 10; 20 and 40 µg/mL.

Test 2:
-S9: 2.5; 5;10; 15; 20 and 30 µg/mL.
+S9: 5; 10; 20; 40 and 80 µg/mL.

Test 3:
-S9: 1.25; 5;10; 15 and 0 µg/mL.
+S9: 2.5; 20; 40; 50; 60 and 70 µg/mL.

Test 4:
+S9: 40; 50; 55; 60; 65 and 70 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: insoluble in water at 50 mg/mL, soluble in DMSO at 500 mg/mL.
- Justification for percentage of solvent in the final culture medium: not reported.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 6 trials among which 3 non activation and 4 activation trials are presented in the study report, the unreported trials were considered to be invalid.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): not reported
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): 2-3 days
- Selection time (if incubation with a selective agent): 10 days
- Method used: agar plates
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: BrdU
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 3 x 10E6
- Criteria for small (slow growing) and large (fast growing) colonies: not performed

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: % relative growth

Rationale for test conditions:

4 to 5 doses selected to be in the range of 0 to 50-90% reduction in cell growth.

Evaluation criteria:

A compound is considered mutagenic for this assay if:
1/ a dose-response relationship is observed over 3 of the 5 dose levels employed.
2/ The minimum increase at the low level of the dose-response curve is at least 2.5 times greater than the solvent and/or negative control values.
3/ The solvent and negative control data are within the normal range of the spontaneous background for the TK locus.
All evaluations of mutagenic activity are based on consideration of the concurrent solvent and negative control values run with the experiment in question. Positive control values are not used as reference points, but are included to ensure that the current cell population responds to direct and promutagens under the appropriate treatment conditions.
Occasionally, a single point within a concentration range will show an increase 2.5 times greater than the spontaneous background. If the increase if a the high dose, is reproducible, and if and additional higher dose level is not feasible because of toxicity, the chemical can be considered mutagenic. If the increase in internal within the dose range and is not reproducible, the increase will normally be considered aberrant. If the internal increase is reproducible, several doses clustered around the positive concentration will be examined to either confirm or reject the reliability of the effect.

Statistics:

None

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: a white precipitate appeared in the growth medium at 78 µg/mL and higher concentrations, whereas the test material appeared to remain soluble at 39 µg/mL.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
- Results from cytotoxicity measurements: cf. attached Result tables.A range of concentrations up to the highly toxic levels of 30 µg/ml (without metabolic activation) and 75 µg/ml (with metabolic activation) were assayed.
- Genotoxicity results: cf. attached Result tables. No effect observed. No differentiation on colony-size was made.

HISTORICAL CONTROL DATA: not reported

Conclusions:

Interpretation of results: negative

Executive summary:

The test substance CBS was negative in a mouse lymphoma assay with L5178Y cells, with S-9 mix up to a dose of 75 µg /ml and without S9-mix up to a dose of 30 µg/ ml; toxic effects were found with and without S-9 mix in high doses (Monsanto Co 1979).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A testing proposal (based on read-across to DCBS - CAS 4979-32-2) is included in this dossier to generate compliant in vivo genotoxicity study. 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

A combined in vivo mammalian somatic cell study: cytogenicity / A combined in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus & in vivo mammalian cell study: DNA damage and/or repair (comet assay)

Type of information:

experimental study planned (based on read-across)

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out :
N,N-dicyclohexylbenzothiazole-2-sulphenamide; CAS 4979-32-2
- Name of the substance for which the testing proposal will be used [if different from tested substance]
N-cyclohexylbenzothiazole-2-sulphenamide ; CAS 95-33-0


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION
- Available GLP studies
No adequate in vivo genotoxicity study available on CAS 95-33-0 or any member of the Category.
- Available non-GLP studies
No adequate in vivo genotoxicity study available on CAS 95-33-0 or any member of the Category.
- Historical human data
No data available.
- (Q)SAR
No reliable data available.
- In vitro methods
Ambiguous/Weak positive results were reported in the in vitro cytogenicity / chromosome aberration study in mammalian cells.
- Weight of evidence
The available dataset is not sufficient to properly assess the genotoxicity profile of the substance.
- Grouping and read-across
The substance is a member of the Sulphenamides (benzothiazoles) Category. Adequate and reliable studies to cover the genotoxicity endpoint are currently not available. Located within section 13.2 of IUCLID there is the category read-across justification document. This document is a scientific evaluation of the collective category data set, identifying testing requirements to ensure suitable coverage of required endpoints for all member substances. This category member has been identified as having a data gap for in vivo genotoxicity testing (MNT/COMET), required in accordance with Annex VIII of Regulation (EC) No 1907/2006. This study will be performed on another category member and will a be used as part of the overall category data set as a source study for read-across.
- Substance-tailored exposure driven testing [if applicable]
Not applicable
- Approaches in addition to above [if applicable]
Not applicable
- Other reasons [if applicable]
Not applicable


CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
According to Regulation (EC) No 1907/2006 Annex VIII column 2 “Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.


FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed
A testing proposal on the Category member CAS 4979-32-2 is included in this dossier to generate compliant in vivo genotoxicity study. An in vivo mammalian erythrocyte micronucleus test (OECD TG 474) combined with an in vivo mammalian alkaline comet assay (OECD TG 489) in rats with oral administration is proposed.
For the comet assay, the following tissues are proposed for analysis: liver (primary site of xenobiotic metabolism), stomach and duodenum (sites of direct contact).
For the micronucleus test, the bone marrow shall be analysed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Species:

rat

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

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) WoE, 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 (OECD 473, GLP) K, rel.2	Chromosomal aberration	Chinese Hamster lung CHL/IU	-S9 +S9	Tested up to 81 µg/mL (cytotoxicity limit)) in DMSO	- S9 (24/48 hrs): negative - S9 (6 hrs): negative + S9 (6 hrs): ambiguous
3(Monsanto, 1979)	hprt (eq. OECD 476, GLP) K, rel.1	Gene mutation	Mouse Lymphoma L5178Y	-S9 +S9	Tested up to 80µg/mL in DMSO(cytotoxicity limit)	- S9 (24/48 hrs): negative - S9 (6 hrs): negative + S9 (6 hrs): ambiguous

 

In vitro data:

Gene mutation assays:

The test substance CBS was evaluated in a GLP- and guideline study (OECD TG 471) bacterial gene mutation test. Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used. Treatment by the pre-incubation method was done for doses up to 5000 µg/plate. Toxicity effects varied in a strain-specific manner; at high doses from 200 µg/plate upward precipitations were observed. The test substance CBS was negative in presence and absence of metabolic activation (MHWJ 1997). These findings were confirmed by another Ames assay, employing Salmonella typhimurium tester strain TA 98, TA 100, TA 1535, TA 1537, TA 1538 and Saccharomyces cerevisiae strain D4 with and without metabolic activation for doses ranging from 0.1 to 500 µg/ plate (Monsanto Co 1976 / Test No.1).

The findings from the bacterial mutation assays were confirmed in a mammalian cell mutation assay. The test substance CBS was negative in a mouse lymphoma assay with L5178Y cells, with S-9 mix up to a dose of 75 µg /ml and without S9-mix up to a dose of 30 µg/ ml; toxic effects were found with and without S-9 mix in high doses (Monsanto Co 1979 / Test No.3). No differentiation on colony-size was made.

 

Chromosomal aberration assay:

However, the test substance CBS was weak positive in an in vitro chromosomal aberration using CHL cells. In the absence of metabolic activation (S-9 mix) no induction of structural chromosomal aberrations was found for doses ranging from 10 to 41 µg/ml; no strong cytotoxicity was induced, but higher doses led to total cytotoxicity; various treatment and sampling times used (24/24 h; 48/48 h and 6/18 h). In the presence of S-9 mix weakly increased frequencies of structural chromosome aberration were found in two experiments after 6 hour treatment and 18 hours sampling. Again, no strong cytotoxicity was induced but higher doses led to total cytotoxicity.

The main results were as followed: experiment I: 41 µg/ml, 81 µg/ml results in 3.5 % and 2.0% aberrant cells (excluded gaps); experiment II: 41 mg/ml, 81 µg/ml results in 1.0% and 3.5 % aberrant cells (excluded gaps).

Since all solvent controls ranged from 0.5 to 1.0 aberrant cells (excluded gaps), this is interpreted as weak evidence for a clastogenic potential. However, the author stated, that trend tests showed no dose dependency for structural chromosomal aberrations (MHWJ 1997 / Test No. 2).

 

In vivo data:

Embryonic mortality of CBS was investigated in rats. However, no reliable data on dominant lethal mutations can be derived because test method and data presentation are inadequate (Aleksandrov 1982).

A testing proposal on the Category member CAS 4979-32-2 is therefore included in this dossier to generate compliant in vivo genotoxicity study. An in vivo mammalian erythrocyte micronucleus test (OECD TG 474) combined with an in vivo mammalian alkaline comet assay (OECD TG 489) in rats with oral administration is proposed.

Justification for classification or non-classification

The test substance CBS was negative in bacterial gene mutation assays (Monsanto Co. 1976, MHWJ 1997); also a mouse lymphoma assay was negative (Monsanto Co. 1979). An in vitro chromosomal aberration assay test gave weak evidence for a clastogenic potential in presence of metabolic activation, but the positive findings was assessed to be ambiguous. The only in vivo test (on embryonic mortality) cannot be assessed adequately due to insufficient data reporting. Further in vivo testing is proposed to conclude on classification or non-classification of CBS.