N,N-dicyclohexylbenzothiazole-2-sulphenamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

GLP study comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

EC Number:

225-625-8

EC Name:

N,N-dicyclohexylbenzothiazole-2-sulphenamide

Cas Number:

4979-32-2

Molecular formula:

C19H26N2S2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

white

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a plastic container
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: not assessed
- 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

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO-K1-BH4 from Dr. Abraham W. Hsie; Biology Division; Oak Ridge National Laboratories; P.O. Box Y; Oil Ridge, Tennessee 37830.
- Suitability of cells: Chemicals capable of inducing mutations have been shown to increase the forward mutation frequency at the hurt locus in Chinese Hamster Ovary Cells.
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination: not reported
- Number of passages if applicable: Cultures of CHO-K1-BH4 cell line were prepared from stock culture known to have a stable spontaneous mutation frequency of 0-10 x 10E-6 mutants per cells, however, values up to 20 x 10E-6 were deemed acceptable.
- Methods for maintenance in cell culture: maintained in frozen aliquots in a Revco Ultra-low Freezer.
- Cell cycle length, doubling time or proliferation index : no data
- Modal number of chromosomes: no data
- 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: culture media not specified; 37°C at 90% humidity in a 5% CO2 air atmosphere.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: lot 4/28/84 from Aroclor 1254 induced rat liver homogenate.
- method of preparation of S9 mix:
MgCl2 6H2O: 8 µmol
CaCl2 2 H2O: 8 µmol
KCl: 33 µmol
glucose-6-phospate: 5 µmol
NADP: 4 µmol
Sodium phosphate buffer: 50 µmol
S-9 fraction: 0.01 mL (1%) : 0.02 mL (2%); 0.05 mL (5%); 0,1 mL (10%)
At least 30 mg protein/mL.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.01 mL (1%) : 0.02 mL (2%); 0.05 mL (5%); 0,1 mL (10%)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported

Test concentrations with justification for top dose:

Preliminary cytotoxicity test: 0.16, 0.5, 1.6, 5.0, 16.6, 50, 166, 500 µg/ml (1, 2, 5 or 10% S9)
Preliminary study: 5, 200, 500 µg/ml (1, 2, 5 or 10% S9)
Main study: 5, 50, 100, 250, 500 µg/ml (5% S9)
(500 µg/mL is the highest soluble concentration in acetone).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: not reported
- Justification for percentage of solvent in the final culture medium: upper limit of solvent volume is 0.5 % as per the study plan

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate in initial experiment, triplicate in confirmatory experiment
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10E5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hrs
- Harvest time after the end of treatment (sampling/recovery times): 19 hrs incubation
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours
- Method used: microwell 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: 6-thioguanine (10E-3 M) for 7-days following expression time
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x 10E5 cells/plate. The colonies were fixed in methyl alcohol, stained with a dilute crystal violet solution, counted and the clone numbers recorded.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in colony forming ability
- Any supplementary information relevant to cytotoxicity: a range-finding for cytotoxicity was performed using a 5-hour treatment at concentrations ranging from 0 to 500 µg/mL +/-S9 mix (1, 2, 5 and 10%). A single replicate per culture was included. Incubation during 19 hours following test substance removal.

METHODS FOR MEASUREMENTS OF GENOTOXICIY:
- Method: mutant frequency (by correcting the total number of mutant clones by the cloning efficiency of the cells at the time of mutant selection)

Rationale for test conditions:

The highest concentration (500 µg/mL) was the highest soluble concentration in the solvent acetone.

Evaluation criteria:

Positive:
a/ the mean mutation frequencies of at least one of the 3 highest test article concentrations, with a mean survival rate of at least 10% are significantly greater than that of the solvent control (p ≤ 0.01 based on pooled inter-group variance); and,
b/ the change in mean mutation frequency with increasing test article concentration exhibits a significant (p ≤ 0.01) linear component of the dose-response relationship up to a maximum toxicity level of 90%.

Negative:
a/ none of the the mean mutation frequencies of any of the 3 highest test article concentrations with a mean survival rate of at least 10% are significantly greater than that of the solvent control (p ≤ 0.01); and,
b/ the change in mean mutation frequency with increasing test article concentration does not exhibit a significant (p ≤ 0.01) linear component of the dose-response relationship up to a maximum toxicity level of 90%.

Statistics:

Dose-response analysis were performed on transformed mutation frequency data by the one-way analysis of variance method outlined by See et Irritation (1981). Dose-response was considered significant if p ≤ 0.01. A computer program was used to analyse the raw data.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test article solution appeared to precipitate slightly at the 500 µg/mL solution

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
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not soluble
- Precipitation and time of the determination: slight precipitate observed at 500 µg/mL.
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: none reported

CYTOTOXICITY STUDY:
At 500 µg/mL, 30.5, 37.7, 24.5 and 43.2% relative initial survival was noted at the 1, 2, 5 and 10% concentrations of S-9 respectively and 23.3% without (0%) metabolic activation.

PRELIMINARY STUDY:
The concentrations selected to yield a 10 to 100% survival were 5, 200 and 500 µg/mL, they resulted in approximate mean relative cell survivals of:
- 88, 60 and 39%, with 1% S9-mix;
- 92, 67 and 54%, with 2% S9-mix;
- 96, 84 and 92%, with 5% S9-mix;
- 97, 69 and 67%, with 10% S9-mix;
- 95, 60 and 19 % without S9-mix.
At these dose levels, there were no increase in the mutant frequencies observed in the treated cultures when compared to the negative controls.

STUDY RESULTS (cf attached Results tables)
- Concurrent vehicle negative and positive control data: appropriate response were obtained.
- Results from cytotoxicity measurements: he approximate mean relative cell survivals at 5, 50, 100, 200 and 500 µg/mL were 95, 76, 62, 42 and 31% without metabolic activation and 91, 82, 63, 58 and 43% with metabolic activation.
- Genotoxicity results: There were no statistically significant increases in the mutation frequencies of the treated cultures when compared to the negative controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: within historical range

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Executive summary:

In a mammalian cell gene mutation assay performed similarly to OECD TG 476 (hprt test) and in compliance with GLP, Chinese Hamster Ovary Cells, clone K1 cultured in vitro were exposed to the test item diluted in acetone up to precipitating concentration (500 µg/mL) in the presence and absence of mammalian metabolic activation (10% rat S9). A cytoxicity assessment was initially performed, followed by a preliminary mutagenicity screen and a main CHO/hprt mammalian cell forward gene mutation assay.

 

The positive controls (Ethylmethanesulfonate and Dimethylnitrosamine) did induce the appropriate response.  

 

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