N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study, but no internationally recognized protocol mentioned. Nevertheless, the study is very well documented and the described protocol is similar to OECD 474.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

EC Number:

407-430-1

EC Name:

N-(1,1-dimethylethyl)bis(2-benzothiazolesulfen)amide

Cas Number:

3741-80-8

Molecular formula:

C18H17N3S4

IUPAC Name:

bis(1,3-benzothiazol-2-ylsulfanyl)(tert-butyl)amine

Details on test material:

- Name of test material (as cited in study report): CP-22595
- Physical state: white powder
- Analytical purity: no data
- Lot/batch No.: 19APP01
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 50 days
- Weight at study initiation: 26-34g (males), 23-28g (females)
- Assigned to test groups randomly: randomized by body weight and assigned to groups by use of a random # table.
- Fasting period before study: yes, 4 hours before dosing
- Housing: stainless steel wire mesh cages, 5 mice of the same sex per cage
- Diet (e.g. ad libitum): Rodent Blox, Lot #022590-1, ad libitum
- Water (e.g. ad libitum): fresh tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 30-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 250 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg
- Lot/batch no. (if required): corn oil, Sigma Chemical Company, Lot #88F-0054
- Purity: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was weighed and diluted with corn oil. Good suspensions were obtained and maintained at all dose levels evaluated.
There were no impurities expected to have been present in the vehicle control which would have affected the outcome of the assay.

Duration of treatment / exposure:

single dose, mice were sacrificed after 24, 48 or 72 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 60 mg/kg bw

Examinations

Tissues and cell types examined:

erytrhopoietic cells of the mouse bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Preliminary dose-range-finding study: test substance was administered orally to 4 groups of CD-1 mice at dose levels of 1000, 2500, 3750 and 5000 mg/kg bw. Due to the pharmacotoxic signs observed in the study, 5000 mg/ kg bw was selected as the high dose in the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): single dosing. Animals sacrificed and bone marrow harvested after 24, 48 and 72 hours.

DETAILS OF SLIDE PREPARATION:
One end of each femur (one proximal, one distal), was opened carefully with a scissors until a small opening to the marrow canal became visible. A 1 mL tuberculin syringe filled with approximately 0.2 mL fetal bovine serum was inserted into the marrow cavity and the marrow was gently flushed (to assure maximum dispersion) into 1.0 mL of fetal bovine serim in a 5 mL round bottom culture tube. The femora were flushed with fetal bovine serum untill all the marrow was out and the bone appeared almost translucent. The suspension was centrifuged at 1000 rpm in a Sorvall RC-5 centrifuge with an HS-4 head for 5 minutes. The supernatant was removed leaving a small amount of fetal bovine serum with the remaining cell button. The button was mixed with a pasteur pipette to assure a homogenous mixture. The marrow smears were made by placing immediately a small drop of the cell suspension near the frosted end of a glass slide pre-cleaned in absolute ethanol and smeared by pulling the cell suspension behind with another pre-cleaned slide at a 45° angle. The slides were quickly dried on a slide warmer set at approximately 56°C, dipped in absolute methanol and air dried.

STAINING:
The slides were air-dried and then stained in Modified Wrights Stain Pak (4481) containing polychrome methylene blue and eosin, using an Ames Hema-Tek 1000 staining machine. Slides were air-dried and coversliped with Permaslip.

CRITERIA FOR SCORING MICRONUCLEI:
Micronuclei are uniform, darkly stained, typically round bodies in the ctoplasm of erythrocytes. Occasionally, micronuclei appear almond or tear drop shaped. Inclusions in PCEs which are reflective, impropertly shapes or stained, or which are not in the focal plane of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are scored as one micronucleated erythrocyte.

SLIDE ANALYSIS:
The slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained. One thousand PolyChromatic Erythrocytes (PCE) per animal were counted for the presence of micronuclei, and the number of micronucleated NormoChromatic Erythrocyets (NCE) present in the optical fields containing these 1000 PCE was also recorded. The data were expressed as the number of micronucleated PCE versus total normal PCE in 1000 total PCE per animal. A total of 1000 PCE and NCE was also counted per animal. These data were expressed as the PCE/NCE ratio.

Evaluation criteria:

Criteria for a valid test:
If the spontaneous rate of micronuclei in the PCE is less than 0.5% and the positive control is statistically greater (p<= 0.05) than the spontaneous rate and at least seven animals per group survived the treatment, the results are deemed acceptable.

Statistics:

Assessment of a test article as positive is based upon its ability to produce a statistically significant increase in the bumber of micronucleated PCE as compared to the vehicle control. One-tailed t tests were used to make pairwise comparisons between each treatment group and its concurrent vehicle control for statistically significant increases in the number of micronucleated PCE. The ratio of PCE/NCE was also calculated based on 1000 erythrocytes for each animal. The proportion of PCE per 1000 erythrocyters per animal was evaluated by pairwise two-tailed t tests after an arcsin transformation was performed. Statistical significance was judged at p <= 0.05 and p<= 0.01 levels. All comparisons were made for each sacrifice time separately comparing treated groups versus the vehicle control group.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
- Dose range: 500, 1000, 2500, 3750 mg/kg bw
- Solubility: no data
- Pharmacotoxic signs: mainly piloerection and ptosis -- effects not dose-related.

RESULTS OF MNT STUDY:
One female dosed with the negative control (corn oil) had decreased body tone at 24 hours.
No pharmacotoxic signs were observed in animals administered the positive control.
In the 5000 mg/kg bw dose group, 5 males and 6 females had piloerection at 24 hours, 3 females had decreased body tone and 4 females abnormal stance and gait.

Statistical analysis of the data for CP-22595 (5000 mg/kg bw) did not produce a significance increase in the number of micronucleated PCEs in the treated group versus their concurrent vehicle control group at any of the sacrifice times evaluated. Animals treated with positive control gave a statistically significant increase in the incidence of micronucleated PCE (p<= 0.01) and a significant depression of the PCE/NCE tratio (p<= 0.05).
Both the negative and the positive control are within the acceptable range of the historical data.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
The results of the test substance were negative for clastogenic effect in the micronucleus test at dose level of 5000 mg/kg bw administered in single oral doses with sacrifice times of 24, 48 and 72 hours. These findings are based upon the inability of the test article to produce a statistically significant increase in the incidence of micronucleated PCEs in the treated groups versus the vehicle control groups under the conditions of this assay.

Executive summary:

In a preliminary dose-range-finding study, the test substance was administered orally to 4 groups of CD-1 mice at dose levels of 1000, 2500, 3750 and 5000 mg/kg bw. Due to the pharmacotoxic signs observed in the study, 5000 mg/kg bw was selected as the high dose in the micronucleus test.

In the MNT, three groups of mice were given single doses of test substance by oral intubation at 5000 mg/kg bw and sacrificed at 24, 48 or 72 hours. Three similar groups of mice administered the vehicle control (corn oil), were evaluated concurrently at each sacrifice interval, serving as the negative control. An additional group of mice was administered cyclophosphamide (CP- at a dose of 60 mg/kg bw and sacrificed at 24 hours, serving as the positive control. Slides were prepared from the bone marrow of the femora and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE/animal. The ratio of polychromatic to normochromatic erythrocytes (NCE) per 1000 erythrocytes was determined for each animal.

Statistical analyses of the data did not indicate a significant increase in the number of micronucleated PCEs in the treated groups versus the vehicle control groups.

In conclusion, the test substance is not clastogenic in the micronucleus test at any of the time intervals evaluated under the experimental conditions of this protocol.