N-tert-butylbenzothiazole-2-sulphenamide

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference 1

Reference Type:

other: cited in OECD SIDS 2003

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

other: OECD SIDS

Title:

SIDS Initial Assessment Report, N-tert-butylbenzothiazole-2-sulphenamide, CAS No 95-31-8

Author:

OECD

Year:

2003

Bibliographic source:

UNEP Publications

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

N-tert-butylbenzothiazole-2-sulphenamide

EC Number:

202-409-1

EC Name:

N-tert-butylbenzothiazole-2-sulphenamide

Cas Number:

95-31-8

Molecular formula:

C11H14N2S2

IUPAC Name:

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

Details on test material:

N-tert-butylbenzothiazole-2-sulphenamide

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

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

Duration of treatment / exposure:

24 and 48 h

Frequency of treatment:

once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.