Benzimidazole-2-thiol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Micronucleated Erythrocyte Frequency in Peripheral Blood of B6C3F1 Mice from Short-Term, Prechronic, and Chronic Studies of the NTP Carcinogenesis Bioassay Program

Author:

Kristine L. Witt

Year:

2000

Bibliographic source:

Environmental and Molecular Mutagenesis

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The rodent bone marrow erythrocyte micronucleus (MN) assay was performed to screen the test chemical in vivo for genotoxicity resulting from clastogenic activity or mitotic damage.

GLP compliance:

not specified

Type of assay:

other: The rodent bone marrow erythrocyte micronucleus (MN) assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzimidazole-2-thiol

EC Number:

209-502-6

EC Name:

Benzimidazole-2-thiol

Cas Number:

583-39-1

Molecular formula:

C7H6N2S

IUPAC Name:

1H-benzimidazole-2-thiol

Details on test material:

- Test material identity: 2-mercaptobenzimidazole (benzimidazole-2-thiol)
- Substance type: Organic
- Physical state: Solid
- Molecular formula: C7H6N2S
- Molecular weight: 150.204 g/mol

Test animals

Species:

mouse

Strain:

B6C3F1

Details on species / strain selection:

No data

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Administration / exposure

Route of administration:

inhalation

Vehicle:

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

Details on exposure:

No data

Duration of treatment / exposure:

90days

Frequency of treatment:

6 hr/day, 5 day/week, over a period of 90 days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Doses / concentrations 2

Dose / conc.:

12.5 mg/m³ air

Doses / concentrations 3

Dose / conc.:

25 mg/m³ air

Doses / concentrations 4

Dose / conc.:

50 mg/m³ air

No. of animals per sex per dose:

Total: 40
0.0 mg/m3: 10 male and 10 female mice
12.5 mg/m3: 10 male and 10 female mice
25.0 mg/m3: 10 male and 10 female mice
50.0 mg/m3: 10 male and 10 female mice

Control animals:

yes, concurrent vehicle

Positive control(s):

No data

Examinations

Tissues and cell types examined:

Blood smear was observed for polychromatic and normochromatic eryhtrocytes and the micronucleus frequency was scores

Details of tissue and slide preparation:

METHOD OF ANALYSIS: Peripheral blood smears were prepared from B6C3F1 mice by the NTP contract laboratory conducting the toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Blood was obtained immediately before or at the time of sacrifice by retro-orbital bleeding; less often, other methods were employed, including heart puncture, puncture of the ventral tail vessels, or tail clip. The micronucleus frequencies in blood obtained by all four of these methods are similar.

DETAILS OF SLIDE PREPARATION: Slides were stained and analyzed at the USDA Western Regional Laboratory, under the direction of Dr. James MacGregor.
Drops of blood were spread on precleaned standard glass microscope slides, air dried, and fixed in absolute methanol for 5 min. Slides stained with acridine orange or
Hoechst 33258/pyronin Y were scored at 6303 or 10003 magnification by epifluorescence microscopy, and slides stained with Giemsa were scored. by Koehler microscopy at 1000X.

Evaluation criteria:

Criteria for identification of MN were those of Schmid [1976] with the additional requirement that MN exhibit the fluorescence emission characteristic of the fluorescent stain used (blue with UV excitation and orange with green [540nm] excitation with Hoechst/pyronin stain, or yellow to greenish yellow with acridine orange stain).

Polychromatic erythrocytes (PCE) were scored by direct manual counting.

Normochromatic erythrocytes (NCE) were scored using a semiautomated method described by Jauhar et al. [1988], in which cell counts were determined by counting a subfield of approximately 1/16th of the full microscope field. Routine micronucleus frequency scores were based on approximately 10,000 NCE or 1000 PCE per sample, and the percentage of PCE among the total erythrocyte population was based on the number of PCE among approximately 10,000 (USDA) erythrocytes.

Although statistical analyses were used as an important aid in evaluating the test results. A decision to classify a test as negative, equivocal, or positive for induction of micronuclei in this in vivo assay was based on a broader evaluation of a number of factors that determined the biological relevance of the results, including the appropriateness of the concurrent control data, the magnitude of the observed response and the presence of a dose-dependent increase in the frequency of micronucleated cells. Test chemicals were evaluated separately in each gender; results from tests in male and female mice were not combined to reach an overall conclusion. Positive control groups were not routinely included in these subchronic MN tests because the toxicity bioassays from which the test animals were obtained did not include positive control groups.

Statistics:

The frequency of micronucleated cells among NCE or PCE was analyzed by a statistical software package that tested for increasing trend over exposure groups using a one-tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group. In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation. Pairwise comparisons between each treatment group and the concurrent solvent control group were performed using an unadjusted one-tailed Pearson x2 test that incorporated the calculated variance inflation factor for the study. Generally, a test was considered positive if (1) the trend test P value was 0.025 or less or (2) the P value for any single exposure group was 0.025/N or less where N is the number of test chemical treatment groups. Trend test P values between 0.025 and 0.05 were considered to be equivocal if accompanied by a monotonic increase in the frequency of micronuclei over the dose range investigated. All other responses were considered to be negative.

The percentage of polychromatic erythrocytes (%PCE) data were analyzed by a standard ANOVA to determine if significant PCE suppression or stimulation occurred.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Systemic toxicity was observed in the female mice at all test concentrations and in the male mice at concentration levels of and above 6.25 mg/m3.

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

The result of the micronucleus test carried out at the end of a 90-day inhalation study must be considered relevant in accordance with OECD guideline No. 474, as in this 90-day study systemic toxicity was observed in the female mice at all test concentrations and in the male mice at concentration levels of and above 6.25 mg/m3, and also because the mice received test substance treatment upto the time of sacrifice.

Applicant's summary and conclusion

Conclusions:

Test substance did not induce an increase in the number of micronucleated polychromatic or normochromatic erythrocytes as compared with the controls.Thus, test substance was devoid of clastogenicity under these experimental conditions.Therefore, it is considered that test substance is non mutagenic.

Executive summary:

The rodent bone marrow erythrocyte micronucleus (MN) assay was performed to screen the test chemical in vivo for genotoxicity resulting from clastogenic activity or mitotic damage. The test chemical was dissolved in suitable solvent and used at dose levels of 0, 12.5, 25 or 50 mg/m³. In none of the groups treated with test substance were any of the male or female mice found to have increased numbers of micronucleated polychromatic or normochrom atic erythrocytes as compared with the controls.Thus, test substance was devoid of clastogenicity under these experimental conditions.Therefore, it is considered that the test substance is non mutagenic.