2-Propyl-4-methyl-6-(1-methylbenzimidazol-2-yl)benzimidazole

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

Study period:

2011-01-13 to 2011-0602

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 474 and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA Pesticide Assessment Guidelines Subdivision F Series 84-2

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guidelines and Japanese MHLW Guidelines on Genotoxicity Testing (Notification No. 1604 of the Pharmaceutical Affair Bureau of MHW, November 1st, 1999)

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: recommendations published by the US Environmental Protection Agency Gene-Tox Program and the Japanese Collaborative Study Group for Micronucleus Testing (Mavournin et al, 1990; CSGMT/JEMS.MMS, 1990)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Propyl-4-methyl-6-(1-methylbenzimidazole-2-yl)benzimidazole

IUPAC Name:

2-Propyl-4-methyl-6-(1-methylbenzimidazole-2-yl)benzimidazole

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Storage condition of test material: at ambient temperature and dark.

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 UK, (Margate, CT9 4LT, UK); female and male CD-1 mice CD (Crl:CD-1)- Age at study initiation: 7 weeks- Weight at study initiation: male 30.3 - 38.8 g, female 24.1 - 28.1 g- Assigned to test groups randomly: yes- Housing: individually- Diet (e.g. ad libitum): ad libitum, except for 2-4 h prior to dosing and 1-2 h after dosing- Water (e.g. ad libitum): ad libitum- Acclimation period: no dataENVIRONMENTAL CONDITIONS- Temperature (°C): 17.6 - 22.4 °C- Humidity (%): 30 - 68%- Air changes (per hr): at least 15 air changes per hour- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: carboxymethyl cellulose (1%, w/w, 10 mL/kg/day)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test item was dissolved in 1% CMC to give the required dosing concentrations, as near to the time of dosing as practicable. The dose volume used for both the vehicle control and test item treated animals was a constant 10 mL/kg body weight.Cyclophosphamide was prepared fresh as a solution in water for irrigation (5 mg/mL). It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg.All formulations were dosed within 2 h of preparation with the exception of toxicity group 4 on day 1.

Frequency of treatment:

at 0 h and 24 h

Post exposure period:

marrow samples taken 24 h after the last treatment

Doses / concentrations

Remarks:

Doses / Concentrations:50, 100, 200 mg/kg/dayBasis:actual ingested

No. of animals per sex per dose:

vehicle control: 5 males and 5 females50 mg/kg: 5 males100 mg/kg: 5 males200 mg/kg: 8 males and 8 femalespositive control: 3 males

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide- Route of administration: oral- Doses: 50 mg/kg/day orally dosed at 0h and 24 h

Examinations

Tissues and cell types examined:

Bone Marrow - Erythrocytes (normochromatic cells and polychromatic cells)

Details of tissue and slide preparation:

Mice were humanely killed by an appropriate method and one femur from each mouse was quickly dissected out and freed of adherent tissue. A small hole was made in the neck of one femur and the marrow flushed, using a syringe fitted with a hypodermic needle, into a centrifuge tube containing a 1:1 (v/v) mixture of foetal calf serum and trisodium citrate (0.8%, w/v) in Sorensen's buffer, (pH 6.8).The tubes were centrifuged to pellet the cells. All but a few drops of supernatant fluid were discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid.DETAILS OF SLIDE PREPARATION:Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube/animal. The smear was left to air dry, fixed in methanol and then stained in Giemsa stain (15%, v/v), to give optimum erythrocyte discrimination.The stained smears were gently rinsed in water for and left to air dry. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium.METHOD OF ANALYSIS:One of the prepared slides for every animal was selected for examination and the coded slides assessed blind by the same operator. At least two thousand (2000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined.As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded. In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis.The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.A suitable binocular microscope was used for the assessment. The scoring was done under a nominal magnification of x 1250 using x 12.5 magnification eye pieces and a x 100 oil immersion objective.

Evaluation criteria:

refer to "Any other information on materials and methods incl. tables"

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Animal Observations:

No animal deaths occurred in the main micronucleus test. No animals displayed signs of toxicity.

Vehicle Control Group:

The numbers of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in mice dosed with the vehicle 1% CMC (10 mL/kg/day), averaged 0.05%. This MN-PCE frequency conformed to the established in-house control range for vehicle treated mice (0.00-0.23% per 5 mice or 0.00-0.18% per 10 mice).

Positive Control Group:

Exposure of mice to the positive control agent cyclophosphamide (50 mg/kg/day), induced large increases in bone marrow micronuclei. The mean MN-PCE frequency for the mice was 1.47%. An evident increase in the number of MN-NCE was also observed. Bone marrow toxicity accompanied these findings as shown by a suppression of the PCE/NCE ratios.

Test Item Group:

There was no indication that the test substance induced bone marrow micronuclei in the treated mice. The highest MN-PCE frequency recorded for the test item was in the mid dose where an incidence of 0.08% was observed. There was no indication of bone marrow toxicity in any of the test item dose groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negativeThe test item did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 200 mg/kg/day in male and female CD-1 mice using a 0 h and 24 h oral dosing and 48 h sampling regimen.