5-amino-6-methyl-1,3-dihydrobenzoimidazol-2-one

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:

2007-09-05 - 2007-11-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-410-9

EC Name:

-

Cas Number:

67014-36-2

Molecular formula:

C8 H9 N3 O

IUPAC Name:

5-amino-6-methyl-2,3-dihydro-1H-1,3-benzodiazol-2-one

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males (mean): 37.3 g; females (mean): 29.8 g
- Assigned to test groups randomly: yes
- Housing: single
- Diet: pelleted standard diet, ad libitum(Harian Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum. (Gemeindewerke, D-64380 Roßdorf)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: PEG400
- Justification for choice of solvent/vehicle: relatively non-toxic for the animals
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in PEG400.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

once

Post exposure period:

48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
actual ingested
24 hour preparation interval

Doses / concentrations 2

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
48 hour preparation interval

No. of animals per sex per dose:

5

Control animals:

yes

Positive control(s):

cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 40 mg/kg bw

Examinations

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A Pre-Experiment was conducted.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 xg) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (Merck, D-64293 Darmstadt)/Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex in the respective test group is usually evaluated in case an animal dies in its test group spontaneously.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results. A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

See "Evaluation criteria"

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

ruffled fur

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: ruffled fur (100 - 2000 mg/kg bw), reduction of spontaneous activity (1000, 2000 mg/kg bw), abdominal position (2000 mg/kg bw), eyelid closure (2000 mg/kg bw)
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: At the highest dose of 2000 mg/kg bw reduction of spontaneous reaction was only seen in the first 24 hours after exposure. Abdominal position and eyelid closure were only seen in two out of 4 animals during the first hour after exposure. Ruffled fur was seen in all animals during the whole examination time of the highest dosing group. On the basis of these data 2000 mg/kg bw were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay):
After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. However, the bedding of the treated animals showed orange colouration, which indicated that the urine of these animals contained the test item, confirming the bioavailability of the test item.
- Appropriateness of dose levels and route: was proven by the pre-test
- Statistical evaluation: was performed with non parametric Mann-Whitney test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative