Registration Dossier

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 Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Isobutylidenediurea, purity 90.1 weight %

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf
- Initial age at start of acclimatization: 8 - 12 weeks
- Weight at study initiation: 35.0 g (SD+- 2.1 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single in Makrolon Type I, with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 3
- Humidity (%): 22 - 70%
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: CMC (carboxymethyl cellulose), 0.5% aqueous suspension
- Justification for choice of solvent/vehicle: The vehicle was chosen to its non-toxicity for the animals.
- Amount of vehicle: All animals received twice a standard volume of 10 ml/kg b.w. orally.
Duration of treatment / exposure:
two applications at an interval of 24 hours. Bone marrow cells were sampled 24 hours after the last application
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
six
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, purity at least 98%
- Justification for choice of positive control: The stability of CPA at room temperature is good . At 25°C only 3.5 % of its potency is lost after 24 hours
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw., Volume administered: 10 ml/kg bw.

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE) were analyzed for micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly
or 2000 mg/kg as the upper limit for non-toxic test articles.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions after double treatment at an interval of 24 hours
without having major effects on survival within 24 hours after the second administration .
The volume to be administered should be compatible with physiological space available .
Three adequate spaced dose levels were applied : 500, 1000, and 2000 mg ISOBUTYLIDENEDIUREA per kg body weight .

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 18 hours before each treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals
(including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight.
The animals received the test article or the vehicle twice at an interval of 24 h, the positive control substance was applied once. Six males were treated per dose group. Sampling of the bone marrow was done 24 hours after the last treatment.


DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with
fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded.
A small drop of the resuspended cell pellet was spread on a slide . The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-791 10 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 as quotient PCEs/NCEs. The analysis was performed with coded slides.
Evaluation criteria:
The study is considered valid as the following criteria are met :
- the vehicle controls are in the range of our historical control data 0.04 - 0.16% PCEs with micronuclei (October 1997 - October 1998; males).
- the positive controls show statistically significant increased values (historical range: 1.27 - 2.82% PCEs with micronuclei (October 1997 - October 1998; males).
- more than 80 % of animals are evaluable
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
Statistics:
non-parametric Mann-Whitney test

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
the animals expressed toxic reactions
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 - 2000 mg/kg bw.
- Clinical signs of toxicity in test animals: reduction of spontaneous activity, eyelid closure, apathy

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no significant increase of PCE's with micronuclei at any dose level tested
- Ratio of PCE/NCE (for Micronucleus assay): vehicle: 2000/1922
500 mg/kg bw.: 2000/1922
1000 mg/kg bw.: 2000/2217
2000 mg/kg bw.: 2000/2017
positive control: 2000/1996
- Appropriateness of dose levels and route: yes
- Statistical evaluation: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Any other information on results incl. tables

The test substance did not induce micronuclei in bone marrow cells of mice when tested in doses up to the limit dose recommended by current guidelines. In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant increase in the frequency of micronuclei. The mean values of micronuclei were in the same range as the vehicle control group and within the laboratory`s historical negative control range (0.04 - 0.16%). 

The highest dose was the maximum guideline-recommended daily dose and was selected on basis of the results from a range-finding study. In the range-finding experiments 3 males were treated with 1000 mg/kg bw (twice at an interval of 24 hours) and 3 males and 3 females each were treated with 1500 or 2000 mg/kg bw, also given twice at an interval of 24 hours. All treated animals showed reduced spontaneous activity at 6 or 24 hours after the first and/or the second treatment and eyelid closure and apathy. With regard to toxicity, no difference between the sexes was noted. 

The test substance had no substantial cytotoxic effects on bone marrow cells. 

The positive and vehicle controls were functional, i.e. they were in the range of the respective historical controls. The stability of the test substance in the vehicle was analytically verified for the study period.

Applicant's summary and conclusion