.beta.-Alanine, N-[3-amino-4-(methylamino)benzoyl]-N-2-pyridinyl-, ethyl ester

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:

06 Dec 2001 to 07 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 3-{1-[3-amino-4-(methylamino)phenyl]-N-(pyridin-2-yl)formamido}propanoate

EC Number:

606-728-0

Cas Number:

212322-56-0

Molecular formula:

C18 H22 N4 O3

IUPAC Name:

ethyl 3-{1-[3-amino-4-(methylamino)phenyl]-N-(pyridin-2-yl)formamido}propanoate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

other: CrlGlxBrlHan : WI (SPF quality)

Details on species / strain selection:

Rats were used since these are suitable animal species for this kind of study and historical control data are available. A non-GLP range finding study with the test article BIBR 1048 Diamin-CDBA 122 BS showed no distinct sex-specific differences in toxicity. Therefore, only male animals were used.

Sex:

male

Details on test animals or test system and environmental conditions:

The animals were housed in groups of 5 animals in Noryl cages, type IV, under standardized air conditioned environment.
Temperature at 17-23 degrees Celsius
Light/darkness cycle: 12/12 hours
Illumination period: 6:00am -6:00pm
Average Illumination: Ca 60lx (dependent on the cage position). During the experimental and observational periods, the light intensity was increased up to 100 lx.
Air circulation: Ca 12 air changes per hour
Temperature and humidity were recorder continuously. The cages were changed weekly. Sterilized wood granules served as bedding. Food Nafag 9433 (Eberle Nafag AG), Gossau/Switzerland) and municipal tap water were provided ad libitum.
The food and water were considered to be free of any contaminants that could have influenced the outcome of the study. The analytical data were filed by the Section Biological Laboratory Service and the Municipal Water Supply Agency, respectively.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

0.5% Cremophor

Details on exposure:

A previous acute oral toxicity study with BIBR 1048 Diamin-CDBA 122 BS caused no marked toxicity up to 5000 mg/kg (Weiglib, 1998). Due to lack of any exposure data such as plasma concentrations, it is unclear to which extent the test article was absorbed. According to existing guidelines a maximum tolerated dose, a dose producing some indications of cytoxicity or the maximum feasible dose should be used for this kind of assay. Using ip injection of 0.5% cremophor suspensions no clinical signs of toxicity were observed at 100 mg/kg (once) and 500 mg/kg BIBR 1048 Diamin-CDBA 122 BS (twice within 24 hrs) in an explorative range-finding study (non-GLP). Higher concentrations could not suspended homogenously. Therefore, the higher dose level of 500 mg/kg and two lower doses of 150 and 50 mg/kg were investigated. A vehicle control (0.5% cremophor) and a positive control group (cyclophosphamide 20 mg/kg) were included. The injected dose volume was 10ml/kg. Animals of all groups were treated twice at an interval of 24 hours.

Duration of treatment / exposure:

24 hrs, 48 hrs

Frequency of treatment:

twice per interval

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day

No. of animals per sex per dose:

5 male rats/group

Control animals:

yes

Positive control(s):

cyclophosphamide, 20 mg/kg

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes
Red Nor-mochromatic erythrocytes

Details of tissue and slide preparation:

Animals were anaesthesized and killed by cervical dislocation 24 hrs post second treatment. The bone marrow of one femur was flushed and suspended in fetal calf serum (FCS). Anucleated Erythrocytic cells were separated from other myeloic cells using cellulose column fractionation. This purification step enables the preparation of slides containing only polychromatic (PE) and normochromatic erythrocytes (NE) without any nucleatic cells or mast cell granules which can occur particularly in rats (Romagna and Staniforth, 1989). Smears were prepared from the sediment after centrifugation of the eluate on cleang and grease-free slides. One slide per animal was stained with May-Grunwald/Giemsa. The slides were mounted with Entellen (Merck, Darmstadt), coded and scored.

Evaluation criteria:

The micronucleus test is considered valid if the positive control chemical induces a statistically significant increase in the frequency of micronucleated PE and a sufficient high number of animals is available for analysis.
The criterion for a positive result is a statistically significant, dose-dependent increase in the frequency of micronucleated polychromatic erythrocytes in treated animals as compared with controls. Additionally, historical control frequencies obtained in similar experiments using this rat strain are taken into consideration.

Statistics:

The statistical analysis of the data was performed using the Fisher-Pitman permutation test (Leimer et al., 1991). This method takes into account the characteristics of the micronucleus assay (the animals is the experimental unit, small absolute incidences with non-normal distributions). All comparisons were made against the vehicle control. A probability of P =<5% was considered statistically significant. All calculations were done in the Biometrics Group, Department of Medical Services.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

This experiment with male rats demonstrated thar BIBR 1048 Diamin-CDBA 122 BS did not damage the chromosome complement (structural and numerical) in vivo when given up to maximal feasible dose levels following intraperitoneal injection.