.beta.-Alanine, N-[[2-[[(2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl)phenylamino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]-N-2-pyridinyl-, ethyl ester, acetate (1:2)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 June 2004 - 01 July 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2004

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The chemical intermediate BIBR 1048 Oxa-Amidin was investigated in a modified
bacterial reverse mutation test as described by Ames et al. (1975). The traditional
Salmonella microsome mutation assay (Ames test) is used extensively as a routine test for
mutagenicity for more than 25 years. The high throughput microtitre-based version, called
Ames II, is based on the same genetic principle (base-pair substitution and frameshift
mutations in the his operon of S. typhimurium) as the traditional Ames test but combined
with the fluctuation method (Gee et al., 1998). Because of its high concordance of 80%,
the Ames II procedure seems an effective screen for identifying bacterial mutagens
(Flueckiger et al., 2004). Due to its explorative character and the lack of regulatory
acceptance these data are, however, intended for internal use only.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

bis(acetic acid); ethyl 3-{1-[1-methyl-2-({[4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl]amino}methyl)-1H-1,3-benzodiazol-5-yl]-N-(pyridin-2-yl)formamido}propanoate

EC Number:

925-312-2

Cas Number:

1184044-93-6

Molecular formula:

C28 H27 N7 O5 x 2 C2 H4 O2

IUPAC Name:

bis(acetic acid); ethyl 3-{1-[1-methyl-2-({[4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl]amino}methyl)-1H-1,3-benzodiazol-5-yl]-N-(pyridin-2-yl)formamido}propanoate

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA7001 - TA7006 and TA98

Metabolic activation:

with and without

Metabolic activation system:

0.2 mL strain mixture and 0.04 mL S9-mix (30%) were used.

Test concentrations with justification for top dose:

1 to 5000 μg/mL medium with and without microsomal rat liver enzymes (Aroclor 1254-induced).

Vehicle / solvent:

dimethylsulfoxide DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

4-nitroquinoline-N-oxide

2-nitrofluorene

Rationale for test conditions:

The experiment is regarded valid, 1. if the vehicle control showed the normal spontaneous
revertant frequency. 2. the diagnostic mutagens caused the expected increase in the
mutation rate.

Evaluation criteria:

The individual test chemicals were classified according to the following
criteria:
1. Negative: ≤8/48 wells Equivocal: 9-12/48 wells Positive: ≥13/48 wells
Historical control range: 0-7/48 wells in ca 220 experiments (1999-up to date)
2. A concentration-dependent increase of revertant wells (mean of triplicate) over the vehicle
control is indicative for a genotoxic activity of the test substance.

Statistics:

1. The pH indicator bromocresol purple turns the colour of the cultures from blue to yellow as the pH drops due to the accumulation of catabolites from the metabolic activity of revertant cells.
2. The number of positive wells (yellow) out of a total of 48 wells is an indication of the frequency of reversion per replicate per dose and was compared to the number of spontaneous revertant wells of the solvent control.
3. Each test point contains 48 wells of a 384-well plate. In each 48-well section, the wells were scored for the number of revertant wells (yellow) and the mean value of the triplicates was calculated

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA7001 - TA7006 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to precipitating concentrations of 5000 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

Based on the described results it is concluded, that BIBR 1048 Oxa-Amidin, when tested up to insoluble concentrations, caused neither base-pair substitutions nor frameshift mutations in bacteria. No evidence of genotoxic activity was observed in a series of S. typhimurium tester strains (TA Mix and TA 98) in the absence and presence of metabolic activation. The test article is, therefore, classified as "Ames II negative".