4-[4-[4-[(3R, 5R)[5-(2,4-difluorophenyl) tetrahydro-5-(1H-1,2,4-triazol-1-yl-methyl)-furan-3-yl]methyloxy]phenyl]-1-piperazinyl]phenyl-2-[(1S,2S)(1-ethyl-2-phenylmethoxypropyl)]-2,4-dihydro-3Htriazol-3-one

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:

November 10th 1998 to December 1st 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[(2S,3S)-2-(benzyloxy)pentan-3-yl]-4-{4-[4-(4-{[(3R,5R)-5-(2,4-difluorophenyl)-5-[(1H-1,2,4-triazol-1-yl)methyl]oxolan-3-yl]methoxy}phenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one

EC Number:

938-153-9

Molecular formula:

C44H48O4N8F2

IUPAC Name:

1-[(2S,3S)-2-(benzyloxy)pentan-3-yl]-4-{4-[4-(4-{[(3R,5R)-5-(2,4-difluorophenyl)-5-[(1H-1,2,4-triazol-1-yl)methyl]oxolan-3-yl]methoxy}phenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one

Details on test material:

White powder

Method

Target gene:

five histidine-requiring strains

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S-9

Test concentrations with justification for top dose:

50-5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:; DMSO;

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

(2-Aminoanthracene)

Positive control substance:

other: 1 ug/plate (TA100 and 2 ug/plate for TA1535 and TA 1537) and 10ug/plate WP2 uvrA (pKM101)

Remarks:

without s-9

Controls 2

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

without S-9

Migrated to IUCLID6: 5ug/plate (TA98)

Controls 3

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

with S-9

Migrated to IUCLID6: 0.22 μg/plate for strain TA98

Controls 4

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

3 μg/plate for strain TA100, 5ug/plate TA 1535 and 2 ug/plate WP2uvrA

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

with S-9

Controls 5

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with s-9

Migrated to IUCLID6: 80 ug/plate(TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation method)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered positive if:
a) test substance should induce a reproducible, dose-related and statistically signficant increase in revertent count in at least one strain of bateria.
b). If a greater than two fold increasein revertant count is observed in 2 experiments

Statistics:

Dunnetts method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

precipitate white powder observed ator above 1500 ug/plate but had no effect on scoring of revertent colinies.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The vehicle (DMSO) control plates gave counts of revertant coloonies within the normal range. All of the positive contol chemicals used in the test induced marked increases in the frequiecy of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated.

Applicant's summary and conclusion

Conclusions:

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 micrograms per plate. A white, powdery precipitate was observed at and above 1500 micrograms per plate, this did not prevent the scoring of revertant colonies.