(+-)-2,4-dihydro-4-[4-[4-(4-methoxyphenyl)-1-piperazinyl]phenyl]-2-(1-methylpropyl)-3H-1,2,4-triazol-3-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005-01-06 to 2005-02-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Only three strains are tested. However, an expert statement is added in the field "any other remarks" to justify the fact that no further testing is necessary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 3 bacterial strains used; the purity of the test substance is unknown and limited methodology is described

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(butan-2-yl)-4-{4-[4-(4-methoxyphenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one

EC Number:

607-685-0

Cas Number:

252964-68-4

Molecular formula:

C23H29N5O2

IUPAC Name:

1-(butan-2-yl)-4-{4-[4-(4-methoxyphenyl)piperazin-1-yl]phenyl}-4,5-dihydro-1H-1,2,4-triazol-5-one

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-7982559-AAA (T001329)
- Physical state: solid (powder)
- Appearance: Grey-brown powder

Specific details on test material used for the study:

Description: Pale purple powder
Batch number: 00434473
Date received: 2004-12-15
Storage conditions: Room temperature in the dark

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, and TA102

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, rat liver homogenate metabolizing system (10% in standard co-factors)

Test concentrations with justification for top dose:

0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl formamide
- Justification for choice of solvent/vehicle: no data

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 for TA100 at 3 µg/plate

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 for TA102 at 0.5 µg/plate

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 for TA98 at 0.2 µg/plate

Controls 4

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

With S9 for TA100 at 1 µg/plate

Controls 5

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 1,8-Dihydroxyanthraquinone (DAN)

Remarks:

with S9 for TA102 at 10 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 for TA98 at 5 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in agar (plate incorporation)

Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix of phosphate buffer. The contents of each test tub
e were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of the test material both with and without S9-mix.

DURATION
- Exposure duration: 3 days
- Selection time: 3 days, simultaneously with exposure

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the growth of the bacterial background lawn

OTHER:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate.

Evaluation criteria:

no data

Statistics:

no data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100, and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

based on maximum recommended dose of the guideline

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: A cream-coloured, flaky precipitate was observed at and above 1500 µg/plate, but this did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: No data were provided on a range-finding test.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle (DMSO) control plates gave counts of revertants colonies witin the normal range. All the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus the sensitivity of the assay and the efficacity of the S9-mix were validated. Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
ambiguous without metabolic activation

The test substance was evaluated for mutagenic potential using the "Ames Test" in S. typhimurium strains TA100, TA98 and TA102 in the absence and presence of metabolic activation. Based on the results and the expert statements, the test substance was considered to be non-mutagenic under the conditions of the study.