1H-Benzimidazole, 6-(2-chloro-5-fluoro-4-pyrimidinyl)-4-fluoro-2-methyl-1-(1-methylethyl)-

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:

16 Janauary 2009 to 21 January 2009

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

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

- Parameters analysed / observed: Two strain mutagenicity assay

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1-(propan-2-yl)-1H-1,3-benzodiazole

EC Number:

941-461-6

Cas Number:

1231930-42-9

Molecular formula:

C15H13CIF2N4

IUPAC Name:

6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-2-methyl-1-(propan-2-yl)-1H-1,3-benzodiazole

Specific details on test material used for the study:

- Source and lot/batch No.of test material: PX2-Z00174-169-3- Storage condition of test material: Room temperature in the dark with desiccant- Analytical purity: 100%

Method

Target gene:

Mutation of the uvrB gene (Salmonella)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Test concentrations with justification for top dose:

The test material was evaluated in the mutagenicity assay at doses of 1.50, 5.00, 15.0, 50.0, 150, 500, 1500, and 5000 micrograms/plate with and without S9. The maximum dose tested of 5000 micrograms/plate; this dose was achieved using a concentration of 100 mg/mL and a 50 micro/L plating aliquot

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dose formulations of the test material and dimethyl sulfoxide- Justification for choice of solvent/vehicle: The volume of Dimetyl Sulfoxide used has been shown not to effect survival or induce mutations in control cultures

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Tester Strain TA98 with and without S9 activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Tester strain TA100 with and without S9 activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation method - the tester strain, test article, and S9 mix (where appropriate) are combined in molten top agar, which is then overlaid onto a minimal bottom agar plate. Following incubation, revertant colonies are counted.DURATION: Plates are incubated for approximately 48-72 hours at 35-39oCSELECTION AGENT (mutation assays): Bacterial reverse mutation assayNUMBER OF REPLICATIONS: All test and control articles were evaluated in duplicate platesDETERMINATION OF CYTOTOXICITY: The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope

Rationale for test conditions:

Tester strain TA98 is reverted from auxotrophy to prototrophy by frameshift mutagens. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations

Evaluation criteria:

Evaluation of Results:For the test material to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentration of test material. Data sets for tester strains TA98 and TA100 were judged postive if the increase in mean revertants at the peak of the dose response was equat to or greater than 2 times the mean vehicle control value. Criteria for a Valid test:All salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa), the deletion in the uvrB gene and the presence of the pKM101 plasmid R-factor. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls as follows (inclusive): TA98, 10 - 50; TA100, 80 - 240The mean of each postive control must exhibit at least a 3.0 fold increase in the number of revertants over the mean value of the respective vehicle control. A minimum of three dose levels is required to evaluate assay data

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

These results indicate that LSN2833975 is negative in the Bacterial Reverse Mutation Assay when tested up to toxicity limiting dose levels with and without S9 metabolic activation, and under the conditions of the study protocol used.

Executive summary:

The objective of this study was to evaluate LSN2833975, for its ability to induce reverse mutations at the histidine locus in Salmonella typhimurium tester strains TA98, TA100.

All criteria for a valid study were met as described in the protocol. The results of the Salmonella Plate Incorporation Mutagenicity Assay indicate that, under the conditions of the study, intermediate LSN2833975 did not cause a positive mutagenic response with either the presence or absence of Aroclor-induced rat liver S9.