5-(2-Fluoro-3-methoxyphenyl)-1-[[2-fluoro-6-(trifluoromethyl)phenyl]methyl]-6-methyl-2,4(1H,3H)-pyrimidinedione

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:

20 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

5-(2-fluoro-3-methoxyphenyl)-1-{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

EC Number:

855-578-4

Cas Number:

1150560-59-0

Molecular formula:

C20H15F5N2O3

IUPAC Name:

5-(2-fluoro-3-methoxyphenyl)-1-{[2-fluoro-6-(trifluoromethyl)phenyl]methyl}-6-methyl-1,2,3,4-tetrahydropyrimidine-2,4-dione

Test material form:

solid: particulate/powder

Method

Target gene:

Histidine locus in several strains of Salmonella typhimurium and tryptophan locus of Escherichia coli strain WP2uvrApKM101

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) was purchased commercially. It was prepared from male Sprague-Dawley rats that were injected (intraperitoneally) with Aroclor(TM) 1254 (200 mg/mL in corn oil), at a dose of 500 mg/kg, five days before sacrifice.

S9 mix was prepared by standard procedures on the day of use and kept on ice until needed.

Test concentrations with justification for top dose:

17, 28, 47, 78, 129, 216, 359, 599 and 998 µg/well

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Controls

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene

Details on test system and experimental conditions:

Inoculation:
Overnight cultures were inoculated into flasks containing culturing broth and the flasks were placed in a shaker/incubator programmed to begin operation (shaking, 130± 25 rpm; incubation, 37 ± 2°C) so that overnight cultures were in late log phase when optical density (OD) monitoring began.

To ensure cultures were harvested at the appropriate phase, the growth of the culture was monitored using a spectrophotometer. An aliquot of each culture was used to determine the OD at 660 nm. Cultures were analyzed by OD so that an OD of 1.5 to 2.5 was reached. This suggested that the bacterial growth was in log phase growth and bacteria were at a concentration of ≥ 1 x 10^9 cells/mL. A cell viability test was conducted concurrent to the bacterial test to ensure that there were an excess of viable cells plated. Once the incubation period has ended, the cultures were removed and were used the same day after the target OD was reached.

Tester Strain Characterization:
All tester strains were checked, at a minimum, for the following phenotypic characteristics.

Tester Strain Media:
Culturing Broth: Nutrient broth was used to grow overnight cultures of the tester strains.
Minimal Bottom Agar Plates: Bottom agar (4-5 mL per well of a 6-well plate) was 1.5% (w/v) agar and 2.0% (w/v) glucose in Vogel-Bonner minimal medium E.
Nutrient Bottom Agar Plates: Nutrient bottom agar (4-5 mL per well of a 6-well plate) was 7.5% (w/v) agar and 12.5% (w/v) nutrient Broth No. 2.
Top Agar for Selection of Revertants: Top agar contained 0.5% (w/v) agar and 0.5% (w/v) NaCl, supplemented with 0.5 mM histidine, 0.5 mM biotin, and 0.5 mM tryptophan.

Test Article Handling:
The test articles were prepared in the selected vehicle (DMSO). Test article formulations were prepared on the same day as use.

Mutagenicity Assay:
Six-well plates were used and each testing condition was conducted in a well of the 6-well plate. Using such wells, the limit dose of 5000 µg/well, suggested by OECD 471, was reduced to 1000 µg/well based on the surface area of the well compared to that of a Petri plate. The assay was performed using the plate incorporation method. In the absence of any solubility limitations, the test article was evaluated in the mutagenicity assay at concentrations 17, 28, 47, 78, 130, 216, 360, 600 and 1000 µg/well. Positive and vehicle controls were also evaluated. All test and positive control article concentrations were evaluated using triplicate wells. Vehicle controls were evaluated using 6-wells.

Frequency and Route of Administration:
Tester strains were exposed to the test articles via the plate incorporation methodology originally described by Maron and Ames. In the plate incorporation methodology, the tester strain, test article, and S9 mix (where appropriate) were combined in molten supplemented top agar, which was then overlaid on minimal bottom agar in a well of a 6-well plate. Following incubation, revertant colonies were counted.

Plating Procedures:
Each 6-well plate was labeled with the study number, date, test article, tester strain, activation condition, and concentrations. Treatments in the absence of S9 were performed by adding 20 µL test or control articles, 100 µL of phosphate buffered saline (PBS) and 25 µL tester strain to sterile tubes containing 0.5 mL molten supplemented top agar (maintained at 47± 2°C). The mixture was overlaid onto the surface of bottom agar wells and gently spread with swirling of the plate. The plates were kept at room temperature until the top agar solidified. After the top agar solidified, the 6-well plates were inverted and incubated for two days at 37± 2°C. When S9 was required, 100 µL S9 mix were used instead of PBS.

Scoring the Plates:
Plates not evaluated immediately following the incubation period were refrigerated (2 to 8°C) until colony counting and bacterial background lawn evaluation could take place.

Bacterial Background Lawn Evaluation:
The condition of the background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control.

Counting Revertant Colonies:
Revertant colonies were counted by hand or automated colony counter. Colony counts were transcribed to a Microsoft Excel spreadsheet. When there was sufficient test article precipitate or other interference that prevented automated analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dephenyl tetrazolium bromide) was used to distinguish bacterial colonies from precipitate particles. These wells were manually counted.

Evaluation criteria:

Criteria for a Positive Response:
A test article was considered to have produced a positive response if it induced a concentration-dependent increase in revertant frequency that was ≥ 2.0-fold vehicle control values for tester strains TA98, TA100, and E. coli, or ≥ 3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.

Criteria for a Negative Response:
A test article was considered to have produced a negative response if no concentration-dependent, ≥ 2.0-fold or ≥ 3.0-fold increases were observed in tester strains TA98, TA100, and E. coli, or TA1535 and TA1537, respectively.

Criteria for an Equivocal Response:
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the
concentration-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response. Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

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

Vehicle controls validity:

valid

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was precipitation beginning at 216 µg/well with the test material in all strains with metabolic activation. There was precipitation beginning at 78 µg/well with the test material in all Salmonella strains and at 130 µg/well with E. coli without metabolic activation.



There was no cytotoxicity present at any concentration of the test material with all five strains with and without metabolic activation.

Mean revertant frequencies for all concentrations of the test material in all five tester strains with and without S9 approximated those observed in the concurrent mean vehicle control cultures.

Any other information on results incl. tables

All positive and vehicle control values were within acceptable ranges. All genetic markers were verified. Therefore, all criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:

The results indicate that the test material was negative (non-mutagneic) in the 6-well 5-strain bacterial reverse mutation assay under the conditions, and according to the criteria of the study plan.