1-[2-fluoro-6-(trifluoromethyl)benzyl]-5-iodo-6-methylpyrimidine-2,4(1H,3H)-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27th September 2018 to 12th October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 476 'In vitro Cell Gene Mutation Test (2013)

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

687-893-6

Cas Number:

1150560-54-5

Molecular formula:

C13H9F4IN2O2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: mouse lymphona cell line L5178Y

For cell lines:
- Absence of Mycoplasma contamination: Confirmed
- Cell cycle and doubling time : Normal
- Periodically checked for karyotype stability: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
temperature 35.6 - 37.9 oC,
humidity 5.0% CO2
Media RPMI plus horse serum (0%, 10% and 20%)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 prepared in-house

Test concentrations with justification for top dose:

Based on the prelimary experiment, the doses of this test were 0.1, 0.26, 0.64, 1.6, 4 and 10ug/plate. Blank control, solvent control (dimethylsulfoxide) and positive controls have been included. Duplicate cultures were made per dose in the test.

Vehicle / solvent:

Dimethylsulfoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10 5
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours with metabolic treatment, 3 hours without metabolic treatment, 3 hours without metabolic treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Method used: microwell plates for the mouse lymphoma assay.
- Selective agent: Trifluorothymidine (TFM), 3ug/ml, incubated for 12 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2000 cells/well

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF)

Evaluation criteria:

When the IMF (Induced Mutant Frequency) in one or several doses is greater than the GEF of 126 x 10 6 and the increase is concentration related and can be replicated, the result is evaluated as positive.
If this criteria is not met, the result is evaluated as negative.

The increase is dose-related or reproducible.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1μg/ml were 25%, 37%, 60%, 96%, 90% and 91%. The  RTG of the cells exposed for 3 hours in the presence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml were 38%, 45%, 56%, 76%, 105% and 107%.The  RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml were 34%, 53%, 61%, 81%, 76% and 80%.

The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 10 6.

Applicant's summary and conclusion

Conclusions:

The results of this study are negative in the three treatment conditions, so it can be concluded that the test item Methyl Iodouracil is non-mutagenic to the cultured L5178Y mouse lymphona cells under the conditions of this study. 

Executive summary:

This study was performed to assess Methy Iodouracil for its ability to cause gene mutations in vitro cultured mammalian cells L5187Y mouse lymphona cells (TK^± -3.7.2C) after being exposed for 3 hours with and without metabolic action and 24 hours without metabolic action respectively. The method was designed to be compatible wth PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 476 'In vitro Cell Gene Mutation Test (2013)'.

L5178Y cells were exposed at 6 doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml in 3 treatment conditions. The positive and solvent (DMSO) controls were included at the same time in each treatment. The dose volume of each dose group and solvent control were 5μg/ml medium in duplicate. Plating Efficiency (PE) of the cells after exposure was determined and the Relative Total Growth (RTG) was calculated to evaluate toxicity. The Mutant Frequency (MF), Induced Mutant Frequency (IMF) of each culture and the Induced Mutant Frequency of small clone (IMF_SC) of the highest dose (10 μg/ml ) and all controls were determined after the inhibition with Trifluorothymidine (TFT). In this test, the results of the solvent and positive control met all validity criteria so the sensitivity of the assay and efficacy of the S9 mixture were validated. 

In three treatment conditions, no test item precipitate was observed in any cell culture at all doses before and after incubation. 

The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1μg/ml were 25%, 37%, 60%, 96%, 90% and 91%. The  RTG of the cells exposed for 3 hours in the presence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml were 38%, 45%, 56%, 76%, 105% and 107%.The  RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of 10, 4, 1.6, 0.64, 0.26 and 0.1 μg/ml were 34%, 53%, 61%, 81%, 76% and 80%.

The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 10⁶.

The results of this study are negative in three treatment conditions above, so it can be concluded that the test item Methyl Iodouracil is non-mutagenic to the cultured L5178Y mouse lymphona cells under the conditions of this study.