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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results from the in vitro mammalian cell gene mutation test, in vitro mammalian chromosome aberration test, and bacterial reverse mutation analysis were negative for genetic toxicity of the test material. Therefore the test material did not meet the GHS criteria to be classified as a genetic toxicant.

Link to relevant study records

Reference

Reference 1

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

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)

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

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

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.

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.

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. 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd August 2018 to 23rd October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: PRC-MEP - Guidelines for Testing of Chemicals (Health Effect, 2nd edition) 471 Bacterial Reverse Mutation Test (2013)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus in in several strains of Salmonella typhimurium (S. typhimurium; TA97a, TA98, TA100, TA102, and TA1535).

Species / strain / cell type:

other: S. typhimurium TA97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Based on the results of a previous preliminary test, six dose levels were selected - 1500, 500, 150, 50, 15 and 5 µg/plate, with and without metabolic activation.

Vehicle / solvent:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfide

Positive controls:

yes

Remarks:

with metabolic action

Positive control substance:

other: 2-Aminoanthracene

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfide

Positive controls:

yes

Remarks:

with metabolic action

Positive control substance:

other: 1,8-Dihydroxyanthraquinone

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfide

Positive controls:

yes

Remarks:

with metabolic action

Positive control substance:

sodium azide

other: Dexon

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfide

Positive controls:

yes

Remarks:

without metabolic action

Positive control substance:

other:

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA97a

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

Remarks on result:

other:

Remarks:

Methyl iodouracil is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Conclusions:

Under the conditions of this study, the results in both the first experiment and validation experiment were negative. Thus the test item Methyl iodouracil is considered to be non-mutagenic in the bacterial reverse mutation assay using histidine requiring tester strains of Salmonella typhimurium.

Executive summary:

The study was performed to evaluate the ability of Methyl iodouracil to induce reverse mutations in the genome of the histidine requiring tester strains of Salmonella typhimurium (S. typhimurium; TA97a, TA98, TA100, TA102 and TA1535) with and without the metabolic activation system (S9).
The method was designed to be compatible with PRC-MEP - Guidelines for Testing of Chemicals (Health Effect, 2nd edition) 471 Bacterial Reverse Mutation Test (2013). 
Five histidine requiring mutant tester strains of S. typhimurium (TA 97a, TA98, TA100, TA102 and TA1535A) were treated with Methyl iodouracil using the standard plate incorporation method and the pre-incubation method at six dose levels, in triplicate, with positive controls and solvent controls, both in the absence and presence of the co-factor suppplemented S9-mix.

Based on the results of the preliminary test (initial toxicity test), previously performed, six dose leves were selected (1500, 500, 150, 50, 15 and 5 µg/plate), with and without metabolic action. Dimethyl sulfoxide (DMSO) was used as solvent. Then the validation experiment was conducted using the same dose levels and solvent as the first experiment. 

In two experiments, the results of the viable count showed that the density of live bacteria in the cultures of each tester strain was within the acceptable range of 0.9 - 9 x 10⁶ colony forming units (CFU/ml). Concurrently all results of the  positive and solvent control values met the requirments of the tests, so the sensitivity of the test and the efficacy of the S9 mix were validated.

In the first experiment there were white precipitates at 5000 and 1500µg/plate dose levels on the MGA plate (monitoring of TA98), before and after incubation, with and without metabolic activation system. In the validation experiment the same results were obtained as in the first experiment. 

In the first experiment, no cytotoxicity was found in any dose level in all tested strains in two treated conditions. In the validation experiment the same results were obtained as in the first experiment. 

In the first experiment, with and without metabolic action, the mean number of revertant colonies at each dose level was two times less than that of the concurrent solvent control in TA97a, TA98, TA100 and TA102 and three times less than that of the concurrent solvent control in TA1535. In the validation experiment the same results were obtained as in the first experiment. 

Under the conditions of this study, the results in both the first experiment and validation experiment were negative. Thus the test item Methyl iodouracil is considered to be non-mutagenic in the bacterial reverse mutation assay using histidine requiring tester strains of Salmonella typhimurium.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

September 27 - October 12, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

From ATCC®CRL-1935

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat-liver S9 was prepared in-house and was used as the metabolic activation system.

Test concentrations with justification for top dose:

Dose selection was determined based on the results of the preliminary test, and the relevant requirements of PRC-MEP the Guidelines for the Testing of Chemicals (Health Effects, Second edition) No.473. The test was conducted respectively in three treatment conditions of exposure for 3 hours with and without metabolic activation, and exposure for 24 hours without metabolic activation. Five doses of 350, 112, 35, 11.2, and 3.5 µg/ml were included in each treatment condition along with the concurrent solvent and positive controls.

Vehicle / solvent:

DMSO(dimethylsulfoxide)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Cyclophosphamide monohydrate

Details on test system and experimental conditions:

A preliminary test for the In Vitro Mammalian Chromosome Aberration Test of the test material was performed in this lab to evaluate the cytotoxicity and solubility in the culture medium of the test item.

The CHL cells were used in the preliminary test, based on the results of test item solubility, DMSO was used as solvent. The highest dose is 350 µg/ml medium, the cells were exposed at 5 doses, including 350, 70, 17, 2.8 and 0.56 µg/ml medium for 3 hours and 24 hours without metabolic activation. The concurrent solvent control (DMSO) was included at each treatment condition in the test. The dose volumes of each dose group and solvent control group were 5 µl/ml medium. In two treatment conditions, the cultures were observed by naked eyes for test-item precipitation at the beginning and end of the treatment. The cells were counted 24 hours after the beginning of cell exposure, and cytotoxicity of the test item were evaluated according to cell survival number. The results of preliminary test showed that without metabolic activation, there was white precipitation of test item in the cell cultures at 350 and 70 µl/ml exposure concentration before and after incubation for 3 hours and only at 350 µl/ml exposure concentration before and after incubation for 24 hours. Moreover, the test item has significant cytotoxicity to CHL cells at 350 µg/ml medium exposure concentrations, and nearly no cytotoxicity at 14 µg/ml medium, and cytotoxicity was dose-related.

Dose selection was determined based on the results of the preliminary test. The test was conducted respectively in three treatment conditions of expsoure for 3 hours iwth and without metabolic activation,and exposure for 24 hours without metabolic activation. 5 doses of 350, 112, 35, 11.2, and 3.5 µg/ml were included in each treatment condition along with the concurrent solvent control (DMSO) and positive controls.

The test items (0.07047g and 0.02253g) were weighed and transferred into the calibrated aseptic tubes, respectively. DMSO was added into the tube and mixed with DMSO thoroughly to a certain volume (here: 1,000 ml) to obtain the test solution with the highest stock concentration of 70 mg/ml and 22.4 mg/ml, respectively. Under aseptic conditions, the test solutions of the other stock concentrations were prepared with 10-fold intervals by dilution respectively.

Approximately 2x 10^5 CHL cells were seeded in the cell culture plate and cultured overnight using 10% MEM. Duplicate cultures were included at each dose level and controls. Before being treated, the cells were washed with Hank's balance salt solution (HBSS) and the mixtures used for each culture during cell treatment were prepared. Test solution and solvent were added to the corresponding mixtures. The finished test mixtures were exposed for 3 h with and without S9 were treated for approx. 3 hours, and the finished test mixtures to be exposed for 24 h without S9 were treated for approx. 24 hours. The cultures were observed by naked eyes for test-item precipitation before and after the treatment. After the treatment, the cells were washed with HBSS and cultured in 10%MEM.

24.5 hours after the beginning of cell treatment, all cells were treated with Colchicine for nearly 2 hours at the final concentration of 1 µg/ml, and harvested. Cells were then harvested, and washed with HBSS solution and treated with 150 µl trypsin solution. Then the medium containing FBS were added to the cultures and mixed repeatedly. After all of these procedures, the cells of each dose and control were counted using a haemocytometer under inverted Microscope and the relative increase in cell count (RICC) were calculated to evaluate the the cytotoxicity.

Considering the cytotoxicity results and the dose-response relationship, the cells exposed at the doses of 350, 112 and 35 µg/ml in two treatment conditions including exposure for 3-4 hours with and without metabolic activation, and at the doses of 350, 112 and 35 µg/ml for 24h without metabolic activation were chosen for chromosome preparation, the current control groups were performed at the same time. The procedures are shown in below: Harvested cells were transferred into tubes to remove the media by centrifugation at 1,000 rpm for 5 minutes. Remove the supernate, add 1 ml of 75 mM KCL hypotonic solution preheated at 37°C and mix. Then incubate samples at 37°C for 30 minutes, and centrifuge at 1,000 rpm for 5 minutes. Remove the supemate, add 1.0 ml freshly prepared Carnoy's mixture, slowly, mix gently with a pipette, and fix for 30 minutes at RT, and centrifuge at 1,000 rpm for 5 minutes. Remove the supernate, add 1 ml Carnoy's mixture, incubate at 2-8°C overnight for 12 hrs.

Centrifuge the samples at 1,000 rpm for 5 minutes, remove the supernate, and add 0.1 ml Carnoy's mixture to get the cell suspension. Drop the cell suspension to clean slide, make at least 2 slides per group, dry them naturally at room temperature. Stain the samples with Giemsa solution for 30 minutes, wash them with water and dry them naturally. Then all slides were independently coded.

Evaluation criteria:

CRITERIA OF POSITIVE RESULT:

Providing that all acceptability criteria below are fulfilled in any of the experimental conditions examined, the result is considered to be clearly positive:

1) At least one of the exposure dose exhibits a statistically significant increase in the percentage of cells with structural chromosomal aberration compared with
the concurrent solvent control (P<0.05);
2) The increase is dose-related or reproducible

CRITERIA OF NEGATIVE RESULT:

Providing that all criteria below are fulfilled in all of the treatment conditions examined, the result is considered to be clearly negative:

None of the exposure doses exhibits a statistically significant increase compared with the concurrent solvent control (P > 0.05)

CRITERIA OF EQUIVOCAL RESULT:

In case the response is neither clearly negative nor clearly positive, additional cells should be scored and the biological relevance of the result should be
considered. If the result is not yet clear, a repeat experiment will be performed using modified experimental conditions. If the result in the repeat experiment is
still equivocal, the test item will be conclude to be equivocal.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results for precipitation: White precipitates were found by naked eyes in the cell cultures at 350 and 112 µg/ml exposure doses of 3 hours with and without metabolic activation, at the beginning and end of the treatment and 24 hours without metabolic activation at the beginning of the treatment, but only at 350 µg/ml exposure doses of 24 hours without metabolic activation at the end of the treatment.

The results for cytotoxicity: the RICC of the cells exposed for 3h in the absence of S9 mix at the doses of 350, 112, 35, 11.2 and 3.5 µg/ml were 51%, 80%, 84%, 92% and 104%; the RICC of the cells exposed for 3 h in the presence of S9 mix at the doses of 350, 112, 35, 11.2 and 3.5 µg/ml were 55%, 77%, 89%, 89% and 90%; the RICC of the cells exposed for 24h in the absence of S9 mix at the doses of 350, 112, 35, 11.2 and 3.5 µg/ml were 71%, 92%, 90%, 99% and 92%.

The results of the microscopic analysis showed that no statistically significant difference (P>0.05) in the percentage of cells with structural chromosomal aberration was observed in all treated cells in each treatment condition as compared with the solvent control, at the same time, statistically very significant differences (P<0.01) were observed in the cells of all positive controls as compared with the solvent controls.

The  results of the  solvent  control showed that the population doubling of cells in each treatment  condition was  more than  1.4,  at  the  same  time,  the  results  of the positive  control  showed that the  percentage  of cells with  structural  chromosomal aberration was  statistically very significant differences  (P<0.01) were observed in the cells of all  positive  controls as  compared with the current solvent controls.  So the  sensitivity  of the assay  and the  efficacy  of the  89  were  validated,  and the test system of this study was considered valid.

Conclusions:

The results of this study in all treatment conditions were negative. Thus, it is considered that the test item, did not cause structural chromosomal aberrations in cultured mammalian cells under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available