3-methyl-1,1-diphenylurea

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.09.-15.02.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

see any other onformation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methyl-1,1-diphenylurea

EC Number:

236-039-7

EC Name:

3-methyl-1,1-diphenylurea

Cas Number:

13114-72-2

Molecular formula:

C14H14N2O

IUPAC Name:

3-methyl-1,1-diphenylurea

Constituent 2

Reference substance name:

131411-72-2

IUPAC Name:

131411-72-2

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: 3-Methyl-1,1-diphenylurea- Physical state: white solid powder- Composition of test material, percentage of components: main component: 3-Methyl-1,1-diphenylurea CAS:13114-72-2 >98% (w/w) impurities: unlisted additives: unlisted-Molecular formula: C14H14N2O-Molecular weight: 226,27-Batch No.: 14015- Expiration date of the lot/batch: 11/2019- Stability under test conditions: stable- Storage condition of test material: in closed container, in dry room at room temperature (at laboratory conditions)

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors.

Test concentrations with justification for top dose:

cytotoxicity: 0.0313, 0.025, 0.125, 0.25, 1.0, 2.5, 5.0 mg.mL-1 (3h treatment)mutagenicity: 0.5, 0.25, 0.125, 0.0625 mg.mL-1 (3h treatment)0.1, 0.075, 0.05, 0.025 and 0.01 mg.mL-1 (24h treatment)

Vehicle / solvent:

DMEM

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

TEST SYSTEMThe lung fibroblasts V79 from male Chinese hamster were used for testing. Frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC). V79 used for experiments: Lot. No.: 10H016The cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened).Cells underwent maximum12 passages after thawing the original culture delivered from cell collection before using for test. Cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants.METHOD OF APPLICATION: in mediumDURATION1st experiment: 10 days, 3h teratment with metabolic activation, 3h without metabolic activation2st experiment: 10 days, 24h teratment without metabolic activation onlyNUMBER OF REPLICATION: 2NUMEBRO OF CELLS EVALUATED: 100 000 cellsSELECTION AGENT (mutation assays): 6-thioguanine 98%, CAS No. 154-24-7, Alfa Aesar, diluted in 0,5% Na2CO3; 5 µg.mL-1 (final concentration) for selection of mutantsDETERMINATION OF CYTOTOXICITY- Method: cloning efficiency

Statistics:

Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 – 240Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity. Mutat. Res. 189, 83 - 91

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: Measuring of pH was performed for treatment solutions used in experiments without metabolic activation. The test substance hardly changed pH of cultivation medium.- Water solubility: As the test substance is insoluble in water it was dissolved in DMSO

Applicant's summary and conclusion

Conclusions:

Under the above-described experimental design, the test substance, Akardit, was non-mutagenic for V79 cells with as well as without metabolic activation.

Executive summary:

The test substance,Akardit,was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on EU methodB.17. Mutagenicity –In Vitro Mammalian Cell Gene Mutation Test, which is analogous to the OECD Test Guideline No. 476 (1997).

V79 hamster fibroblast were used for testing.

The test substance was dissolved in dimethyl sulfoxide in concentration 25 mg.mL^-1. Concentrations tested for cytotoxicity in 3-hour experiment were 0.25, 0.125, 0.0625 and 0.0313 mg.L^-1and other 3 concentrations were tested as a suspension in DMEM (1.0, 2.5 and 5.0 mg.mL^-1). No toxicity was observed in any dose.

First mutagenicity experiment (3 hour treatment) with the test substance followed then. Four concentrations of test substance dissolved in dimethyl sulfoxide were used-0.0625,0.125, 0.25 and 0.5 mg.mL^-1. Each concentration was tested in two independent runs. Experiments were performed with as well as without metabolic activation.

Results of the first experiment were negative, so the modification of experiment was performed with extended treatment time of 24 hours and lower concentrations considering toxicity of the test substance shown in 24 hours cytotoxicity experiment (0.01, 0.025, 0.05, 0.075 and 0.1 mg.mL^-1). The test was performed without metabolic activation only; each concentration was tested in replicate. Also the second experiment gives no evidence of the mutagenicity of test substance.

In the arrangement given above, the test substance, Akardit, was non-mutagenic for V79 cells without as well as with metabolic activation.