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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07.06.-15.07.2016
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.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-dimethyl-1,3-diphenylurea
EC Number:
210-283-4
EC Name:
1,3-dimethyl-1,3-diphenylurea
Cas Number:
611-92-7
Molecular formula:
C15H16N2O
IUPAC Name:
1,3-dimethyl-1,3-diphenylurea
Test material form:
solid: particulate/powder

Method

Target gene:
gene for histidine or tryptophan synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
supernatant of rat liver and a mixture of cofactors.
Test concentrations with justification for top dose:
15, 50, 150, 500, 1500 μg
30, 100, 300, 1000, 3000 μg
Selection of doses/toxicity: 2 mL of dimethyl sulfoxid was added to 100 mg of the test substance to
reach the maximum dose recommended in guidelines - 5000 μg per plate
Vehicle / solvent:
Solvent: Dimethyl sulfoxide, Merck, Lot. No.: K 47112152 544), exp. 31/10/2018
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylenediamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine, 9-aminoacridine hydrochloride monohydrate
Details on test system and experimental conditions:
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 01022001220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.

Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection
of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: two series

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Selection of doses/toxicity: 2 mL of dimethyl sulfoxid were added to 100 mg of the test substance to reach the maximum dose recommended in guidelines - 5000 µg per plate
(per 0.1 mL). The test substance was well soluble and formed clear colourless solution.
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with
the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible doseresponse
effect occurs and/or a doubling of the ratio Rt/Rc is reached.
Statistics:
For the evaluation of results, the modified two-fold increase rule was used, which is compatible with the
application of statistical methods:
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 - 417
Claxton 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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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 TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
For toxicity experiment, the starting solution (5000 µg/0.1 mL) was diluted to concentration series 10-5000 µg per plate. The concentration row was tested for toxicity
in strain TA 98 without metabolic activation.
Toxicity of the test substance or precipitation was observed starting with concentration of 2500 µg per. After adding of application forms to top agar, turbidity occurred starting with the sane concentration.

Controls: Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of DMSO. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

Applicant's summary and conclusion

Conclusions:
Under the above-described experimental design, the test substance, Methylcentralit, was non mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains with as well as without metabolic activation.
Executive summary:

The test substance Methylcentralit was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrAstrain were used. The test substance was diluted in dimethyl sulfoxide and assayed in doses of 15 - 3000µg per plate, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the above-described experimental design, the test substance,Methylcentralit,was non-mutagenicforall the used bacterial strains with as well as without metabolic activation.