3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

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:

26 March 2018 - 13 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Cas Number:

2102522-55-2

Molecular formula:

C20H18N2O4S

IUPAC Name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: Off-white powder

Specific details on test material used for the study:

Stability: Not reactive to light nor air.
Solubility in water: less than 0.1 mg/mL
Solubility in DMSO: 50 mg/mL and more
Solubility in acetone: 100 mg/mL and more
Stability in solvent DMSO: no exothermic reaction nor generation of gas.
Storage conditions: Room temperature.

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Source of TA100: The Division of Mutagenesis, National Institute of Hygienic Sciences, japan (The Division of Genetic and Mutagenesis, Biological Safety Research Center, National Institute of Health Sciences, Japan at present); April 15, 1982.
- Source of other TA strains: Dr. Ames, U.C. Berkley, CA, U.S.A.; January 20, 1988. Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 1987.

- Storage: The bacterial suspension and DmSO (spectrophotometric grade) were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided into 0.3 mL aliquots and then frozen and stores at -85 to -80 °C.

- Reason for choice of strains: These strains are very sensitive to mutagens and are the most commonly used in bacterial reverse mutation assays.

- Characterisation: As characteristic test, the number of viable cells, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Source: Dr. Ames, U.C. Berkley, CA, U.S.A.; January 20, 1988. Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Japan; November 24, 1987.

- Storage: The bacterial suspension and DmSO (spectrophotometric grade) were mixed in a ratio of 0.8 mL to 0.07 mL. The mixture was subdivided into 0.3 mL aliquots and then frozen and stores at -85 to -80 °C.

- Reason for choice of strains: These strains are very sensitive to mutagens and are the most commonly used in bacterial reverse mutation assays.

- Characterisation: As characteristic test, the number of viable cells, amino acid requirement, UV sensitivity, rfa mutation, presence or absence of the drug resistance factor (R-factor plasmid) and positive control test (Dose-relation) were confirmed, and good strains were used as test strains.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 mix
- Source of S9: Oriental Yeast, Co., Ltd.
- Composition of S9 mix per 1 mL: Water (0.9 mL), S9 (0.1 mL), MgCl2 (8.0 μmol), KCl (33.0 μmol), Glucose-6-phosphate (5.0 μmol), NADPH (4.0 μmol), Na-Phosphate buffer, pH 7.4 (100.0 μmol).
- Concentration or volume of S9 mix in the final culture medium: 0.5 mL of S9 mix added to plates with metabolic activation.

Test concentrations with justification for top dose:

PRELIMINARY TEST
1.2, 4.9, 20, 78, 313, 1250 and 5000 μg/plate.

MAIN TEST
2.4, 4.9, 10, 20, 39, 78 μg/plate (TA98, TA100 and TA1537 without metabolic activation)
10, 20, 39, 78, 156, 313 μg/plate (TA1535 without metabolic activation)
39, 78, 156, 313, 625, 1250 μg/plate (WP2 uvrA without metabolic activation)

39, 78, 156, 313, 625, 1250 μg/plate (TA98, TA100, TA1535 and WP2 uvrA with metabolic activation)
2.4, 4.9, 10, 20, 39, 78 μg/plate (TA1537 with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the solubility of the test material in water was < 0.1 mg/mL, the solubility test was performed with DMSO. The test material was dissolved at 50 mg/mL in DMSO, and neither exothermic nor generation of gas was observed. Therefore, DMSO was used as a solvent.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191: 2 -Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine . 2HCL

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2AA: 2 - Aminoanthracene

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- One minimal glucose agar plate was used for each dose level in the preliminary test, and three minimal glucose agar plates were used for each dose level in the two main tests which were performed at the same doses.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: The preincubation method was used for all tests.
For tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution or the negative control solution.
For tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer.
The mixture was pre-incubated in a water bath at 37 °C for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. And 0.1 mL of the positive control was carried out equally.
For the sterility test, 0.1 mL of the test solution of the maximum concentration and 0.5 mL of the S9 mix were put into each tube, 2.0mL of top agar were then added to the tube and the contents of each tube were poured over the surface of the minimal glucose agar plate.
These operations were conducted under lamps with ultraviolet absorbent filter.
As top agar, the 0.5 mM biotin-0.5 mM L-histidine solution and the 0.5 mM L-tryptophan solution were added to the soft agar solution (0.6 % Agar and 0.5 % NaCl) by volume of 1/10, for the S. typhimurium TA strains and the E. coli strain, respectively.
All plates were incubated at 37 °C for 48 hours, and the number of revertant colonies were counted.
Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.

COUNTING PROCEDURE
The number of revertant colonies were counted visually due to the precipitate of the test material on the plates.
The revertant colonies of positive controls were counted with a colony counter.
- Model: Colony Analyzer CA-11
- Manufacturer: System Science Co., Ltf.
- Correction: Count loss correction
- Coefficient: 1- 100 colonies: x 1.11; 101 - 400 colonies: x 1.16; 401 - colonies: x 1.25

Evaluation criteria:

MAIN TESTS
- If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test material was judged to be positive.

Statistics:

The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA100,m TA 1535, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY STUDY
Growth inhibition by the test material was observed at:
> 78 μg/plate in S. typhimurium TA100 and TA98 without metabolic activation, and S. typhimurium TA1537 both with and without metabolic activation.
> 313 μg/plate in S. typhimurium 1535 without metabolic activation.
> 1250 μg/plate in E. coli WP2 uvrA both with and without metabolic activation and S. typhimurium TA100, TA 1535 and TA 98 with metabolic activation.
Precipitate of the test material on the plates was observed at > 78 μg/plate without metabolic activation and at > 1250 μg/plate with metabolic activation.

MAIN STUDY
- In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation.
- The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean ± 3SD) in historical data, indicating that the study was performed correctly.
- The precipitate of the test material on the plates was observed at 78 μg/plate and more without metabolic activation, and at 1250 μg/plate and more with metabolic activation.
- In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.

Remarks on result:

other: Mutagenicity was judged negative.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the mutagenicity of the test material on the applied bacterial strains was judged negative.

Executive summary:

The mutagenicity potential of the test material was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA according to the standardised guidelines OECD 471, under GLP conditions. 

In this study, neither an increase in the number of revertant colonies more than twice in comparison with that of the negative control nor a dose-related response was observed in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. 

It was therefore concluded the test material is not mutagenic to the applied bacterial strains, under the conditions of this assay.