1,2-Benzenediol, 4-[(4-methylphenyl)sulfonyl]-

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:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Principles of method if other than guideline:

/

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-hydroxyphenyl)benzenesulfonamide

EC Number:

654-333-7

Cas Number:

5471-90-9

Molecular formula:

C12 H11 N O3 S

IUPAC Name:

N-(4-hydroxyphenyl)benzenesulfonamide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

White powder; 96.45% purity

Method

Target gene:

thymidine kinase, TK +/-, locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

/

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment 1:
4-hour without S9: 18.75, 37.5, 75, 150, 200, 250 μg/mL
4-hour with S9 (2%): 18.75, 37.5, 75, 100, 150, 200 μg/mL
Experiment 2
24-hour without S9: 9.38, 18.75, 37.5, 75, 150 μg/mL
4-hour with S9 (2%): 18.75, 37.5, 75, 100, 150 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (solvent); Ethylmethanesulphonate (EMS) (vehicle)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) treatment groups were used as the vehicle controls.

True negative controls:

no

Positive controls:

yes

Remarks:

Ethylmethanesulphonate (EMS) Sigma batch BCBK5968V

Details on test system and experimental conditions:

Experiment 1
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (9.38 to 300 μg/mL in the absence of metabolic activation, and 9.38 to 250 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix if required, 0.2 mL of the treatment dilutions, (0.2 mL or 0.15 mL for the positive controls) and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

Experiment 2
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL cultures in R10 medium for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1% S9 final concentration) at eight dose levels of the test item (9.38 to 300 μg/mL in the absence of metabolic activation, and 9.38 to 250 μg/mL in the presence of metabolic activation), vehicle and positive controls. To each culture vessel was added 2 mL of S9 mix if required, 0.2 mL of the treatment dilutions, (0.2 mL or 0.15 mL for the positive controls) and sufficient R0 medium to give a final volume of 20 mL (R10 was used for the 24 hour exposure group).
The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT will be considered):
1. The majority of the plates are analysable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
3. The total suspension growth of the solvent control following 4 hour treatment, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour treatment the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency: range of 50 – 170 x 10-6 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10-6 mutant frequency per survivor are not acceptable and will be repeated.
5. Positive control chemicals (EMS and CP) should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control. The positive controls should ideally yield an absolute increase in total MF, that is an increase above spontaneous background MF (an induced MF [IMF]), of at least 300 x 10-6 cells.
6. The upper limit of cytotoxicity observed in the positive control culture should be = the experimental cultures i.e. the relative total growth and percentage relative suspension growth should be greater than 10 % of the concurrent selective control group.
7.The highest concentration of the test item should be 10 mM or 5000 μg/mL, unless limited by toxicity or solubility of the test item. If toxicity occurred, the highest concentration should lower the relative total growth and/or percentage relativetotal growth and/or percentage relative suspension growth to 10 to 20% of survival.

Statistics:

No specific statistics
The cell counts obtained immediately post treatment and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth.
Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method.
For each culture, the relative cloning efficiency, RCE, was calculated
MF per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).

Results and discussion

Test results

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:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In VitroMammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods.......

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

Experiment 1 (top)

Experiment 2 (bottom)

Group	Concentration of CH03951/BK(μg/mL) plated for mutant frequency
4-hour without S9	18.75, 37.5, 75, 150, 200, 250
4-hour with S9 (2%)	18.75, 37.5, 75, 100, 150, 200

Group	Concentration of CH03951/BK(μg/mL) plated for mutant frequency
24-hour without S9	9.38, 18.75, 37.5, 75, 150
4-hour with S9 (2%)	18.75, 37.5, 75, 100, 150

Results........

The maximum dose levels used in the Mutagenicity Test were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle controls had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked

increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.