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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The OECD 401 test (Ames) showed no indications for genetic toxicity. Additional in vitro tests with a structural analogue (CH03951), i.e. a lymphocyte gene mutation and micronucleus test, further showed no indications for genetic toxicity.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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

Target gene:

thymidine kinase, TK +/-, locus

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)

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).

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Principles of method if other than guideline:

/

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Micronuclei

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

blood from non-smoking volunteer who had been screened for suitability.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0, 75, 150, 300, 400, 500, 600 (microgram CH 03951/mL) in experiment 1; 4 hours with and without S9
0, 37.5, 75, 150, 225, 300, 400 (microgram CH 03951/mL) in experiment 2; 24 hours without S9

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcine

Details on test system and experimental conditions:

blood was drawn from non smoking volunteer. The cell-cycle time for the lympocytes was determined using BrdU (bromodeoxyuridine) incorporation. The average generation time (AGT) was appr. 16 hours under typical experimental exposure conditions.
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented 'in house' with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at appr. 37°C with 5%CO2 in humidified air. Phytohaemagglutinin (PHA) stimulated division of lympocytes.
Test item was weighed, dissolved in DMSO and seiral dilutions prepared.
Duplicate lymphocyte cultures were established for each dose.
At the end of the cytochalasin B treatment period the cells were centrifugated, culture medium was drawn off and discarded, and the cells resuspended in MEME. Cells were treated with a mild hypotonic solution (0.375 KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v).

Evaluation criteria:

Staining by 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
Negative control: frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the range of the laboratory historical control data.
Positive control: all positive control chemicals must induce positive responses. Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9 mix.

Statistics:

Chi-squared Test: frequencty of cells with micronuclei was compared with concurrent vehicle control value.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item did not induce any statistically significant increases in the number of wells with micronuclei, in the 24-hour exposure group in the presence or absence of metabolic activation.

Conclusions:

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non aneugenic to human lympocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Methods.......

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 4-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 2% final concentration). Experiment 2, used a 24-hour exposure in the absence of metabolic activation and was performed concurrently with the exposure groups of Experiment 1. At the end of the exposure period, the cell cultures were washed and then incubated for a further 28 hours in the presence of Cytochalasin B.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:

Exposure Group	Final concentration of test item (μg/mL)
4-hour without S9	75, 150, 300, 400, 500, 600
4-hour with S9 (2%)	75, 150, 300, 400, 500, 600
24-hour without S9	37.5, 75, 150, 225, 300, 400

Results.......
All vehicle (dimethyl sulphoxide) controls had frequencies of cells with micronuclei within the

range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that induced approximately 50% reduction in CBPI or greater.

Conclusion

The test item, CH03951/BK, was considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.

 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally they posses a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of uvrB-Bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin.
Strains TA98 and TA100: R-factor plasmid pKM101
E. coli: mutation in tryptophan + uvrA- DNA repair deficiency

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 microgram/plate) were assayed in triplicate.

Vehicle / solvent:

dimethyl sulphoxide: the test item was fully soluble at 50 mg/mL.

Untreated negative controls:

yes

Remarks:

2AA for TA100, TA1535 and TA1537; BP for TA98

Positive controls:

yes

Remarks:

ENNG for WP2uvrA, TA100 and TA1535; 9AA for TA1537, 4NQO for TA98

Details on test system and experimental conditions:

All plates were incubated at 37°C +- 3°C for appr. 48 hours.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

/

Statistics:

/

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Interpretation of results (migrated information):
negative

CH04008 was considered to be non-mutagenic under the conditions of this test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Based on the OECD 471 test, CH04008 does not have mutagenic properties.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

See attached documents.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
See attached documents.


3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
See attached documents.

4. DATA MATRIX
See attached documents.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

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

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

Conclusions:

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

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

See attached documents.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]
See attached documents.


3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]
See attached documents.

4. DATA MATRIX
See attached documents.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Principles of method if other than guideline:

/

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test item did not induce any statistically significant increases in the number of wells with micronuclei, in the 24-hour exposure group in the presence or absence of metabolic activation.

Conclusions:

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non aneugenic to human lympocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available