Sodium 4-hydroxybenzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay with sodium 4-hydroxybenzenesulfonate

Sodium 4-hydroxybenzenesulfonate, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. The study was conducted according to OECD Guideline 471 and in compliance with GLP. Water was used as the vehicle. In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate. In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. These results indicate sodium 4-hydroxybenezenesulfonate was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date: 11 April 2017, Study Completion Date: 13 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

None

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

updated and adopted 21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Identification: FAT 93588/A TE
- Chemical Name: Sodium 4-hydroxybenzenesulphonate
- Batch/Lot Number: 0041847900
- CAS No.: 825-90-1
- Purity: 98.5%
- Molecular Weight: 196.16 g/mol
- Expiration Date: 29-June-2018
- Physical State: Solid
- Color: White
- Description: White powder

Target gene:

Salmonella typhimurium histidine auxotrophs: TA98, TA100, TA1535 and TA1537, tryptophan auxotrophs: Escherichia coli WP2 uvrA

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

15.0, 50.0, 150, 500, 1500 and 5000 μg per plate

Vehicle / solvent:

Water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: Negative controls were also considered as solvent controls

Positive controls:

yes

Positive control substance:

other: See in the 'remarks' below

Details on test system and experimental conditions:

Test System
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause base pair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and base pair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base pair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Salmonella tester strains were derived from Dr. Bruce Ames’ cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Solubility Determination
Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in water at a concentration of approximately 50 mg/mL in the solubility test conducted at BioReliance.

Preparation of Tester Strain
Overnight cultures were prepared by inoculating from the appropriate frozen permanent stock into a vessel, containing 30 to 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at 125 to 175 rpm and incubating at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Identification of Test System
Each plate was identified by the BioReliance study number and a code system to designate the treatment condition, dose level and test phase, as described in detail in BioReliance's Standard Operating Procedures.

Exogenous Metabolic Activation
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254 (200 mg/mL in corn oil) at a dose of 500 mg/kg, five days before sacrifice.

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance as specified below:
Strains TA1535 and TA1537
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
Strains TA98, TA100 and WP2 uvrA
Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value and above the corresponding acceptable vehicle control range.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Sterility Results

No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.

Preliminary Toxicity Assay

In the preliminary toxicity assay conducted at dose levels of 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate in water, the maximum dose of 5000 μg per plate was achieved using a concentration of 50.0 mg/mL and a 100 μL plating aliquot. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation.

Mutagenicity Assay

Based upon the results of the preliminary toxicity assay, the dose levels selected for the mutagenicity assay were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Conclusions:

FAT 93588/A was found to have no mutagenic potential in this bacterial reverse mutation assay.

Executive summary:

The test substance, FAT 93588/A TE, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. The study was conducted according to OECD Guideline 471 and in compliance with GLP. Water was used as the vehicle.

In the preliminary toxicity assay, the dose levels tested were 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333 and 5000 μg per plate. No precipitate was observed. Toxicity as reduction in revertant count was observed at 5000 μg per plate with tester strain TA1537 in the absence of S9 activation. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

In the mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

These results indicate FAT 93588/A TE was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Sodium 4 -hydroxybenzensulfonate was considered to be not mutagenic based on the outcome of a bacterial reverse mutation assay, hence no classification according to the CLP (Regulation EC No. 1272/2008) criteria is warranted.