Reaction mass of sodium 4-hydroxybenzenesulfonate and disodium 4-hydroxybenzene-1,3-disulfonate and sodium sulfate

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:

From October 10 to November 14, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 4-hydroxybenzene-1,3-disulfonate 4-hydroxybenzene-1-sulfonate sulfate

EC Number:

939-154-7

Cas Number:

not available

Molecular formula:

C6H5NaO4S - C6H4Na2O7S2 - Na2SO4

IUPAC Name:

pentasodium 4-hydroxybenzene-1,3-disulfonate 4-hydroxybenzene-1-sulfonate sulfate

Test material form:

solid: particulate/powder

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Dr. G.P. Meshram genotox services
- method of preparation of S9 mix: from wistar male rats induced with Aroclor 1254 in corn oil
- concentration or volume of S9 mix and S9 in the final culture medium: 0.1 mL of S9 mix (5% v/v S9 mix for initial toxicity-mutation test and 10% v/v S9 mix for confirmatory mutation test) prepared for treatment was added aseptically to 2 mL of the top agar
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): performed

Test concentrations with justification for top dose:

Toxicity test at 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the absence and presence of S9
Confirmatory mutation test at 156.25, 312.5, 625, 1250, 2500, 5000 µg/plate in the absence and presence of S9

Vehicle / solvent:

distilled water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

In presence of S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

In absence of S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate in the toxicity test and triplicate in the main test
- Number of independent experiments: 1 toxicity test and 1 main study

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; plate incorporation method

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h incubation period at 37 ± 1 ºC

Evaluation criteria:

Cytotoxicity Evaluation Criteria
Six analysable doses were available to evaluate assay data. Cytotoxicity was not detectable as a decrease in the number of revertant colonies per plate and/or by a thinning of the bacterial background lawn was not observed.

Assay Evaluation Criteria
A result was considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without any metabolic activation system.
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 negative control value.
Strains TA98, TA100, and TA102
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 negative control value.
Statistical analysis was used as an aid in the evaluation of dose-response.
As the response did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) and so was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for tester strains TA1537, TA1535, TA98, TA100, and TA102, separately, to assess the dose-dependent nature of any increase in revertant colonies.

Results and discussion

Test results

Species / strain:

other: TA1537, TA1535, TA98, TA100, and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Negative Control

Results of this study indicate that values of the negative control in all strains were within the historical range for respective strains.

Positive Controls

2-Aminoanthracene was used as a positive control in the presence of the metabolic activation system for tester strains during the initial toxicity test and the confirmatory mutation test. Large historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of the metabolic activation system. The batch of S9 used in this study, characterised by the supplier with benzo(a)pyrene, required a metabolic activation system by microsomal enzymes.

Positive controls exhibited a clear increase in the number of revertants when compared with that of the concurrent negative controls. This demonstrated the efficiency of the test system and the suitability of procedures employed in this assay.

Any increase in revertants was not observed in TA100 (initial toxicity-mutation test and confirmatory mutation test) treated with 2-aminoanthracene, in the absence of the metabolic activation, but a clear increase was observed in the presence of the metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity-Mutation Test

A normal bacterial background lawn was observed up to the tested concentration of 5000 µg/plate, in all tester strains (TA1537, TA1535, TA98, TA100, and TA102) in the absence and presence of the metabolic activation system (5% v/v S9 mix).

No reduction in the number of revertant colonies was observed at a tested concentration up to 5000 µg/plate in the absence and presence of the metabolic activation (5% v/v S9 mix) in all tester strains.

Results revealed that there was no positive mutagenic effect in any tester strain, up to the tested concentration of 5000 µg/plate in the absence and presence of the metabolic activation (5% v/v S9 mix), when compared with that of the negative control.

Statistical analysis did not reveal any significant effect in tester strains TA1537, TA1535, TA98, TA100, and TA102 in the absence of the metabolic activation and tester strains TA1535, TA98, TA100 and TA102 in the presence of the metabolic activation (5% v/v S9 mix).

In tester strain TA1537 (in the presence of the metabolic activation), the statistical analysis showed correlation at 5%, however the mean value was within the historical control data, hence it was biologically non-significant.

Based on the results of the initial toxicity mutation test, six concentrations (156.25, 312.5, 625, 1250, 2500, 5000 µg/plate) of SELLASOL HH-F were selected for the confirmatory mutation test, in the absence and presence of the metabolic activation system (10% v/v S9 mix) for all tester strains.

Confirmatory Mutation Test

As recommended by the OECD guideline, the confirmatory experiment was conducted with a modification in study parameters to confirm the negative results obtained in the initial toxicity mutation test. In the confirmatory mutation test, the concentration spacing was modified, using a factor of 2 and the concentration of S9 mix was increased to 10% v/v.

A normal bacterial background lawn was observed up to the tested concentration of 5000 µg/plate, in all tester strains (TA1537, TA1535, TA98, TA100, and TA102) in the absence and presence of the metabolic activation system (10% v/v S9 mix).

No reduction in the number of the revertant colonies was observed up to the tested concentration of 5000 µg/plate, in the absence and presence of the metabolic activation (10% v/v S9 mix) in all tester strains.

Results revealed that there was no positive mutagenic effect in the tester strains TA1537, TA1535, TA98, TA100, and TA102, up to the tested concentration of 5000 µg/plate, in the absence and presence of the metabolic activation (10% v/v S9 mix), when compared with that of the negative control.

Statistical analysis did not reveal any significant effect in any tester strains.

Applicant's summary and conclusion

Conclusions:

Non mutagenic

Executive summary:

This study was performed to evaluate the mutagenic activity of the test substance using the bacterial reverse mutation test on five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100, and TA102).

The test item was tested in two independent experiments in the absence and presence of metabolic activation. Bacterial cultures were exposed to the test item at 8 concentration levels (two plates/concentration) ranging from 1.5 to 5000 µg/plate in the initial toxicity-mutation test, using the plate incorporation method.

Normal bacterial background lawn was observed up to the concentration of 5000 µg/plate, in all tester strains (TA1537, TA1535, TA98, TA100, and TA102) in the absence and presence of the metabolic activation system (5% v/v S9 mix).

No increase in the number of revertant colonies (no mutagenic effect) was observed in the absence and presence of the metabolic activation system (5% v/v S9 mix) in any tester strain. To confirm the negative results obtained in the initial toxicity mutation test, the confirmatory mutation test was conducted, using the plate incorporation method with an increased S9 concentration, i.e., 10% v/v S9 mix and concentration spacing modification.

Bacterial cultures were exposed to the test substance at 6 concentration levels (three plates/concentration) ranging from 156.25 to 5000 µg/plate for tester strains TA1537, TA1535, TA98, TA100, and TA102 in the absence and presence (10 % v/v S9 mix) of the metabolic activation. The revertant colonies were scored after 48 hours of incubation at 37 ± 1 °C.

The test item did not induce any significant increase in the number of revertants, in trials with and without S9 mix, in any tester strain. Values for the negative control were within the historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met. Based on the results of this study, under specified experimental conditions, the test substance is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.