6-amino-4-hydroxynaphthalene-2-sulphonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Well documented bacterial reverse mutation assay, but not conducted in accordance with current guideline and only two strains tested.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

as adopted 1983-05-26

Principles of method if other than guideline:

The mutagenicity evaluation was performed using the Salmonella/microsome test, also termed the Ames Test, as described by Ames et al. (1973a, 1975) and Maron and Ames (1983). In this case, only two strains (TA 100 & TA 98) were tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

6-amino-4-hydroxynaphthalene-2-sulphonic acid

EC Number:

202-000-8

EC Name:

6-amino-4-hydroxynaphthalene-2-sulphonic acid

Cas Number:

90-51-7

Molecular formula:

C10H9NO4S

IUPAC Name:

6-amino-4-hydroxynaphthalene-2-sulphonic acid

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 100 & TA 98

Additional strain / cell type characteristics:

other: pKM 101

Metabolic activation:

with and without

Metabolic activation system:

induced S9

Test concentrations with justification for top dose:

0, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water (test item); DMSO (positive controls)
- Justification for choice of solvent/vehicle: The used solvent was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.

Controls

Untreated negative controls:

yes

Remarks:

vehicle

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: nitrofuranthion; 4-nitro-1,2-phenylene diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 30 sec
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): his-negative agar

NUMBER OF REPLICATIONS: 4 plates per strain, dose, ± S9

DETERMINATION OF CYTOTOXICITY
- Method: The toxicity of the substance was assessed in three ways. The first was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with background growth. (The same applies to the signs "c", "v", "p", "n" or "%", which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 100 and TA 98 this increase should be about twice the amount of negative controls. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 100 & TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
At all doses, the substance had a strain-specific bacteriotoxic effect. Nevertheless, they could all be used for assessment purposes.

Applicant's summary and conclusion

Conclusions:

The mutagenicity evaluation was performed using the Salmonella/microsome test, also termed the Ames Test, as described by Ames et al. (1973a, 1975) and Maron and Ames (1983). The testing was sufficiently documented, positive and negative controls gave the appropriate response, but only two strains (TA 100 & TA 98) were tested. A dose-dependent and biologically relevant increase of the mutant figures to the double of the negative controls could not be observed with any of the two strains used. This applied to the examinations with and without the S-9 mix. In consequence, the substance is considered to be non-mutagenic under the conditions of this test.

Executive summary:

In a reverse gene mutation assay in bacteria (Ames Test as described by Ames et al. (1973a, 1975) and Maron and Ames (1983)), histidine-auxotrophic strains TA 100, and TA 98 of S. typhimurium were exposed to gamma acid in deionized water at concentrations of 0, 625, 1250, 2500, 5000 µg/plate in the presence and absence of mammalian metabolic activation (induced S9) via plate co-incubation.

The test substance was tested up to limit concentration 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

At all doses, the substance had a strain-specific bacteriotoxic effect. Nevertheless, they could all be used for assessment purposes. None of the two strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the repeat tests.

Although the study has not been conducted according to the current guideline and only two strains have been tested, it is used in a weight of evidence approach as it shows that the purified substance obtains different results than the substance with a higher content of impurities (T50334404).