Sodium 2,4-diamino-5-(4-(2-sulfoxyethanesulfonyl)phenylazo)benzenesulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

12 June 1996 to 04 July 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted to recent EU test guidance in compliance with GLP and reported with a GLP certificate. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

27624-67-5

EC Number:

608-116-9

Cas Number:

27624-67-5

IUPAC Name:

27624-67-5

Details on test material:

see below

Method

Target gene:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 92/69/EEC.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

50 to 5000 ug/plate

Vehicle / solvent:

- Vehicle: dimethyl sulphoxide
- Justification for choice of solvent/vehicle:

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) 9-Aminoacridine (9AA) 4-Nitro-o-phenylenediamine (4NOPD) 4-Nitroquinoline-1-oxide (4NQO)

Details on test system and experimental conditions:

Five concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.

Test Material and Vehicle Controls
Known aliquots (0.1 ml) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar at 45⁰C, 0.1 ml of the appropriately diluted test material or vehicle control and either 0.5 ml of the S9 liver microsome mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material with and without S9-mix.

Positive Controls
Without Activation: A known aliquot (0.1 ml) of one of the positive control solutions (ENNG, 9AA, 4NQO or 4NOPD) was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of phosphate buffer was added to the tube, the contents mixed and poured onto the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated, in triplicate, for each tester strain.

With Activation: A known aliquot (0.1 ml) of 2AA solution was added to a test tube containing 2.0 ml of molten, trace histidine supplemented, top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of S9-mix was added to the tube, the contents mixed and poured onto the surface of a Vogel-Bonner Minimal agar plate. This procedure was then repeated, in triplicate, for each tester strain.

Incubation and Assessment of Plates
All of the plates were incubated at 37⁰C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 μg/plate due to intense colouration from the test material

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 μg/plate. In this case the limiting factor was the maximum recommended dose.

Statistics:

All data are statistically analysed using the methods recommended by the UKEMS (5) and normally Dunnett's method of linear regression is used to evaluate the result.

Results and discussion

Test results

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No toxicity was exhibited to any of the strains of Salmonella used. An intense test material induced colouration was observed at 5000 μg/plate, this did not, however, interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies of bacteria were recorded for any of the strains of Salmonella used, at any dose level with or without metabolic activation.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

The results are read across from the free acid form. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

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

Executive summary:

The results are read across from the free acid form. Read across is applicable based on the content of sodium ions. The presence of these is determined by the pH of the isolation of the dyestuff itself. Therefore the substance to be registered is deemed to be a mixture of free acid, mono and di sodium salts. Upon comparison of the NMR-Spectra of the substance to be registered and the read across chemical it is evident that the chemical shifts as well as the integrations are the same, hence it is difficult to quantify free acid, mono and di variants. The CAS number proposed for the substance to be registered covers the sodium element. The associated free acid has a unique CAS number; this is referenced in the substance identity, as does the disodium variant, which is also referenced.

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 92/69/EEC. Study conducted in compliance with GLP and reported with a GLP certificate.

 The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range.

 

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

 

The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels to any of the strains of Salmonella tested. The test material was, therefore, tested up to the maximum recommended dose of 5000 μg/plate. An intense test material induced colouration was observed at 5000 μg/plate, this did not, however, interfere with the scoring of revertant colonies.

 

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test