2-Naphthalenesulfonic acid, 4-hydroxy-3-[2-[2-methoxy-5-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-7-[(sulfomethyl)amino]-8-[2-[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]diazenyl]-, sodium salt (1:5)

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Test Guideline 111.1

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

464-700-1

EC Name:

-

Cas Number:

607724-42-5

Molecular formula:

Hill formula: C28H24N5Na5O23S7 CAS formula: C28H29N5O23S7.5Na

IUPAC Name:

pentasodium 4-hydroxy-3-(2-{2-methoxy-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-8-(2-{2-sulfonato-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-7-[(sulfonatomethyl)amino]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Red F01-0481

Specific details on test material used for the study:

- pH value in water: approx. 5.5
- Solubility in water: > 100 g/L
- Stability and homogeneity in the solvent: guaranteed for 4 h in deionized water by HPLC analysis
- Concentration of stock solution: 50 mg/mL

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Plate incorporation test:
with metabolic activation (10% rat liver):
50, 160, 500, 1600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1600 and 5,000 µg/plate

Preincubation test:
with metabolic activation (30% hamster liver):
16, 50, 160, 500, 1600 and 5,000 µg/plate
without metabolic activation:
16, 50, 160, 500, 1600 and 5,000 µg/plate

Vehicle / solvent:

- Vehicle used: deionized water
- solvent used for positive controls: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (10% rat liver) for all strains

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE:
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. The highest concentration in the first mutation experiment was 50 mg/mL of the test substance in the chosen solvent, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 mL, 2 mM).
The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 48°C:
0.5 mL S9-mix (if required) or buffer
0.1 mL of an overnight nutrient broth culture of the bacterial tester strain
0.1 mL test substance solution (dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approximately 30°C. After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (1.5% (w/v) agar, Vogel-BonnerE medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer'srequirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test substance is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test substance at complete bacterial background lawn

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and peincubation test)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the plate incorporation test toxicity was observed only with the strain TA 1537 with metabolic activation at concentrations of 500 to 5000 µg/plate and without S9-mix at a concentration of 5000 µg/plate. In the preincubation test no toxicity was seen.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY: Test substance was dissolved in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in all experiments. For the preincubation test the concentration of 16 µg/plate was additionally included in the treatment series. Test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.
In the plate incorporation test toxicity was observed only with the strain TA 1537 with metabolic activation at concentrations of 500 to 5,000 µg/plate and without S9-mix at a concentration of 5,000 µg/plate. In the preincubation test no toxicity was observed in the absence and in the presence of hamster liver metabolic activation.

MUTAGENICITY:
Plate incorporation test: The test substance did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix. No dose-dependent effect was obtained.
Preincubation test: In the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival the test substance did not cause a significant or dose dependent increase in the number of revertant colonies under the experimental conditions described.

All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated. The number of revertant colonies of the positive control in the preincubation test with the strain TA 1537 in the presence ofS9-mix was even slightly above ofthe historical control data range, but the criteria for the positive response were clearly fulfilled and the validity not influenced.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

STERILITY CHECKS AND CONTROL PLATES:

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range. The number of revertant colonies of the solvent and negative controls with the strain WP2uvrA in the presence of S9-mix in both experiments was marginally below the historical control data range, which had no influence on the validity of the assay.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

The test substance was investigated for its potential to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japan: Guidelines for Screening Mutagenicity Testing of Chemicals, Test Guidelines 111.1, in compliance with GLP.

Two independent mutagenicity studies were conducted, one with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was assessed at 50 - 5,000 µg/plate in the standard plate test and 16 - 5,000 µg/plate in the modified preincubation test.

The substance did not cause a significant increase in the number of revertant colonies at any dose level in the absence or presence of metabolic activation system in either of the tests.

Under the study conditions, the substance was found to be non-mutagenic in the bacterial reverse mutation assay.