Reaction product of disodium 4,4'-dinitrostilbene-2,2'-disulphonate, p-[(4-amino-2,5-xylyl)azo]benzenesulphonic acid, 3-[(4-amino-2-methoxyphenyl)azo]-4-hydroxybenzenesulphonic acid, copper (II) sulfate

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

Study period:

From September 15, 1993 to December 16, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test conducted according to internationally accepted testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1994

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 OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 15-[(Z)-2-{4-[(E)-2-{4-[(E)-2-{2,5-dimethyl-4-[(E)-2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]-2-sulfophenyl}ethenyl]-3-sulfophenyl}diazen-1-yl]-10,12-dioxa-2,3-diaza-11-cupratricyclo[11.4.0.0^{4,9}]heptadeca-1(17),2,4,6,8,13,15-heptaene-6-sulfonate

EC Number:

943-210-6

Molecular formula:

Not applicable. UVCB Substance

IUPAC Name:

tetrasodium 15-[(Z)-2-{4-[(E)-2-{4-[(E)-2-{2,5-dimethyl-4-[(E)-2-(4-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl]-2-sulfophenyl}ethenyl]-3-sulfophenyl}diazen-1-yl]-10,12-dioxa-2,3-diaza-11-cupratricyclo[11.4.0.0^{4,9}]heptadeca-1(17),2,4,6,8,13,15-heptaene-6-sulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

RANGE FINDING TEST: 20.58, 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plateMUTAGENICITY EXPERIMENTS: 61.73, 185.19 ,555.56 ,1666.67, 5000.00 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the substance for the positive control or the solvent for the negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5 % agar supplemented with 2 % salts of the Vogel-Bonner Medium E and 2 % glucose). The top agar was composed of 0.6 % agar and 0.6 % NaCl. It was supplemented with 10 % of 0.5 mM 1-histidine and 0.5 mM (+)biotin dissolved in water.DURATION- Incubation period: 48 hours at 37 ± 1.5 °C in darkness

Statistics:

In deviation to the OECD guideline a statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended. No appropriate statistical method is available.

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:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: yesADDITIONAL INFORMATION ON CYTOTOXICITY: The test material exerted no toxic effect on the growth of the bacteria

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

In the original experiment performed without metabolic activation on strain TA 98, treatment with test substance led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. In the original experiment carried out with activation on strain TA 102 a marginal increase in the number of back-mutants was registered at the concentration of 1666.7 µg/plate only.

In the confirmatory experiment performed without metabolic activation, treatment of strain TA 98 with test item led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 555.6 to 5000 µg/plate. With strain TA 1537, a marginal increase in the number of back-mutants was registered at the concentrations of 1666.7 and 5000 µg/plate.

In the confirmatory experiment carried out with activation, a slight increase in the number of back-mutants was registered with strain TA 98 at the concentration of 5000 µg/plate, and a marginal increase in the number of back-mutants was observed with strain TA 102 at the concentrations of 555.6 and 1666.7 µg/plate and with strain TA 1537 at the concentrations of 185.2 and 555.6 µg/plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results: positiveBased on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance exerted a weak mutagenic action on strain S. typhimurium TA 98 and the metabolites of the test substance exerted a marginal mutagenic action on strain S. typhimurium TA 102.

Executive summary:

This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium.

Concentrations tested

The concentration range of test substance to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, the substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. An independent repetition of the experiments was performed with the same concentrations.

Toxicity test/Range finding test

In the experiment without and with metabolic activation, test item exerted no toxic effect on the growth of strain TA 100.

Mutagenicity test, original experiment

In the experiment performed without metabolic activation on strain TA 98, treatment with test substance led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 1666.7 and 5000 µg/plate. No effects were observed with the other strains. In the experiment carried out with activation on strain TA 102 a marginal increase in the number of back-mutants was registered at the concentration of 1666.7 µg/plate only. No effect occurred on the other strains.

Mutagenicity test, confirmatory experiment

In the experiment performed without metabolic activation, treatment of strain TA 98 with test item led to a slight, concentration dependent increase in the number of revertant colonies at the concentrations of 555.6 µg/plate and above. With strain TA 1537, a marginal increase in the number of back-mutants was registered at the concentrations of 1666.7 and 5000 µg/plate. No effect was seen with the other strains. In the experiment carried out with activation, a slight increase in the number of back-mutants was registered with strain TA 98 at the concentration of 5000 µg/plate, and a marginal increase in the number of back-mutants was observed with strain TA 102 at the concentrations of 555.6 and 1666.7 µg/plate and with strain TA 1537 at the concentrations of 185.2 and 555.6 µg/plate. No effect occurred on the other strains. In the mutagenicity tests with metabolic activation, normal background- growth was observed with all strains. The number of revertant colonies was not reduced. The test material exerted no toxic effect on the growth of the bacteria.

Conclusion

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test substance exerted a weak mutagenic action on strain S. typhimurium TA 98 and the metabolites of the test substance exerted a marginal mutagenic action on strain S. typhimurium TA 102.