reaction mass of: pentasodium bis[6-anilino-3,5'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III);tetrasodium [6-anilino-3,5'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato][6-anilino-5'-sulfamoyl-3-sulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III);trisodium bis[6-anilino-5'-sulfamoyl-3-sulfonatonaphthalene-2-azobenzene-1,2'-diolato]cobaltate(III)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

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:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-290-0

EC Name:

-

Cas Number:

508202-43-5

Molecular formula:

Not applicable

IUPAC Name:

tricobalt(3+) dodecasodium tris(2-[2-(2-oxido-5-sulfamoylphenyl)diazen-1-yl]-6-(phenylamino)-3-sulfonatonaphthalen-1-olate) tris(2-[2-(2-oxido-5-sulfonatophenyl)diazen-1-yl]-6-(phenylamino)-3-sulfonatonaphthalen-1-olate)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 MIX

Test concentrations with justification for top dose:

-Pre-experiment: 20.6, 61.7,185.2, 555.6,1666.7 and 5000 µg/plate
On basis of the pre-experiment results:
- Main tests: 312.5, 625,1250, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without metabolic activation (S9)

Controls 2

Untreated negative controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation (S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The plates with the selective agar (minimal agar plates) were made in-house. Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The agar used was Select AGAR, GIBCO BRL, Switzerland. Glucose was delivered from Merck, Germany [D(+)glucose, anhydrous]. The Vogel-Bonner Medium E was prepared in-house.
The overlay agar contained per litre:
6.0 g GIBCO BRL Select Agar
6.0 g NaCl (Merck, Germany)

Sterilisation was performed at 121° C in an autoclave, cooled down to 50°C and dispensed into glass bottles. On day of test performance the aga was molten in a water bath and 10% aliquots (v/v) of 0.5 mM L-histidine / 0.5 mM d-biotin for Salmonella strains or 0.5 mM tryptophan, dissolved in bidistilled water, for E.coli strains were added sterile filtered.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant If the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative controls such an increase Is not considered biologically relevant.

Statistics:

A statistical analysis was not required. The use of statistical methods concerning this particular test system was not generally recommended

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli strain WP2 uvrA.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

-Pre-experiment: normal background growth was observad with both strains(S. typhimurium TA 100 and E. coli WP2 uvrA). The number of revertant colonies was not reduced at any concentrations tested. The test item did not precipitate on the surface of the agar plates.
-Main tests: The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No reduction in the number of revertant colonies occurred at any concentration tested in comparison with the respective solvent control. The test item did not precipitate on the surface of the agar plates. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any concentration level, neither in the presence nor in the absence of an metabolic activation system. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

The substance was considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations, according to the OECD Guideline 471 (1983) and method B.14 EEC-Directive 2000/32/EC. The plate incorporation test (first experiment with and without metabolic activation, second experiment without activation) and the pre-incubation test (second experiment with metabolic activation) were performed using Salmonella typhimurium strains TA 100, TA 1535, TA 98, TA 1537, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Negative (solvent) and positive control treatments were included for all strains. Each concentration and the controls were tested in triplicate. The test item was dissolved in dimethyl sulfoxide (DMSO) and tested at five concentrations from 312.5 to 5000 µg/plate. Each strain was additionally tested In the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. Previously, a pre-experiment for toxicity was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay.From the results obtained, the highest concentration suitable for the first mutagenicity test was selected. Both in the first and the second mutagenicity main tests, with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with substance at any concentrations. In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced. The test item did not precipitate on the surface of the agar plates. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base-pair changes or frameshifts in the genome of the strains used.