Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

according to the previous version of the OECD Guideline (1983), four strains were used instead of five strains

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

December 29, 1992.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)

EC Number:

419-210-2

EC Name:

Pentasodium bis{7-[4-(1-butyl-5-cyano-1,2-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridylazo)phenylsulfonylamino]-5'-nitro-3,3'-disulfonatonaphthalene-2-azobenzene-1,2'-diolato} chromate (III)

Cas Number:

178452-71-6

Molecular formula:

C66H48CrN16Na5O28S6

IUPAC Name:

chromium(3+) pentasodium bis(7-{4-[2-(1-butyl-5-cyano-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridin-3-yl)diazen-1-yl]benzenesulfonamido}-2-[2-(5-nitro-2-oxido-3-sulfonatophenyl)diazen-1-yl]-3-sulfonatonaphthalen-1-olate)

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 µg/plate
According to the results of pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. The maximum concentration was 5000.0 µg/plate.

Vehicle / solvent:

-Vehicle used: water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

With metabolic activation (rat liver S9 mix)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-aminoanthracene

Remarks:

With metabolic activation (hamster liver 59 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
-Selective Agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium.
-Overlay Agar
-The overlay agar contains per litre:
6.0 g Merck Agar
6.0 g NaCl
10.5 mg L-Histidine * HCl * H2O
12.2 mg Biotin

NUMBER OF REPLICATIONS: 3

-OTHER: Sterilisations were performed at 121° C in an autoclave

Evaluation criteria:

A test substance was considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test substance producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A test substance was considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

Noappropriate statistical method was available.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

PRE-TEST

The plates with the test item showed normal background growth up to 5000.0 ng/plate in strain TA 98 and TA 100, respectively. Thus the same dose levels were used in the main test.

MAIN TESTS

Toxic effects, evidenced by a reduction in the number of revertants, occurred at the higher concentrations with and without metabolic activation in the strains TA 1537 and TA 98 in experiment II.

The plates incubated with the test substance showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Applicant's summary and conclusion

Conclusions:

Tthe substance was considered to be non-mutagenic in this Salmonella typhimurium 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 92/69. Two experiments were performed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100: the plate incorporation test with Aroclor 1254-induced rat liver S9 mix (experiment I) and the pre-incubation test with uninduced hamster liver S9 mix (experiment II). The two independent experiments were performed 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 water and tested at six concentrations ranging from 33.33 to 5000.0 μg/plate. Previously, a pre-experiment for toxicity was carried out with strains S. typhimurium TA 100 and TA 98 to determine the highest concentration to be used in the mutagenicity assay.

Toxic effects, evidenced by a reduction in the number of revertants, occurred at the higher concentrations with and without metabolic activation in the strains TA 1537 and TA 98 in experiment II. The plates incubated with the test item showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with test item at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.