Disodium [3-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2-)]chromate(2-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from March 29 to May 2, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was carried out according to an internationally accepted testing guideline, even if it is an earlier version (ver.1983) that includes only four strains and not five as in the last update (ver.1997).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

ver. 1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Brown 298

IUPAC Name:

Acid Brown 298

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Regular checking of the properties of the strains with regard to membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in CCR according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

Concentration range in the range finding test: from 3.3 to 5000.0 µg/plate
Concentration ranges in the mutagenicity test: from 33.3 to 5000.0 µg/plate (active ingredient)

Vehicle / solvent:

- Vehicle: water
- Justification for choice of solvent/vehicle: the test substance is very soluble in water

Controls

Untreated negative controls:

yes

Remarks:

solvent alone

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-Aminoanthracene and 4-nitro-o-phenylene-diamine

Remarks:

Sodium azide was dissolved in distilled water and tested without metabolic activation; 4-NOPD was dissolved in DMSO and tested without metabolic activation; 2-AA was dissolved in DMSO and tested with metabolic activation;

Details on test system and experimental conditions:

- Characterisation of the Salmonella typhimurium Strains:
The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent.
Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the
strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
In summary, the mutations of the TA strains used in this study can be described as follows:
TA 1537: his C 3076; rfa-; uvrB-; frame shift mutations
TA 98: his D 3052; rfa-; uvrB-;R-factor frame shift mutations
TA 1535: his G 46; rfa-; uvrB-; base-pair substitutions
TA 100: his G 46; rfa-; uvrB-;R-factor base-pair substitutions

- Storage:
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

- Precultures:
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl.
The bacterial culture was incubated in a shaking water bath for 10 hours at 37° C.

- 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. Sterilisations were performed at 121° C in an autoclave.

- Overlay Agar:
The overlay agar contains per litre:
6.0 g Merck Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H20
12.2 mg biotin
Sterilisations were performed at 121° C in an autoclave.

Statistics:

No appropriate statistical method is available.

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100.
The plates with the test article showed normal background growth up to 5000.0 fig/plate in strain TA 98 and TA 100, respectively.

MAIN EXPERIMENTS
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.

RESULTS
Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 2500.0 µg/plate without S9 mix and at 5000.0 µg/plate with S9 mix in experiment I as well as at 2500.0 and 5000.0 µg/plate without S9 mix and at 2500.0 and 5000.0 µg/plate with S9 mix in experiment II.
The plates incubated with the test article showed normal background 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 the substance at any concentration 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 significance.
In the strains TA 98 and TA 100 dose-dependent increases in revertant colony numbers were observed beginning at 1000.0 µg/plate up to the highest investigated dose. However, the obtained mutation factors (1.9 in strain TA 98 and 1.5 in strain TA 100, respectively as the highest) did not reach the mutation factors (2.0 for strain TA 100 and 3.0 for strain TA 98, respectively) recommended for a mutagenic response and additionally in the normally more sensitive pre-incubation assay (experiment II) no indication of a mutagenic response was obtained in both strains. Therefore, the effects obtained in experiment I are considered not to be biologically relevant.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Remarks on result:

other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

The test substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used, both with and without metabolic activation.

Executive summary:

The test substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium, according to the OECD Guideline 471 (1983).

The test article was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the strains TA 1535, TA 1537, TA 98, and TA 100.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test article was tested at the following concentrations: 33,3; 100; 333,3; 1000; 2500 and 5000.0 µg/plate (active ingredient).

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