Sodium bis[3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzene-1-sulphonamidato(2-)]chromate(1-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From January 21, 1994 to March 21, 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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 gene mutation assay

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

Test concentrations with justification for top dose:

20.58, 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the substance was dissolved in DMSO by slightly warming. The test substance was soluble up to the concentration of 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO.

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 mix

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

Remarks:

without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Inoculates from frozen master copies were set up monthly. They were grown in liquid nutrient broth medium (NB-medium) overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

OTHER:
A range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control according to a Standard Operating Procedure of Genetic Toxicology. The highest concentration applied was 5000.0 ug/plate. The five
lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5 °C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative
control was used.

Evaluation criteria:

Assay acceptance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study
Director.

Criteria for a positive response
The test substance will be considered to be positive in the test system if the following condition is met:
• At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested. Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended

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 determined

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitates or aggregates were noted.


RANGE-FINDING/SCREENING STUDIES: Six concentrations of test substance ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and
without metabolic activation. Normal background growth was observed. The highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and 5000.0 µg/plate without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Arithmetic Mean and Standard Deviation (SD) of colony counts obtained in 75 separate experiments over the period of January 01, 1993 to December 31, 1993 and acceptable ranges for mean colony counts of spontaneous revertants.

In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with test substance did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment

In the experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with test item no increase in the incidence of either histidine-prototrophic mutants was observed in comparison with

the negative control.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Conclusions:

The substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Executive summary:

Similar Substance 1 was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimunum. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537.

The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in DMSO and tested at five concentrations in the range of 61.7 to 5000.0 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 61.7 to 5000.0 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. In both experiments, performed with and without metabolic activation, none of the tested concentrations of test item led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that tested substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008) a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms.

The test substance is not capable to induce permanents mutation inside the genetic structure, therefore according to the ECHA guidance R.7a R.7.7 -1 Flow chart of the mutagenicity testing strategy, no further testing at this level are necessary and no classification is warranted according to the CLP Regulation (EC n. 1272/2008)