Disodium 3,3'-[cyclohexylidenebis[(2-methyl-4,1-phenylene)azo]]bis(4,6-dihydroxynaphthalene-2-sulphonate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

other: read across from similar substance

Adequacy of study:

key study

Study period:

From January 21, 1994 to April 15, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is conducted according to internationally accepted testing guideline and in compliance with the GLP Principles.

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 OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Principles of method if other than guideline:

SOP No. 30 50 01

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-Aminoanthracene,

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)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 µg/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.NUMBER OF REPLICATIONS: 3

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitates or aggregates were notedADDITIONAL INFORMATION ON CYTOTOXICITY: The numbers of revertant colonies were occasionally slightly reduced at the highest concentration of 5000 µg/plate.RANGE-FINDING/SCREENING STUDIES: Six concentrations of test item) 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 numbers of revertant colonies were not reduced. From the results obtained 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 revenants.ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative
It is concluded that the substance and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.

Executive summary:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. 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 bidistilled water 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The experiment was in compliance with the GLP principles and conducted according to the guideline OECD 471 and EU Method B.13/14 and EPA § 798.5265

 

The test was performed in two independent experiments both with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. ln both mutagenicity tests normal background growth was observed with all strains at all concentrations.

Justification for selection of genetic toxicity endpoint
The study is conducted according to internationally accepted testing guideline and in compliance with the GLP Principles.

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).