Tetrasodium 7,7'-(carbonyldiimino)bis[4-hydroxy-3-[(6-sulphonato-2-naphthyl)azo]naphthalene-2-sulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative

Additional information

In Vitro:

Ames test:

The key study was performed according to OECD TG 471 in compliance with GLP (BASF AG 40M0402/064062 (2006)). The test article (same dye as the registered substance plus monoethanolamine, CAS no.: 85959-62-2, chemical name: 7,7-(Carbonyldiimino)bis(4-hydroxy-3-((6-sulpho-2-naphthyl)azo)naphthalene-2-sulphonic) acid, sodium salt, compound with 2-aminoethanol; dye concentration: 61.5% w/w) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (Ames standard plate test and Prival preincubation test). STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. Water was used as vehicle. DOSE RANGE: 23 μg - 8 350 μg/plate (SPT); 23 μg - 8 350 μg/plate (PIT). TEST CONDITIONS: Ames standard plate test (SPT) with and without metabolic activation (Aroclor-induced rat liver S9 mix) and Prival preincubation test (PIT) with and without metabolic activation (uninduced hamster liver S9 mix). SOLUBILITY: No precipitation of the test substance was found. TOXICITY: No bacteriotoxic effect was observed, except in the experiments using TA 1537 where slightly decreased numbers of his+ revertants were observed from 835 μg/plate onward (SPT) and from 575 μg/plate onward (PIT). MUTAGENICITY: An increase in the number of his+ or trp+ revertants was not observed either in the Ames standard plate test or in the Prival preincubation test without S9 mix or after the addition of a metabolizing system. CONCLUSION: According to the results of the present study, the test article is not mutagenic in the Ames standard plate test and in the Prival preincubation test under the experimental conditions chosen here.

 

In support hereof an Ames test ( Ciba/CCR 188201 (1990)) is avalable which was performed using a guideline similar to OECD guideline 471 (1981) and the principles of GLP. The purity of the test material was 96.4%. Distilled water was used as vehicle. In the presence and absence of rat liver S-9 microsomal activation system and at doses of 10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate, S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 did not show an increased number of revertant colonies. In addition, there was no indication of toxicity to bacteria. Therefore, no evidence of a mutagenic potential was associated with the test substance.

In a second supporting Ames test (Ciba/Microtest 2S5RECGC.022 (1989)) with the test material (purity: 84.4% free acid), performed according to OECD 471, in the presence and absence of rat liver S-9 microsomal activation system and at doses of 8, 40, 200, 1000 and 5000 µg/plate (vehicle distilled water), S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 did not show an increased number of revertant colonies. In addition, there was no indication of toxicity to bacteria. Therefore, no evidence of a mutagenic potential was associated with the test substance.

 

Gene mutation/chromosomal aberration test:

The test item Analogue substance 1 was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 5000 µg/mL (with and without metabolic activation) was selected as the highest concentration. In experiment ll 5000 µg/mL (with metabolic activation) and 2500 µg/mL (without metabolic activation) were selected as the highest concentrations. Experiment 1 with and without metabolic activation and experiment ll with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I, with and without metabolic activation: 10, 50, 100, 200, 400, 600, 1800 and 5000 µg/mL

Experiment ll, with metabolic activation: 30, 70, 150, 350, 700, 2l00, 3600 and 5000 µg/mL; without metabolic activation: 10, 20, 50, 100, 200, 600, 1200 and 2500 µg/mL

Precipitation of the test item was noted in all experiments.

Growth inhibition was observed in experiment I and ll without metabolic activation.

ln experiment I with metabolic activation the relative total growth (RTG) was 93.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 5000 µg/mL with a RTG of 65.0%.

In experiment II with metabolic activation the relative total growth (RTG) was 71.5% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 2500 µg/mL with a RTG of 12.9%.

In experiment I and ll no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories [13, 14, 15]) was not exceeded by the induced mutant frequency at any concentration.

No dose~response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

ln conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Analogue substance 1 is considered to be non-mutagenic and non-clastogenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5l78Y cells.

Moreover, the test item Analogue substance 2 was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro, in the absence and presence of metabolic activation by S9 mix, according to the OECD guideline 473.

In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosome aberrations. The following dose levels were evaluated, with and without S9 mix: at 7 h 1.0 mg/mL; at 18 h 0.5; 2.5; 5.0 mg/mL; at 28 h: 1.0 mg/mL
Under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

Short description of key information:

The test substance is considered not to be mutagenic and not to be clastogenic, with and without metabolic activation, in the in vitro gene mutation/chromosomal aberration test (according to OECD TG 476 and OECD 473) conducted with the read across substances Analogue substance 1 and analogue substance 2. Furthermore the test substance is considered non-mutagenic, with and without metabolic activation,  in the Ames test (bacterial reverse mutation assay according to OECD TG 471).

Justification for classification or non-classification

Based on the available in vitro genotoxicity tests, the test substance does not need to be classified for genotoxicity according to the Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.