pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test was performed to investigate the potential of Bayscript Black TP LXS 51134 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471.
Because the test item is an azo-dye, a special test design according to Prival and Mitchell (1982) was applied as proposed in OECD TG 471. The assay was performed in three independent experiments. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and experiment II and IIa were performed with non-induced hamster liver S9 mix.

During the mutagenicity test and under the experimental conditions reported (OECD TG 471, modified version for azo-dyes), the test item did induce gene mutations by frameshifts in the genome of the strain TA 98 in the presence of non-induced hamster liver S9 mix.
Therefore, Bayscript Black TP LXS 51134 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Modification according to Prival and Mitchell (1982) (as proposed in OECD TG 471 for azo dyes):
Prival. M.J. and V.D. Mitchell (1982) Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9 Mutation Res. 97, 103-116

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix:from phenobarbital/beta-naphthoflavone incuded rats, prepared by ICCR-Rossdorf GmbH
hamster S9 mix: from uninduced Syrian golden hamsters, prepared by ICCR-Rossdorf GmbH

Test concentrations with justification for top dose:

10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate.

Vehicle / solvent:

On the day of the experiment, the test item was dissolved in deionized water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

for cultures with hamster S9 mix

Positive control substance:

congo red

other: 2-aminoanthacene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

for cultures with rat S9 mix

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium and Escherichia coli reverse mutation assays. Because the test item is an azo-dye, a special test design according to Prival and Mitchell (1982) was applied as proposed in OECD TG 471.
Experiment I was performed as a plate incorporation assay with induced rat liver S9 mix as an exogenous metabolic activation system. Since a negative was obtained in this experiment, experiment II was performed as a pre-incubation assay with non-induced hamster liver S9 mix. In experiment II a single positive effect was observed in strain TA 98 in the presence of S9 mix. To validate this effect this part of experiment II was repeated as a pre-incubation assay with adequately spaced concentrations. Since the data of the solvent and negative control of strain TA 100 in the presence of S9 mix were invalid, this part of experiment II repeated. The repeated parts are reported as experiment IIa.
Each concentration, including the controls, was tested in triplicate. The test item was tested as solution in water at the following concentrations in both experiments:
10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Based on the minor toxic effects and the desnse coloring of the overlay agar on the incubated agar, observed in experiment I, seven concentrations were tested in experiment II. 5000 μg/plate were chosen as maximal concentration in experiment II.
The concentration range included two logarithmic decades. The following concentrations were tested in experiment II:
10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
To validate a single positive effect observed in experiment II in strain TA 98 in the presence of S9 mix this part of experiment II was repeated as a pre-incubation assay with adequately spaced concentrations. Furthermore the data of the solvent and negative control of strain TA 100 in the presence of S9 mix were invalid and therefore repeated (reported as experiment IIa). The following concentrations were tested in experiment IIa:
Strain TA 98 (with S9 mix): 33; 100; 200; 400; 800; 1600, and 3200 μg/plate
Strain TA 100 (with S9 mix): 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in the presence of hamster liver S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 100, and the Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in the presence and absence of hamster liver S9 mix

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in the presence and absence of rat liver S9 mix

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment II a substantial and dose depended increase in revertant colony numbers was observed following treatment with Bayscript Black TP LXS 51134 in strain TA 98 in the presence of non-induced hamster liver S9 mix. The threshold of twice the number of the corresponding solvent control was exceeded at 333 μg/plate solely. To validate this positive effect and to proof its reproducibility strain TA 98 with S9 mix was repeated as a pre-incubation assay with adequately spaced concentrations. In the repeated experiment again an increase in revertant colony number was observed. The threshold of twice the number in revertant colony count of the corresponding solvent control was exceeded from 100 to 800 μg/plate. At the next higher concentrations the number in revertant colony count decreased below the significant threshold of twice the number of the corresponding solvent control.

Conclusions:

Bayscript Black TP LXS 51134 is considered to be mutagenic in this bacterial reverse mutation assay (frameshift mutations in the genome of strain TA 98 in the presence of non-induced hamster liver S9 mix).

Executive summary:

This study was performed to investigate the potential of Bayscript Black TP LXS 51134 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471.
Because the test item is an azo-dye, a special test design according to Prival and Mitchell (1982) was applied as proposed in OECD TG 471. The assay was performed in three independent experiments. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and experiment II and IIa were performed with non-induced hamster liver S9 mix.

During the mutagenicity test and under the experimental conditions reported (OECD TG 471, modified version for azo-dyes), the test item did induce gene mutations by frameshifts in the genome of the strain TA 98 in the presence of non-induced hamster liver S9 mix.
Therefore, Bayscript Black TP LXS 51134 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) the conduction of an in-vivo Comet assay is proposed to evaluate potential mutagenic activity in vivo.

Justification for classification or non-classification

To assess the relevance of the positive outcome of an bacterial mutagenesis test (Ames Test) for the in vivo situation an in vivo Comet-assay is proposed. As long as the results of this test are not available, the registered substance is considered as 'inconclusive' with regard to mutagenicity.