Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

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:

Experimental start date 18 April 2017 Experimental completion date 01 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

EC Number:

256-851-5

EC Name:

Tetrasodium 3,3'-[[6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)azo]]bisnaphthalene-1,5-disulphonate

Cas Number:

50925-42-3

Molecular formula:

C39H30N10Na4O13S4

IUPAC Name:

tetrasodium 3,3'-({6-[(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl}bis[imino(2-methyl-4,1-phenylene)diazene-2,1-diyl])dinaphthalene-1,5-disulfonate

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid

Specific details on test material used for the study:

Identification: Bayscript Gelb BR
CAS Number: 50925-42-3
Chemical Name: 1,5-Naphthalenedisulfonic acid, 3,3-[[6](2hydroxyethyl)amino-1,3,5-triazine-2,4-diyl]bis[imino(2-methyl-4,1-phenylene)-2,1-diazenediyl]]bis-, sodium salt (1:4)
Empirical Formula: C39H30N10Na4O13S4
Molecular Weight: 1066.9 g/mol
Physical state/Appearance: Brown solid
Expiry Date: 11 July 2018
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated impurity content of the test item.

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

A liver microsomal preparation (S9) prepared from hamsters not pre treated with any enzyme inducers.

Test concentrations with justification for top dose:

Experiment 1 – Pre-Incubation Method
The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were used.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate. Six concentrations of the test item (15, 50, 150, 500, 1500 and 5000 µg/plate) were used.

Vehicle / solvent:

The solvent (vehicle) control used was sterile distilled water.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

3 µg/plate for TA100, 5 µg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

80 µg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/plate for TA98

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

without metabolic activation

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

0.5 µg/plate for TA102

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/plate for TA102

Positive control substance:

other: 1, 8-Dihydroxyanthraquinone

Remarks:

with metoblic activation

Controls 8

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

50 µg/plate for TA100 and TA98

Positive control substance:

congo red

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Test Item Preparation and Analysis
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. Formulated concentrations were adjusted to allow for the stated impurity content (13.9%) of the test item. All formulations were used within four hours of preparation.

Test for Mutagenicity: Experiment 1 – Pre-Incubation Method
The Prival-Mitchell modification to the standard Ames Test is necessary for the testing of azo dyes which can contain mutagenic aromatic amines which are not readily detected using the standard method (Prival and Mitchell (1982)). The modification differs in five key areas from the standard plate incorporation Ames Test:
• Uninduced hamster liver S9 rather than induced rat liver S9.
• 0.15 mL of S9 rather than the maximum of 0.05 mL of S9 in the standard Ames Test.
• The use of flavin mononucleotide (FMN), nicotinamide adenine dinucleotide (NADH), four times the standard amount of glucose-6-phosphate, and the inclusion of exogenous glucose 6 phosphate dehydrogenase in the co-factor mix.
• A 30 minute pre-incubation prior to the addition of the molten top agar.
• Vogel-Bonner plates containing 0.5% glucose instead of the standard 2% glucose.

Only the test item concentrations, vehicle and the positive control, Congo Red, were dosed using the Prival Mitchell modification.

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 g/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the Prival and Mitchell method for testing azo dyes in the presence of flavin mononucleotide and hamster liver S9.

Without Metabolic Activation
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation or vehicle. Each mixture was shaken gently at 37 ± 3 ºC for 30 ± 3 minutes. Then, 2 mL of molten, trace histidine supplemented, top agar was added to each tube. The mixture was vortexed and poured onto Vogel-Bonner minimal agar plates containing 0.5% glucose. Each concentration of the test item, appropriate positive control, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of hamster S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer. In addition, 0.1 mL of TA98 or TA100, 0.5 mL of uninduced hamster liver S9 and 0.1 mL of Congo Red at 50 µg/plate was dispensed into dosing tubes, incubated and overlaid onto 0.5% glucose Vogel Bonner plates as previously described.

The standard Ames positive controls were dosed using the pre-incubation method (previously described) where 0.1 mL of bacterial culture was mixed with 0.5 mL of rat liver S9-mix (phenobarbitone/B naphthoflavone) or phosphate buffer and 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

The negative (untreated) controls were dosed using the plate incorporation method where 0.1 mL of bacterial culture was mixed with 2 mL of amino-acid supplemented top agar before overlaying onto 2% glucose Vogel-Bonner agar plates.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.


Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.

Six test item concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test item.

Without Metabolic Activation
Testing was performed as described in Experiment 1

With Metabolic Activation
Testing was performed as described in Experiment 1

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

S. typhimurium 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

Test results 5

Species / strain:

S. typhimurium TA 102

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:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A yellow test item induced colouration was noted from 50 µg/plate, becoming orange at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Applicant's summary and conclusion

Conclusions:

Bayscript Gelb BR was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated withBayscript Gelb BR using the Ames pre-incubation method (Prival Mitchell Modification for Azo Compounds)at up to eight dose levels, in triplicate, both with and without the addition of a hamster liver homogenate metabolizing system (30% liver S9 in modified co‑factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the induced rat liver and the uninduced hamster liver S9-mixes were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. A yellow test item induced colouration was noted from 50 µg/plate, becoming orange at and above 500mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2. 

Conclusion

Bayscript Gelb BRwas considered to be non-mutagenic under the conditions of this test.