[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

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:

April May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

Ames plate pre-incubation method (Prival and Mitchell modification)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

EC Number:

824-263-3

Cas Number:

2196165-14-5

Molecular formula:

C28H21N9O7S2.xNa.yK.zLi

IUPAC Name:

[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Method

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:

Hamster liver homogenate metabolizing system (30% liver S9 in modified co-factors)

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the OECD TG 471 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.

Vehicle / solvent:

Sterile distilled water ; Supplier: BAXTER; Batch number, (purity), expiry: 17H09BB1A, (N/A), expiry Jul 2020.
The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

congo red

mitomycin C

other: 2-Aminoanthracene (2AA) and 1,8-Dihydroxyanthraquinone (DAN)

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 – The Prival-Mitchell Modification to the Ames Test
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.

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/beta-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). Manual counts were performed at and above 500 µg/plate (absence of S9) and 1500 µg/plate (presence of S9) because of test item induced coloration. Sporadic manual counts were also performed due to spreading colonies which prevented an accurate automated count.

Test for Mutagenicity: Experiment 2 – The Prival-Mitchell Modification to the Ames Test
The second experiment was not performed because the OECD 471 test guideline permits non repetition of the experiment when a clear, positive response is obtained in the first experiment. Therefore, a second, confirmatory experiment (employing the Prival-Mitchell modification) was not required.

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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and TA102 or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5.Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

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

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

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 the experiment 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 S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any test item dose level, either in the presence or absence of metabolic activation (30% liver S9 in modified co factors). Toxicity, in terms of reductions in revertant colony frequency, were noted to the majority of tester strains at the upper test item dose levels.
A black test item colouration was noted from 50 µg/plate (absence of S9) and 150 µg/plate (presence of S9) with an associated precipitate observed by eye from 500 µg/plate (absence of S9) and 1500 µg/plate (presence of S9). These observations did not prevent the scoring of revertant colonies.
The test item induced substantial increases in the frequency of TA100, TA102, TA98 and TA1537 revertant colonies in the presence of S9 only (30% liver S9 in modified co factors). The increases for TA98 were particularly large and showed a dose-related response following a bell-shaped curve with a maximum 15.4 fold increase over the concurrent vehicle control noted at 150 µg/plate. Furthermore, the individual colony counts from 5 µg/plate exceeded the maxima in-house untreated/vehicle historical data for the strain. The responses for TA100, TA102 and TA1537 were smaller but 2 fold increases were noted and in many instances individual colony counts were in excess of the in-house maxima historical control counts for each strain. A smaller response was also noted for TA98 in the absence of S9, however revertant colony counts were within the in-house untreated/vehicle control range.
Experiment 2 (The Prival-Mitchell Modification to the Ames Test)
The second experiment was not performed because the OECD 471 test guideline permits non repetition of the experiment when a clear, positive response is obtained in the first experiment. Therefore, a second, confirmatory experiment (employing the Prival-Mitchell modification) was not required.

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ (employing the Prival-Mitchell Modification) using strains of Salmonella typhimurium (OECD TG 471) the test item induced large, statistically significant and dose-related increases in the frequency of TA100, TA102, TA98 and TA1537 revertant colonies at the majority of the dose levels used predominantly with metabolic activation (30% liver S9 in modified co factors). Under the conditions of this test the test item was considered to be mutagenic.

Executive summary:

 

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, the ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with the test item using the Ames plate pre-incubation method (Prival and Mitchell modification) at 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 pre-determined and was 1.5 to 5000 µg/plate. 

The OECD 471 test guideline permits non-repetition of the experiment when a clear positive response is obtained in the first experiment, therefore, with the Sponsor’s approval, testing was suspended at the end of Experiment 1.

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 S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any test item dose level, either in the presence or absence of metabolic activation (30% liver S9 in modified co‑factors), in the first mutation test. Toxicity, in terms of reductions in revertant colony frequency, were noted to the majority of tester strains at the upper test item dose levels.

A black test item colouration was noted from 50 µg/plate (absence of S9) and 150 µg/plate (presence of S9) with an associated precipitate observed by eye from 500 mg/plate (absence of S9) and 1500 µg/plate (presence of S9). These observations did not prevent the scoring of revertant colonies.

The test item induced substantial increases in the frequency of TA100, TA102, TA98 and TA1537 revertant colonies in the presence of S9 only. The increases for TA98 were particularly large and showed a dose-related response following a bell-shaped curve with a maximum 15.4 fold increase over the concurrent vehicle control noted at 150 µg/plate. Furthermore, the individual colony counts from 5 µg/plate exceeded the maxima in-house untreated/vehicle historical data for the strain. The responses for TA100, TA102 and TA1537 were smaller but 2 fold increases were noted and in many instances individual colony counts were in excess of the in-house maxima historical control counts for each strain. A smaller response was also noted for TA98 in the absence of S9, however revertant colony counts were within the in-house untreated/vehicle control range.

The test item was considered to be mutagenic under the conditions of this test.