4,4'-[(6-chloro-1,3,5-triazine-2,4-diyl)diimino]bis[5-hydroxy-6-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic] acid, sodium salt

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:

18 July 2003 to 28 August 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

incorporating Privall Mitchell preincubation test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

JSCL TG III.1 Gene mutation test with bacteria

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

405-900-0

EC Name:

-

Cas Number:

111211-40-6

Molecular formula:

C29H22ClN7Na4O19S6

IUPAC Name:

tetrasodium 5-{[4-chloro-6-(4-{[2-(sulfonatooxy)ethyl]sulfonyl}anilino) -1,3,5-triazin-2-yl]amino}-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl) diazenyl]naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Method

Target gene:

Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: tryptophan dependent

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat and hamster liver

Test concentrations with justification for top dose:

50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

deionised water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

w/o S9 TA100, TA1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

w/o S9 TA1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

w/o S9 TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

w/o S9 WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with rat S9 10%: TA98, TA100, TA1535, TA1537, WP2uvrA

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with hamster S9 30% TA98

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with hamster S9 30%: TA100, TA1535, TA1537, WP2uvrA

Details on test system and experimental conditions:

Test groups
plate incorporation test:
with metabolic activation (10 % rat liver): 50,160, 500, 1600 and 5000 μg/plate
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate

preincubation test:
with metabolic activation (30 % hamster liver): 50, 160, 500, 1600 and 5000 μg/plate
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate

Control groups
negative controls:
a: untreated controls
b: solvent controls (0 μg/plate)

positive controls:
- without metabolic activation: sodium-azide for strain TA 100 and TA 1535, 9-aminoacridine for strain TA 1537, 2-nitrofluorene for strain TA 98, 4-nitroquinoline-N-oxide for strain WP2uvrA
- with metabolic activation (10 % rat liver): 2-aminoanthracene for all strains
- with metabolic activation (30 % syrian golden hamster liver): 2-aminoanthracene for strain TA 100, TA 1535, TA 1537 and WP2uvrA, congo red for strain TA 98

Formulation of test compound: dissolved in deionised water at appropriate concentrations immediately before use.

Formulation of reference compounds
Sodium-azide dissolved in deionised water final concentration: 1.0μg/plate for strain TA 1535, 2.0μg/plate for strain TA 100
9-aminoacridine dissolved in DMSO final concentration: 50.0μg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5μg/plate for strain AT 98
4-nitroquinoline-N-oxide dissolved in DMSO final concentrations: 2.0μg/plate (plate inc.), 0.5μg/plate (preinc.) for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations: (10% (v/v) rat liver S9-mix): 1.5μg/plate for strains TA 98, TA 100, TA 1535 and TA 1537, 20.0μg/plate for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations (30% (v/v) hamster liver S9-mix): 1.0μg/plate for strain TA 100, TA 1535 and TA 1537, 30.0μg/plate for strain WP2uvrA
Congo red dissolved in deionised water final concentration: 250μg/plate for strain TA98
The frozen stock solutions of each compounds were diluted progressively up to the final concentration on the day of treatment.

Source of bacteria: stock cultures in the bank of “Genetic Toxicology”, Aventis Pharma Germany, ProTox prepared from the original bacteria strains.

Test organism: Salmonella typhimurium strains – TA 98 hisD3052 rfa uvrB +R, TA 100 hisG46 rfa uvrB +R, TA 1535 hisG46 rfa uvrB, TA 1537 hisC3076 rfa uvrB and Escherichia coli WP2uvrA pKM101

Experimental conditions in vitro: approx. 37°C in an incubator.

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction of Spraque Dawley rat liver induced with Aroclor 1254 was obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, [phone]. The protein content for every batch was guaranteed by a Quality Control & Production Certificate by the supplier. Also for every batch of S9 an independent validation was performed in the laboratory with a minimum of two different mutagens, e.g. 2-aminoanthracene and benzo(a)pyrene, to confirm metabolic activation by microsomal enzymes.
The S9 fraction of Syrian golden hamster liver was prepared by the department conducting the study according to Prival et. al (1982). Male Syrian golden hamsters (7-8 weeks old), were supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany. Liver preparations were performed from the liver of non pretreated Syrian hamsters. The livers were removed from 10 male Syrian hamsters (7-8 weeks old) using cold sterile solutions at approx. 0 to 4 °C and glassware, and were then pooled and washed in approx. 150 mM KC1 (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KC1. The homogenate was centrifuged at approx. 9000g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g. 2-aminoanthracene and congo red, to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed at room temperature immediately before each test. One volume of Moltox. S9 fraction (batch no. 1530 for the plate incorporation test, protein concentration 36.1 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the rat liver S9-mix were:

8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
4mM NADP
100 mM phosphate buffer pH 7.4

According to the modification proposed by Prival (8) the test substance and the tester strains were
preincubated for 20 to 30 minutes with 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction (batch no. 2002/1 for the preincubation, protein concentration
45 g/l) were mixed with 7 volumes of the S9 cofactor solution.

This preparation is termed S9-mix. The hamster liver S9-mix consists of:

8 mM MgCl2
33 mM KC1
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4mM NADP+
2 mM NADH
2 mM FMN (Riboflavine-5’-phosphate-sodium-salt)
100 mM phosphate buffer pH 7.4

Bacteria
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. coli was obtained from E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored in liquid nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeability, ampicillin resistance, tetracyclin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.

ASSAY PROCEDURE
An independent mutation test was performed using the plate incorporation method. When results were negative or equivocal, a second test was conducted. This included a preincubation step if the first test was clearly negative. Preincubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in deionised water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approx. 30 °C.

After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose).
After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

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

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

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

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

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

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

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

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

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:

STERILITY CHECKS AND CONTROL PLATES
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.
The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled.
The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.

SOLUBILITY AND TOXICITY
The substance was dissolved in deionized water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were used in all experiments.
The substance did not precipitate on the plates up to the highest investigated dose of 5000μg/plate.

The substance proved to be not toxic to the bacterial strains.

MUTAGENICITY
The number of colonies per plate with each strain as well as mean values of 3 plates were given.

Plate incorporation test:
The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.
Preincubation test:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increase in the number of revertant colonies under the experimental conditions described.

Applicant's summary and conclusion

Conclusions:

The results lead to the conclusion that the substance is not mutagenic in the absence and presence of metabolic activation using the standard Ames Test procedure (plate incorporation test) and the preincubation test as described.

Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: 21-July-1997 and U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, Aug-1998andEC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14andJapanese Substance Control Law (JSCL) Test Guideline III.l Gene Mutation Test with bacteria.The study is based on the Principles of Good Laboratory Practice (GLP).

 The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. For all studies, the substance was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000 μg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control. All positive controls gave the expected increase in the number of revertant colonies. The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled. The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.

The substance did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate. In both studies toxicity was not observed either with or without metabolic activation.

 In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence or presence of the rat liver activation system (10 % (v/v)). Also in the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.

Summarizing, it can be stated that the substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.