tetrasodium 3-amino-4-{[4-({4-fluoro-6-[phenyl(2-{[2-(sulfonatooxy)ethyl]sulfonyl}ethyl)amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazenyl}-5-hydroxynaphthalene-2,7-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 19, 2004 to October 29, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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)

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:

other: Japanese Substance Control Law (JSCL) Test Guideline 111.1 Gene Mutation Test with bacteria

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-630-8

EC Name:

-

Cas Number:

2138836-26-5

Molecular formula:

Hill formula: C29H23FN8Na4O16S5

IUPAC Name:

tetrasodium 3-amino-4-[(1E)-2-[4-({4-fluoro-6-[phenyl({2-[2-(sulfonatooxy)ethanesulfonyl]ethyl})amino]-1,3,5-triazin-2-yl}amino)-2-sulfonatophenyl]diazen-1-yl]-5-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Plate incorporation test:
with metabolic activation(i.e.,10% rat liver):
50, 160, 500, 1,600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate
Preincubation test:
with metabolic activation (i.e., 30% hamster liver):
50, 160, 500, 1,600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate

Vehicle / solvent:

Deionized water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (10% rat liver) for all strains

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Controls 7

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE

An independent mutation test was performed using the plate incorporation method as results were negative, a second test was conducted. This included a preincubation step which 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 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/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 (i.e., 0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (i.e., 2.5 mL, 2.0 mM). The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 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 substance solution (i.e., dissolved in deionized water)

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

After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (i.e., 1.5% (w/v) agar, Vogel-Bonner E medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (i.e., 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. Slight deviations from the laboratory's normal control range can be accepted if the positive controls show no correlated deviation and if requirements for a negative response are clearly fulfilled.

- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range. Slight deviations from the laboratory's normal control range can be accepted if the negative/solvent controls show no correlated deviation and if requirements for a positive response are clearly fulfilled.

Criteria for a positive response

A test substance is classified as mutagenic if it has either of the following effects:

a) 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

b) 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 substance 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:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

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:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

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:

SOLUBILITY AND TOXICITY

Test 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 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in all experiments.

Test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.

Test substance proved to be not toxic to the bacterial strains in all experiments.

MUTAGENICITY

In both independent mutation tests test substance was tested for mutagenicity with the stated concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates were given.

Plate incorporation test:

The test substance 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 and absence of hamster liver S9-mix (i.e., 30 % (v/v)) using the preincubation method according to Prival the test substance did not cause a significant increase in the number of revertant colonies under the experimental conditions described.

All positive controls produced significant increases in the number of revertant colonies. Thus the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Any other information on results incl. tables

STERILITY CHECKS AND CONTROL PLATES

 

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance and S9-mix sterility check plates. Control plates (i.e., background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range

 

In the plate incorporation test the number of revertant colonies of the solvent control with the strain TA 1537 in the absence of S9-mix was marginally above the historical control data range, which did not influence the validity of the study. The number of revertant colonies of the solvent control in the absence of S9-mix (i.e., plate incorporation test) and of the solvent and negative control in the presence of S9-mix (i.e., plate and preincubation test) with the strain WP2uvrA was below the historical control data range. This indicated rather higher sensitivity of this strain than usual, because all positive controls fulfill the required criteria completely and were within the expected range. The validity of the study is therefore not influenced.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay with and without metabolic activation.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.13/14 and Japanese Guideline, in compliance with GLP.

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 (i.e., Prival test). The studies were performed in the absence and presence of a metabolizing system derived from a rat or hamster liver homogenate. The substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5,000 µg/plate in both assays.

Positive controls showed an expected increase in the number of revertant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix. In the plate incorporation test, the test substance exposure did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (i.e., rat liver S9-mix (10% (v/v)). Also, in the preincubation test no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (i.e., hamster liver S9 -mix (30% (v/v)).

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.