Disodium 6-(4,6-dichloro-1,3,5-triazin-2-ylamino)-1-hydroxy-2-(4-(2-(sulfonatooxy)ethylsulfonyl)phenylazo)naphthalene-3-sulfonate

Genetic toxicity: in vitro

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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:

1999-02-17-1999-03-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

Test organism:
Salmonella typhinium strains:
TA98 hisD3052 rfa uvrB + R
TA 100 hisG46 rfa uvrB +R
TA 1535 hisG46 rfa uvrB,
TA 1537 hisC3076 rfa uvrB
and
E. coli WP2 uvrA pkm101

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Bacteria:
The strains of Salmonella typhimurium were obtained from professor B.N. Ames, University of California, U.S.A. The strain of E. coli was obtained from the national Collection of Industrial Bacteria, Aberdeen, Scotland.
Bacteria were grown overnight in nutrient broth (25 g Oxid 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 at approx. -80 °C. The different bacterial strains are checked half-yearly with regard to their respective biotin, histidine requirements, membrane permeability, ampicillin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens. All criteria for a valid assay were fulfilled.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from induces rat liver and uninduced hamster liver

Test concentrations with justification for top dose:

Concentration range (all tests): 50, 160, 500, 1600, and 5000 µg/plate

Vehicle / solvent:

Solvent: distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: 2-aminoanthracene, 1-methyl-3-nitro-1-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation; in suspension;

Test System:
Test Groups: PLATE INCORPORATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (10 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate
PREINCUBATION TEST
a) Without metabolic activation: 50; 160, 500, 1600 and 5000 µg/plate
b) With metabolic activation (30 % rat liver): 50, 160, 500, 1600 and 5000 µg/plate


CONTROL GROUPS:
Negative controls:
a) Untreated control
b: solvent control
Positive controls:
a) without metabolic activation (Sodium-azide for Strain TA 100 and TA 1535, 9 aminoacridine for strain TA 1537, 2-Nitrofluorene for strain 98, MNNG for strain WP2uvrA and 4-NQO for strain WP2uvrA
b): with metabolic activation (10 % rat liver)
2-aminoanthracene for all tester strains
c)-with metabolic activation (30 % Syrian golden hamster liver and preincubation)
2-aminoanthracene for strain TA 100, TA 1535 and TA 1537
Congo red for Strain TA98

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

DURATION
- Preincubation period: 30 min preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction was mixed with 7 volumes of the S9 cofactor solution.

Evaluation criteria:

Assay considered valid if:
-Solvent control data are within the laboratory´s normal control range for the spontaneous mutant frequency
-Positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory´s normal range
Test compound is classified as mutagenic if has either 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 test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system 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 laboratories control range. Test compound proved to be not toxic to the bacterial strains. In all independent mutation tests, the test substance was tested for mutagenicity with concentrations as described in table above. Number of colonies per plate with each strain as well as mean values of 3 plates is given.

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 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 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 3

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 4

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

Test results 5

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: NA

- Water solubility:
The test compound did not precipitate on the plates until the highest investigated dose of 5000 µg/plate
The test substance proved to be not toxic to the bacterial strains.
HISTORICAL CONTROL DATA (see table under "overall remarks”)

Any other information on results incl. tables

STERILITY CHECKS AND CONTROL PLATES

Sterility of S9-mix and the test material and S9-mix sterility check plates. Control plates gave the expected number of colonies, i.e. values were within the laboratory's historical range.

SOLUBILITY AND TOXICITY

The test compound 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 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in all experiments.

The test compound proves to be not toxic to the bacterial strains.

MUTAGENICITY:

In all independent mutation tests, the test substance was tested for mutagenicity with the same concentration.

 

AMES 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 in the presence of rat liver S9-mix in either mutation test. No dose dependant effect was obtained.

PRIVAL TEST:

In the presence of hamster liver S9-mix (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.

Applicant's summary and conclusion

Conclusions:

The test substance is not mutagenic in the standard plate test; Ames Test as well as in the preincubation method according to Prival.

Executive summary:

The substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

Two independent mutagenicity studies were conducted one standard plate test ( Ames Test) and 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. Additionally, a repeat of the preincubation test was performed with the strain WP2uvrA in the absence of S9 -mix.

For all studies, the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels. Doses for all 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 range and similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies, except in the first preincubation test with the strain WP2uvrA in the absence of S9-mix, where the positive control showed not the expected increase in the number of revertant colonies. This questionable effect was caused by a decomposition of N-Methyl-N-nitrosoguanidine (MNNG). In a repeat of this strain with an alternative positive compound 4 -Nitroquinoline-N-Oxide (4 -NQO) the sensitivity of the assay could be demonstrated.

Toxicity: In the plate incorporation test and also in the preincubation test toxicity was not observed either with or without metabolic activation.

Ames Test:

Mutagenicity: in the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with the test substance did not result in relevant increases in the number of revertant colonies.

 

Prival 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 increases in the number of revertant colonies with any of the tester strains under experimental conditions described.

Therefore, the test substance is not mutagenic in the standard plate test (Ames Test) as well as in the preincubation method according to Prival at the investigated dose levels.