reaction mass of: tetrasodium 7-(4-(4-fluoro-6-(4-(2-sulfonatoethylsulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate;tetrasodium 7-(4-(4-hydroxy-6-(4-(2-sulfonatoethylsulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

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:

25 March 1998 to 03 April 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

In addition to the standard plate incorporation test (Ames Test) a modified protocol using preincubation with hamster S9 supplemented with flavine mononucleotide was used. This protocol has been proposed by Prival for assessing the mutagenic activity of azo dyes.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

427-650-1

EC Name:

-

IUPAC Name:

reaction mass of: tetrasodium 7-(4-(4-fluoro-6-(4-(2-sulfonatoethylsulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate and tetrasodium 7-(4-(4-hydroxy-6-(4-(2-sulfonatoethylsulfonyl)phenylamino)-1,3,5-triazin-2-ylamino)-2-ureidophenylazo)naphthalene-1,3,6-trisulfonate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Reaktivgelb FD 08064

Method

Target gene:

Reverse mutation to histidine prototrophy using histidine auxotrophic mutants.

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix aus Rattenleber und S9-Mix aus Hamsterleber

Test concentrations with justification for top dose:

a: without metabolic activation: 4, 20, 100, 500, 2500, 5000 ug/plate
b: with metabolic activation (10 % rat liver): 4, 20, 100, 500, 2500, 5000 ug/plate
c: with metabolic activation (30 % Syrian golden hamster liver and preincubation): 4, 20, 100, 500, 2500, 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: double-distilled water
- Justification for choice of solvent/vehicle: Standard laboratory reagent with history of use.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

a: untreated control b: solvent controls (0 ug/plate)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: See below

Remarks:

5 substances used for +ve control

Details on test system and experimental conditions:

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 TA 98

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 andTA 1537
Congo red for strain TA 98

Formulation of test compound:

Dissolved in double-distilled water at appropriate concentrations immediately before use

Formulation of reference compounds:

sodium-azide dissolved in double-distilled water final concentration: 1.0 ug/plate for strain TA100 and TA 1535

9-aminoacridine dissolved in DMSO final concentration: 50.0 ug/plate for strain TA 1537

2-nitrofluorene dissolved in DMSO final concentration: 2.5 ug/plate for strain TA 98

2-aminoanthracene dissolved in DMSO final concentrations (10 % rat liver S9-mix (v/v)): 0.5 ug/plate for strains TA 100 and TA 98 1.0 ug/plate for strains TA 1535 and TA 1537

Final concentrations (30 % Syrian golden hamster liver S9-mix (v/v)): 1.0 ug/plate for strain TA 100 0.5 ug/plate for strain TA 1535 2.5 ug/plate for strains TA 1537

Congo red dissolved in DMSO final concentration: 500.0 ug/plate for strain TA 98

The frozen stock solutions of each compound were diluted progressively up to the final concentration on the day of treatment.

Source of biological material:

Stock cultures in the bank of "Genetic Toxicology", Hoechst Marion Roussel Deutschland GmbH, Lead Optimization, Department of oxicology/Pathology prepared from the original bacterial 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

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

Preparation and storage of a liver homogenate fractions (S9)

The S9 fraction was prepared by the department conducting the study according to Ames et. al (1975) and Prival et. al (1982). Male Sprague Dawley rats (200-300 g) and male Syrian golden hamster (7-8 weeks old), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany. Liver preparations were performed from the liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrian hamsters. Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 Sprague Dawley rats or from 10 male Syrian hamsters (7-8 weeks old) at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. 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 dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix

Sufficient S9 fraction was thawed immediately at room temperature before each test. One volume of S9 fraction (batch no. 97/16 for both experiments, protein concentration 25.6 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 S9-mix of the rat liver were:

246.0 mg MgCI2 8 mM
162.65 mg KCI 33 mM
170.1 mg glucose-6-phosphoric acid
disodium salt dihydrate = 5 mM
333.36 mg NADPH 4 mM
213.76 mg NADH 4 mM

Na2HPO4/NaH2PO4 - phosphate buffer pH 7.4 = 100

According to the modification proposed by Prival (5) using 30 minutes preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction (batch no. 97/7 for both experiments, protein concentration 30.0 g/l) was mixed with 7 volumes of the S9 cofactor solution. This preparation is termed S9-mix. The S9-mix consists of:

8 mM MgCI2
33 mM KCI
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NADP+
2mM NADH
2mM 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..
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 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 as described below.

Assay procedure
Two independent mutation tests for each of the two protocols (Ames, Prival) were 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 first mutation experiment also assessed the toxicity of the test substance in order to select a suitable range of dose levels for the second mutation test. A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawns were used as toxicity indicators. Thinning of the bacterial lawns was evaluated microscopically.

Parallel to the second mutation experiment, precise toxicity testing was performed as follows using dose levels selected on the basis of toxicity results in the first test: 0.1 ml of the different concentrations of the test compound were thoroughly mixed with 0.1 ml of 106 dilution of the overnight culture of TA 100 (designated TA 100 D for the Ames Test and TA 100 HD for the Prival modification) and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

a)- with 10 % (v/v) rat liver S9-mix or buffer and the Salmonella strains TA 98, TA 100, TA 1535 and TA 1537
For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml 10 % (v/v) rat liver S9-mix (if required) or 1/3 nutrient broth-buffer mix 0.1 ml of an overnight nutrient broth culture of the bacterial tester strain 0.1 ml test compound solution (dissolved in double-distilled water)

After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal (histidine deficient) 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+ revertants) were counted with an automatic colony counter (Artec counter Model 880).

b)- with 30 % (v/v) Syrian golden hamster S9-mix and preincubation and the Salmonella strains TA 98, TA 100, TA 1535 and TA 1537
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S9-mix were incubated at approx. 30 °C for approx. 30 minutes. Subsequently, 2 ml of soft agar containing of 100 ml agar (0.6 % (w/v) agar + 0.5 % (w/v) NaCI) and 10 ml amino-acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) was added. After mixing, the samples were poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approximately 30 seconds. After incubation for 48 hours at 37 °C in the dark, colonies (his* revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter. A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.

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 induced increases in the mutation frequency which were both statistically 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:
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 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.

The test results must be reproducible.

Statistics:

Comparison with the testing laboratorie's historical range.

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

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.

Solubility and toxicity

The test compound was dissolved in double-distilled water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provides a final concentration of 5000 ug/plate. Further dilutions of 2500, 500, 100, 20 and 4 ug/plate were used in all experiments.
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 ug/plate.
The test compound proved to be not toxic to the bacterial strains.

In the toxicity test with a dilution of tester strain TA 100 (designated TA 100 D and TA 100 HD), which was performed in parallel with the second mutation experiment, no toxicity was found either in the absence or in the presence of metabolic activation up to the highest investigated dose of 5000 ug/plate.

Mutagenicity

In both independent mutation tests Reaktivgelb FD 08064 was tested for mutagenicity with the same concentrations as described in section 6.2. The number of colonies per plate with each strain as well as mean values of 3 plates are given.

Ames 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 in the presence of rat liver S9-mix in either mutation test. No dose-dependent 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 compound 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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Both Ames and Prival tests

Applicant's summary and conclusion

Conclusions:

The results lead to the conclusion that Reaktivgelb FD 08064 is not mutagenic in the absence and presence of rat S9-mix (10 % (v/v)) using the standard Ames Test procedure. Also in the presence of hamster liver S9-mix (30 % (v/v)) and preincubation the test compound did not induce a significant increase in the number of revertant colonies.

Executive summary:

The substance did not cause mutagenic effects in either the Ames nor Prival tests conducted. The substance is not considered therefore to be potentially mutagenic on the basis of the study results.