Reaction mass of 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 and trisodium 5-({4-chloro-6-[4-(vinylsulfonyl) anilino]-1,3,5-triazin-2-yl}amino)-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-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:

11 October to 20 October 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

84/449/EWG, B.14, Prival Modification

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

Details on test material:

- Name of test material (as cited in study report): Reaktiv-Rot F-66813 FW

Method

Target gene:

TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift
TA1538 hisD3052 Frameshift

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine dependent

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat and hamster liver

Test concentrations with justification for top dose:

Preliminary test: 4 to 10000 µg/plate
Main test: 4 to 5000 µg/plate

Vehicle / solvent:

At the day of the experiment the test substance was dissolved in Aqua bidest at appropriate concentrations. Two independent experiments were performed for each protocol (Ames, Prival).

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqua bidest

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

w/o S9 Migrated to IUCLID6: TA100, TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

w/o S9 Migrated to IUCLID6: TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

w/o S9 Migrated to IUCLID6: TA1538, TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with rat S9 10%: TA98, TA100, TA1535, TA1537, TA1538; with rat S9 30%: TA98, TA100, TA1535, TA1537

Controls 5

Untreated negative controls:

no

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, TA1538; with rat S9 30%: TA98, TA100, TA1535, TA1537

Controls 6

Untreated negative controls:

no

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:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzidine

Remarks:

with hamster S9 30%: TA98

Controls 8

Untreated negative controls:

no

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

Details on test system and experimental conditions:

Preparation and storage of a liver homogenate fraction ("S-9")
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrien hamsters. Male Sprague Dawley rats (200 -300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5-6 Sprague Dawley rats or from 5-6 male Syrian golden hamsters (7-8 weeks old) are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KC1. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at -80 °C for not longer than three months.

Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room tempera¬ture. One or three volumes of S-9 fraction is mixed with nine or seven volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are:

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

According to the modification proposed by Prival (5) using 30 minutes preincubation in the presence of 30 % Syrian golden hamster S-9 Mix. The S-9 Mix consists of:

8 mM MgCl2
33 mM KCI
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NAPD+
2 mM NADH
2 mM FMN (Riboflavin-5’-phosphate-Na-salz)
100 mM phosphate buffer pH 7.4

Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98.

Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with five or four tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically. In combination with the main experiments, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is 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).

Mutagenicity test
Two independent experiments which each of the two protocols (Ames, Prival) were performed.

a) - with 10 % rat liver S-9 Mix or buffer and the strains TA 100, TA 1535, TA 1537, TA 1538 and TA 98 - with 30 % rat liver S-9 Mix and the stains TA 100, TA 1535, TA 1537 and TA 98

Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The follow¬ing ingredients are added (in order) to 2 ml of molten top agar at 45 °C:

0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml 10 % or 30 % rat liver S-9 Mix or buffer

After mixing, the liquid is poured into a petridish with minimal agar (1.2 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

b) with 30 % Syrian golden hamster S-9 Mix and preincubation

0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at 30 °C for the duration of 30 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

Positive controls
Positive control plates were included for each strain. The following substances were used as positive controls.

a) without metabolic activation:
Na-azide: TA 100, TA 1535
9-Aminoacridine: TA 1537
2-Nitrofluorene: TA 1538, TA 98

b) with rat liver S-9 Mix (10 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537, TA 1538
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537, TA 1538

c) with rat liver S-9 Mix (30 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537

d) with hamster liver S-9 Mix (30 %):
2-Aminoanthracene: TA 100, TA 1535, TA 1537
Benzidine: TA 98
Congored: TA 98

Evaluation criteria:

No data

Statistics:

No data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

10% rat S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

10% rat S9, 30% rat S9, 30% hamster S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

10% rat S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

10% rat S9, 30% rat S9, 30% hamster S9

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Reaktiv-Rot F-66 813 FW was tested for mutagenicity with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the absence and presence of a metabolic activation systems. S-9 Mix from Sprague Dawley rats (10 % or 30 %) and from Syrien golden hamsters (30 %) were used. The number of colonies per plate with each strain as well as mean values of 3 plates, corrected to the next whole number are given.

Sterility checks and control plates
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Toxicity test
The test compound was tested at doses of 4 to 10000 microgram/plate and proved to be not toxic to the bacterial strains. For mutagenicity testing 5000 microgram/plate was chosen as the highest dose in the main experiments.

Mutagenicity test with Reaktiv-Rot F-66 813 FW
Ames-Test:
The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat S-9 Mix (10 %). No dose dependent effect was obtained.

Prival-Test:
In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: preliminary test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It can be stated that Reaktiv-Rot F-66 813 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Executive summary:

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:OECD Guidelines for testing of chemicals 471Genetic Toxicology : Salmonella typhimurium, Reverse Mutation Assay, Adopted : May 26th, 1983andEEC Directive 79/831 Annex V, 4.3.1. This study was conducted in compliance with the principles of good laboratory practice (GLP).

 

Reaktiv-Rot F-66 813 FW was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA  1538 and TA 98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 microgram/ plate to 5000 microgram/plate was used.

 

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive con­trol compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. 5 000 microgram/plate was chosen as top dose level for the mutagenicity study.

Ames Test:

Mutagenicity: In the absence of the metabolic activation system the test com­pound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 %), treatment of the cells with Reaktiv-Rot F-66 813 FW did not result in relevant increases in the number of revertant colonies.

Prival Test:

In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival Reaktiv-Rot F-66 813 FW did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

 

Summarizing, it can be stated that Reaktiv-Rot F-66 813 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.