Benzenesulfonic acid, 2-[2-[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]diazenyl]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:2)

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:

21 April 1993 to 29 April 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU & OECD test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

79/831/EEC and incorporating the Prival-Mitchell refinement for azodyes.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

The study describes experiments performed in a short term test using the pro¬cedure of the Salmonella/mammalian-microsome-mutagenicity test - Standard plate test (Ames Test) and a modified preincubation test (Prival modification) to assess the mutagenic potential of the test-material in histidine dependent strains of Salmonella typhimurium.

By the use of liver homogenate the test takes into account the mammalian metabo¬lism of the compound to be tested. The requirement for metabolic activation was investigated by incorporating into the test an activation system by nicotin¬amide-adenine dinucleotide phosphate (NADP+)-cytochrom P450 dependent mixed function oxidase enzymes of the liver. The 9000 g supernatant of rat liver homo¬genate has been shown to be very useful in metabolic activation of foreign com¬pounds. The animals were pretreated with Aroclor 1254 as an inducer of several drug metabolizing enzymes.

In the Ames Test with Salmonella typhimurium strains the effect of the test, com¬pound upon the number of back mutations to histidine prototrophy using histidine auxotrophic mutants is investigated. The strains TA 100 and TA 1535 were origi¬nally derived by a substitution mutation, the strains TA 1537 and TA 98 by frame shift mutation from histidine prototrophic bacteria. All four Salmonella strains are deficient in the complete structure of their lipopolysaccharide layer and in DNA excision repair system. TA 98 and TA 100 possess a modified postreplica-tion DNA repair system which frequently causes an increase in the rate of mutations.

In addition to the standard plate incorporation test (Ames Test) a modified pro¬tocol using preincubation with hamster S-9 supplemented with flavin mononucleotid was used. This protocol has been proposed by Prival for assessing the mutagenic activity of azodyes.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

See below

Method

Target gene:

None

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 appilcable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat liver (Ames) S9-Mix from hamster liver (Privall-Mitchell)

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Solvent: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: See below.

Details on test system and experimental conditions:

In addition to the standard plate incorporation test (Ames Test) a modified protocol using preincubation with hamster S-9 supplemented with flavin mononucleotid was used. This protocol has been proposed by Prival (5) for assessing the mutagenic activity of azodyes

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 pretreafed Syrian 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 approximatly 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 (/ - 8 weeks old) are removed and pooled, washed in 150 mM KC1 (approx. 1 ml/g wet. livers). The washed livers are cut into small pieces and homogenized in three volumes of KCI. The homogenate is centrifuged at approx. 9000 g for 10 minutes. The supernatant is the S-9 fraction, it is divided into small portions, rapidly frozen and stored at approx, -80 ⁰C for not longer than six months.

Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room temperature. 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

Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at approx. 37 °C. The suitable amount of bacterid in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at approx, -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 100, TA 1535. TA 1537 and TA 98. Identification of the different bacterial strains is performed periodically and all criteria for a valid assay were achieved .

Toxicity experiments and dose, range finding
Preliminary toxicity tests were performed with 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 evaluated 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 106 dilution of the overnight culture of TA 100 and plated with histidine and biotm 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 for 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 and TA 98

Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0 6 % agar, 0.6 % NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top agar at approximatly 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 % rat liver S-9 Mix or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for approximate 48 hours at approx. 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 approx. 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 approx. 48 hours at approx. 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: Sodium-azide; TA 100, TA 1535 9-Aminoacridine. TA 1537 2-Nitrofluorene-. TA 98
b) with rat liver S-9 Mix (10 %): 2-Aminoanthracene. TA 98, TA 100. TA 1535. 1A 1537
c) with hamster liver S-9 Mix (30 %): 2-Aminoanthracene: TA 100, TA 1535. TA 1537 Benzidine: TA 98 Congo red: TA 98

Evaluation criteria:

A test article is classified mutagenic if either of the following conditions is achieved:
a test article 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 bacteria I background lawn
a test article induces a dose-related increase in the mean number of rever tants 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 article at complete bacterial background lawn.
The test results must be reproducible.

Statistics:

As above under evaluation criteria.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS
Reaktiv-Gelb F-66 923 FW was tested for mutagenicity with Salmonella typhirnunurn strains TA 98, TA 100, TA 1535 and TA 1537 in the absence and presence of a metabolic activation systems. S-9 Mix from Sprague Dawley rats (10 %) and from Syrian 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 10 000 microgram/plate and proved to be not toxic to the bacterial strains.
For mutagenicity testing 5 000 microgram/plate was chosen as the highest dose in the main experiments.

Mutagenicity test
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. No dose dependent effect was obtained.
Prival-Test:
In the presence of 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'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation in both the Ames and Prival-Mitchell modification tests.

It is concluded that Reaktiv-Gelb F-66 923 FW is not mutagenic in the absence and presence of rat S-9 Mix (10 X) using the standard Ames Test procedure. Also in the presence of hamster liver S-9 Mix (30 %) and preincubation the test compound did not induce a significant increase in the number of revertant colonies.

Executive summary:

Study conducted to EU test guidance 78/831/EEC and OECD test guideline 471 in compliance with GLP.

 

Reaktiv-Gelb F-66 923 FW was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 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. 5000 microgram/plate was chosen as top dose level for the mutagenicity study.

 

a) 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-Gelb F-66 923 FW did not result in relevant increases in the number of revertant colonies.

 

b) Prival Test:

In the presence of hamster liver S-9 Mix (30%) using the preincubation method according to Prival Reaktiv-Gelb F-66 923 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-Gelb F-66 923 FW is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Privall.