Disodium 1,1'-isopropylidenedi-p-phenylene bis[2-[[5-amino-3-methyl-1-(3-sulphonatophenyl)-1H-pyrazol-5-yl]azo]benzenesulphonate]

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:

11 January 1994 to 26 April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only four tester strains tested, no tester strain to detect cross-linking mutatens included.

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 1,1'-isopropylidenedi-p-phenylene bis[2-[[5-amino-3-methyl-1-(3-sulphonatophenyl)-1H-pyrazol-5-yl]azo]benzenesulphonate]

EC Number:

276-882-8

EC Name:

Disodium 1,1'-isopropylidenedi-p-phenylene bis[2-[[5-amino-3-methyl-1-(3-sulphonatophenyl)-1H-pyrazol-5-yl]azo]benzenesulphonate]

Cas Number:

72828-69-4

Molecular formula:

C47H42N10O12S4.2Na

IUPAC Name:

disodium 1,1'-isopropylidenedi-p-phenylene bis[2-[[5-amino-3-methyl-1-(3-sulphonatophenyl)-1H-pyrazol-5-yl]azo]benzenesulphonate]

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test material: FAT21015/B (Polar Gelb 4G roh trocken)
Batch No.: 1
Purity: Approx 80%
Appearance: Yellow-orange powder
Expiry date: October 1998
Storage: Room temperature

Method

Target gene:

histidine requiring strains of Salmonella typhimurium

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat-liver post mitochondrial supernatant (S9 fraction)

Test concentrations with justification for top dose:

Concentration range in the range finding test: 20.6 to 5000 µg/plate
Concentration ranges in the mutagenicity tests: Original experiment 61.7 to 5000 µg/plate. Confirmatory experiment: 61.7 to 5000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: With S9: 2-Aminoanthracene for strains TA 100, TA 102, TA 98 and TA 1537, Cyclophosphamide for strain TA 1535; without S9: sodium azide for TA 100 and TA 1535, mitomycin-C for TA 102, 2-nitrofluorene for TA 98 and 9-aminoacridine for TA 1537

Details on test system and experimental conditions:

Source of strains
The histidine-auxotrophic strains of Salmonella typhimurium (TA 98, TA 102, TA 1535, TA 1537) were obtained from Prof. B. Ames, Berkeley, USA. Strain TA 100 was obtained from Dr. M. Schiipbach, Hoffmann-La Roche Limited, Basel, Switzerland.

Preparation of the bacterial cultures:
Inoculates from frozen master copies were set up monthly. They were grown in liquid nutrient broth medium (NB-medium) overnight and then plated on NB-agar. After incubation, single colonies were taken from the plates, grown overnight in liquid NB-medium and then used for the experiment.

Control of the genotype of the strains:
The characteristics of the strains were checked monthly. Histidine-auxotrophy of the Salmonella strains was demonstrated by the requirement for L-histidine. The presence of the rfa character was assayed by the sensitivity for cry stal-violet. The deletion of the uvrB gene (TA 98, TA 100, TA 1535 and TA 1537) was demonstrated by the sensitivity for UV-light. The Salmonella strains containing the R-factor (TA 98 and TA 100) were additionally checked for ampicillin resistance. The strain TA 102 was additionally checked for tetracycline resistance (presence of multicopy plasmid pAQl). 'W The presence of the uvr+ gene (TA 102) was demonstrated by the resistance against UV light. Furthermore, all strains were checked for their characteristic reversion properties with known mutagens (positive controls).

Preparation of the metabolic activation mixture:
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male RAI rats (Tif: RAIf [SPF]), reared at the Animal Farm of CIBA-GEIGY Limited, Sisseln, Switzerland. The animals were treated with Aroclor 1254 (500 mg/kg, i.p.) 5 days prior to sacrifice. The livers were homogenized with 3 volumes of 150 mM KCl. The homogenate was centrifuged for 15 minutes at 9000x g and the resulting supernatant (S9 fraction) was stored at approximately -80°C for no longer than one year. The protein contents of the S9 fractions were 33.37 and 35.81 mg/ml.

Solubilisation of the test substance:
FAT 21015/B was suspended in DMSO by slightly warming. The highest concentration was 50 mg/ml. Lower concentrations of the test substance were obtained by appropriate dilution of the stock solution with DMSO. No precipitates or aggregates were noted.

Setting up of the test plates:
0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl and was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water.

Preliminary range finding test:
A range finding test was carried out with strain TA 100 with and without metabolic activation at six concentrations of the test substance and one negative control. The highest concentration applied was 5000 µg/plate. The five lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the colonies and determining the background lawn. One plate per test substance concentration and negative control was used.

Mutagenicity test:
The mutagenicity test was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, TA 1537 with and without metabolic activation. Each of the five concentrations of the test substance, a negative and a positive control were tested, using three plates per test substance concentration and controls. The highest concentration applied was determined in the preliminary range finding test and the four lower concentrations decreased by a factor of three. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. Thereafter, they were evaluated by counting the number of colonies and determining the background lawn.

Negative and positive controls:
The solvent alone was used as the negative control.

Colony counting and scoring of the plates:
Colonies were counted electronically using an Artek Colony Counter (Fisher Scientific), or manually where minor agar damage or test chemical precipitates might have interfered with automating counting. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means for all mutagenicity assays were calculated and included in the Results section.

Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Rationale for test conditions:

None

Evaluation criteria:

The test substance will be considered to be positive in the test system if the following condition is met:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the strains tested.
Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test results

Key result

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range finding test:
Six concentrations of FAT 21015/B (Polar Gelb 4G roh trocken) ranging from 20.6 to 5000 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

Mutagenicity test, original experiment:
In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 21015/B (Polar Gelb 4G roh trocken) did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment:
In the experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 21015/B (Polar Gelb 4G roh trocken) no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Remarks on result:

other: negative with all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

FAT 21015/B was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

A study was performed to determine mutagenic effects of FAT 21015/B in vitro in histidine-requiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 102, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in DMSO and tested at five concentrations in the range of 61.7 to 5000 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 21015/B led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Based on the results of these experiments and standard evaluation criteria, it is concluded that FAT 21015/B (Polar Gelb 4G roh trocken) and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.