Disodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2-)]chromate(2-)

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:

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

Test guideline 1

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 mutagens have been included

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 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2-)]chromate(2-)

EC Number:

258-027-0

EC Name:

Disodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-4-hydroxybenzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2-)]chromate(2-)

Cas Number:

52587-68-5

Molecular formula:

C38H25CrN8O7S.2Na

IUPAC Name:

Disodium [4-(hydroxy-kO)-3-{[5-(hydroxy-kO)-3-methyl-1-phenyl-1H-pyrazol-4-yl]diazenyl-kN1}benzenesulfonato(3-)][1-({2-(hydroxy-kO)-5-[phenyldiazenyl]phenyl}diazenyl-kN1)naphthalen-2-olato(2-)-kO]chromate(2-)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

None

Specific details on test material used for the study:

Identification: FAT 21095/C
Purity: 80 %
Batch no.: 34
Appearance: Black powder
Expiry date: July 1999
Storage: Room temperature.

Method

Target gene:

histidine-prototrophic 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):

Except for strain TA 100 all strains from Prof. B. Ames, Berkeley, USA. Strain TA 100 from Dr. M. Schüpbach, Hoffmann-La Roche Limited, Basle, Switzerland.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction.

Test concentrations with justification for top dose:

- Concentration range in the range finding test: 20.58, 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate

- Concentration ranges in the mutagenicity tests:
Original experiment: 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate
Confirmatory experiment: 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Bidistilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation:TA 100, TA 98, TA 1537

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation:TA 1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation: TA 100, TA 1535.

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation: TA 98.

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation: TA 1537

Details on test system and experimental conditions:

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 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 according to a Standard Operating Procedure of Genetic Toxicology. 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 1535, TA 1537 with and without metabolic activation according to Standard Operating Procedures of Genetic Toxicology. 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.

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 or strong coloration of the agar plates 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.

Evaluation criteria:

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.

Criteria for a positive response
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

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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range finding test
Six concentrations of FAT 21095/C 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. The numbers of revertant colonies were reduced at the upper concentrations. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 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 1535 and TA 1537 with FAT 21095/C 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 1535 and TA 1537 with FAT 21095/C 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. In the experiments without and with activation performed on strain TA 100, the numbers of revertant colonies were slightly reduced at the upper concentrations. The test substance exerted a weak toxic effect on the growth of this strain.

Applicant's summary and conclusion

Conclusions:

Based on the findings of the study, FAT 21095/C and its metabolites did not induce any gene mutation in the strains of Salmonella typhimurium.

Executive summary:

A key study was performed to determine the mutagenicity of FAT 21095/C with purity of about 80 % in Salmonella typhimurium strains according to OECD Guideline 471 (Bacterial Reverse Mutation Assay). The following strains of Salmonella typhimurium were used: TA 98, TA 100, 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 dissolved in bidistilled water 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 five concentrations in the range of 61.7 to 5000 µg/plate. 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 21095/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Based on the findings of the study, FAT 21095/C and its metabolites did not induce any gene mutation in the strains of Salmonella typhimurium.