Sodium bis[2-chloro-4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-hydroxybenzenesulphonamidato(2-)]chromate(1-)

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:

17 February 1994 to 21 March 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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only four tester strains used; no tester strain to detect cross-linking mutagens 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:

Sodium bis[2-chloro-4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-hydroxybenzenesulphonamidato(2-)]chromate(1-)

EC Number:

266-577-8

EC Name:

Sodium bis[2-chloro-4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-hydroxybenzenesulphonamidato(2-)]chromate(1-)

Cas Number:

67109-27-7

Molecular formula:

C32H24Cl2CrN10O8S2.Na

IUPAC Name:

sodium bis[2-chloro-4-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-hydroxybenzenesulphonamidato(2-)]chromate(1-)

Test material form:

not specified

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 11
- Expiration date of the lot/batch: December 31, 1998
- Appearance: Brown solid
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:

The S. typhimurium histidine (his) reversion system measures his- = > his* reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

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:

Experiment 1Without metabolic activation): 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate.
Experiment 2 (With metabolic activation): 123.46, 370.37, 1111.11, 3333.33 and 10000 µg/plate.

Vehicle / solvent:

Dimethylsulfoxide (DMSO).

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:

TA 100, TA 98 and TA 1537: With metabolic activation in DMSO solvent

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 1535: With metabolic activation in Cyclophosphamide solvent

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535: Without metabolic activation in bidistilled water

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98: Without metabolic activation in DMSO solvent

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537: Without metabolic activation in DMSO solvent

Details on test system and experimental conditions:

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.

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.

Evaluation 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.

- 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 revenants per plate above that of the negative control at any concentration for one or more of the strains tested.

Statistics:

A statistical analysis of the test data was not performed.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 100, TA 98 and TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

slight toxic effect on the growth of the bacteria.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Range finding test:
Six concentrations of FAT 20028/D (Irgalan Rot 2GL roh feucht) ranging from 20.6 to 5000.0 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 reduced at the highest concentration both in the absence and in the presence of S9. The highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and 5000.0 µg/plate without metabolic activation. Precipitates of the test substance were visible on the plates at 1666.67 and 5000 µg/plate.

- 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 20028/D 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 confirmatory experiment concentrations up to 10000 µg/plate were tested. This concentration corresponds to about 5000 ug/plate of pure compound. 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 20028/D 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 reduced with some of the strains at the highest concentration of 5000 or 10000 µg/plate. Therefore, the test substance exerted a slight toxic effect on the growth of the bacteria. Precipitates of the test substance were formed on the plates at concentrations down to 1666.67 µg/plate (original experiment) and 1111.11 µg/plate (confirmatory experiment).

Applicant's summary and conclusion

Conclusions:

FAT 20028/D and its metabolites did not induce gene mutations in Salmonella typhimurium strain.

Executive summary:

The bacterial mutation assay was performed with FAT 20028/D to detect gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium. This experiment has been performed in compliance with GLP, and was done according the OECD Guideline 471 (Bacterial Reverse Mutation Assay). FAT 20028/D, identified as a brown solid, purity ca. 50 %, batch no. 11, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium. 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 DMSO and tested at five concentrations in the range of 61.73 to 5000.0 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 123.46 to 10000.0 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 (2-Aminoanthracene and cyclophosphamide). In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 20028/D 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 on standard evaluation criteria, it is concluded that FAT 20028/D and its metabolites did not induce gene mutations in the strains of Salmonella typhimurium used.