Sodium bis[3-[[1-(3-chlorophenyl)-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxybenzenesulphonamidato(2-)]cobaltate(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:

From the 5th of January, to the 9th of March, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Test conducted according to internationally accepted testing guidelines

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

277-554-7

EC Name:

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

Cas Number:

73612-40-5

Molecular formula:

C32H24Cl2CoN10NaO8S2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Method

Target gene:

Genetic changes which cause them to revert to a non-histidine-requiring state and thus to grow in the absence of exogenous histidine.

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Range finding test:
20.6 to 5000.0 ug/plate
Mutagenicity tests:
Original experiment
625.0 to 10000.0 ug/plate
Confirmatory experiment
37.0 to 3000.0 ug/plate
According to the purity óf the test chemical the corrected highest concentration is about 2500 ug/plate
According to the purity of the test chemical the corrected highest concentration is about 5000 ug/plate
According to the purity of the test chemical the corrected highest concentration is about 1500 ug/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-Aminoanthracene

Remarks:

with metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Details on test system and experimental conditions:

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 crystal-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). 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 centrifiiged 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 content of the S9 fraction was 35.81 mg/ml.

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 according to Standard Operating Procedures of Genetic Toxicology. The highest concentration applied was 5000 ug/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.

MAIN 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 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 two in the original experiment and by a factor of three in the confirmarory experiment. 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.

EVALUATION
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.

Evaluation criteria:

A test is considered acceptable if the mean colony counts of the negative control values of all strain 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.
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 moreof the strains tested. Generally a concentration-related effect should be demonstrable.

Statistics:

Means for all mutagenicity assays were calculated.
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.
Arithmetic Mean and Standard Deviation (SD) of colony counts obtained in 75* separate experiments over the period of January 01, 1993 to December 31, 1993 and acceptable ranges for mean colony counts of spontaneous revenants.

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

Positive controls validity:

valid

Additional information on results:

Range finding test:
Normal background growth was observed up to the concentration of 1666.67 ug/plate in the experiment without metabolic activation. At the highest concentration the background growth was reduced. The number of revertant colonies was slightly diminished at the highest concentration in the experiments with and without metabolic activation. From the results obtained, the highest concentration suitable for the mutagenicity test was selected tobe 10000.0 ug/plate with and without metabolic activation.

Mutagenicity test, original experiment
The substance did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control. After treatment of the cultures of strain TA 102 without metabolic activation, a marginal increase in the number of revertant colonies was observed at the lowest concentration tested (625.0 ug/plate). Due to a growth-inhibiting effect of the test material, the number of colonies diminished again at the upperconcentrations. No effect occurred with strain TA 102 in the experiment with metabolic activation.

Mutagenicity test, confirmatory experiment
No increase in the incidence of histidineprototrophic mutants was observed in comparison with the negative control. With strain TA 102 a
slight increase in the number of back-mutants was observed at the concentration of 333.3 ug/plate.
In these experiments a marginal increase in the number of back-mutants of strain TA 102 was also observed in the part with metabolic activation.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. However, due to a growth-inhibiting effect of the test material on the bacteria, in the experiments without metabolic activation the numbers of revertant colonies were reduced at the concentrations of 2500.0 ug/plate and above with all strains. In the experiments with metabolic activation, this effect was less pronounced. In the original experiment with metabolic activation the number of revertant colonies of strain TA 100 was slightly above the acceptable range. This does, however, not affect the quality of the test results. 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: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:

Marginal mutagenic effect

Executive summary:

The substance was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium, according to OECD471, EU B.13/B.14.

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 dissolved in DMSO and tested at five concentrations in the range of 625.0 to 10000.0 ug/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 37.0 to 3000.0 ug/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 the experiments performed without metabolic activation with strain TA 102, a slight increase in the number of back-mutants was observed at concentrations of 625.0 and 333.3 ug/plate. A similar effect with this strain in the experimental part with metabolic activation was not reproducible. No effect at all occurred with the other strains.

As a conclusion, the substance exerted a marginal mutagenic effect in this test system.