Sodium 2-[[4-[3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl]phenyl]sulphonyl]ethanesulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Remarks:

source of read across

Adequacy of study:

key study

Study period:

From November 12, 1985 to February 06, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 02

IUPAC Name:

Similar Substance 02

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98

Details on mammalian cell type (if applicable):

Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2/liter) at 37 °C. The suitable amount of bacteria in the cell suspension is thecked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The comound is tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coli WP2uvrA. Identification of the different bacterial strains is performed periodically.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2/liter) at 37 °C. The suitable amount of bacteria in the cell suspension is thecked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The comound is tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538 and E. coli WP2uvrA. Identification of the different bacterial strains is performed periodically.

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate

Test concentrations with justification for top dose:

EXPERIMENT 1: 0, 0.8, 4, 20, 100 and 500 µg/plate
EXPERIMENT 2: 0, 4, 20, 60, 100, 200 and 500 µg/plate
EXPERIMENT 3: 0, 0.8, 4, 20, 100 and 500 µg/plate

Vehicle / solvent:

- Solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: M-methyl-N'-nitro-N-nitrosoguanidine // 2-Aminoanthracene

Details on test system and experimental conditions:

Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.5 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine is replaced by trypbophan (2.5 ml, 0.5 mM). The following ingredients are added (in order) to 2 ml of motten top agar at 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 S9 Mix (if required) 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 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

CITOTOXICITY - DOSE RANGE FINDING
The first test was performed with all 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 controlled microscopically.
In combination with the first and third experiment, 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 10^-6 dilution of the overnight culture of TA 100 and plate with histidine and biotin rich top agar (3 plates per dose). In addition, toxicity testing was also performed in TA 98 and TA 1538 in the second experiment. The solvent control is compared with the number of colonies per ptate in the presence of the test compound.

PREPARATION OF LIVER HOMOGENATE FRACTION (S9)
Male Sprague Dawley rats (200 - 300 g) receive a single intraperitoneal injection of Aroclor t254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using-cold steiile solution and glassware. The livers from at least 5 - 6 animals are removed and pooled, washed in 150 mM KCI (ca 1 ml/g wet livers).
Sufficient S9 fraction is thawed immediately before each test at room temperature. One volume of S9 fraction is mixed with 9 volumes of the S9 cofactor solution and kept on ice until used. The concentrations of the different compounds in the S9 Mix are: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4mM NADP+, 100 mM phosphate buffer pH 7.4.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 1537, TA 1538, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains neither in the absence nor presence of S9 Mix in two independent experiments. A very small increase in the number of revertant colonies just below the level of toxicity was obtained with TA 1538 and TA 98 in the presence of exogenous metabolic activation. Therefore the test was repeated with TA 1538 and TA 98 in the presence of S9 Mix.
The small increases in the number of revertant colonies never exeeded 2 times the control values and were of no toxicological significance.

TOXICITY TEST
The test compound was tested at doses of 4 to 10000 µg/plate and proved to be toxic to the bacteria at doses of 20 or 100 µg/plate. Thinning of the bacterial lawn and a reduction in the number of colonies has been observed at these doses. Therefore the test could be peformed only with reduced sensitivity. Visible precipitation of the test compound on the plates has been observed at 20 µg/plate.
For mutagenicity testing 500 µg/plate was chosen as the highest dose.

STERILE CHECKS AND CONTROL PLATES
Sterility of S9 Mix and the test compound was indicated by the absence of contamination on the test material and S9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Applicant's summary and conclusion

Conclusions:

The test item resulted to be non-mutagenic in the AMES test performed.

Executive summary:

The substance was tested for mutagenicity with the strains TA 100, TA 1535, 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The rnutagenicity studies were conducted in the absence and in presence of a metabolizing system derived from rat liver homogenate. A dose range of 5 different doses from 0.8 µg/plate to 500 µg/plate was used. Control plates without mutagen showed that the number of colonies was similar to that described in the literature.

All the positive control compounds gave the expected increase in the number of revertant colonies.

The test compound proved to be toxic to most of the bacterial strains at 20 µg/ptate. On the basis of the preliminary test results the top dose level did not exceed 500 µg/plate.

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 a metabolic activation system, treatment of the cells with test item did not result in relevant increases in the number of revertant colonies. Very slight increased nunbers of revertants with the strain TA 1538 and TA 98 were of no toxicological significance.

Summarizing, it can be stated that the test compound is not mutagenic in the bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.

Conclusion

The test item resulted to be non-mutagenic in the AMES test performed.