m-(4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl)benzenesulphonic acid

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

m-(4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl)benzenesulphonic acid

EC Number:

204-303-0

EC Name:

m-(4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl)benzenesulphonic acid

Cas Number:

119-17-5

Molecular formula:

C10H10N2O4S

IUPAC Name:

3-(3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl)benzene-1-sulfonic acid

Details on test material:

- Name of test material (as cited in study report): Pyrazolsäure 3 TF

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced Rat liver S9-mix

Test concentrations with justification for top dose:

Plate incorporation test:
without metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate
with metabolic activation: 50, 160, 500, 1600 and 5000 µg/plate

Preincubation test:
without metabolic activation:16, 50, 160, 500, 1600 and 5000 µg/plate and 5, 16, 50, 160, 500, 1600 and 5000 µg/plate (TA 1535)
with metabolic activation: 16, 50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

Suspended in deionized water at appropriate concentrations immediately before use.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 1 µg/plate, dissolved in water, for TA 100 and TA 1535

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9

Migrated to IUCLID6: 50 µg/plate, dissolved in DMSO, for TA 1537

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: 2.5 µg/plate, dissolved in DMSO, for TA 98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 2 µg/plate (plate incorp.), 0.5µg/plate (preincub.), dissolved in DMSO, for WP2uvrA

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 0.5 µg/plate (TA 98, TA 100); 1.0 µg/plate (TA 1535, TA 1537); 10.0 µg/plate (WP2uvrA), dissolved in DMSO

Remarks:

with S9

Details on test system and experimental conditions:

Two independent mutation tests were performed unless clearly positive or dose-related activity was observed in the first test. Where results were negative or equivocal, a second test was conducted. This included a pre-incubation step if the first test was clearly negative.
Pre-incubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used.
The highest concentration in the first mutation experiment was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used. Suitable dose levels used in the second experiment may be different depending on any toxicity seen in the first experiment. A reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:

0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound suspension (suspended in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 minutes at approx. 30 °C.
After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v)-glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ and trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for the counter's sensitiveness.

Evaluation criteria:

Criteria for a valid assay:

The assay is considered valid if the following criteria are met:

- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency

- the positive controls induce increases in the mutation frequency which are significant and within the laboratory's normal range


Criteria for a positive response:
A test compound is classified as mutagenic if it has either of the following effects:

a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn

b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility and toxicity

The test compound was suspended in deionized water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in the plate incorporation test.

The test compound did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate.

In the plate incorporation test dose-related toxicity was observed at concentrations of 1600 µg/plate and above in the presence of metabolic activation with the tester strains TA 100, TA 1537 and TA 98 and at 5000 µg/plate with the strain TA 1535.

In the absence of metabolic activation, toxicity was observed at concentrations of 1600 µg/plate and greater (TA 100, TA 1537 and WP 2uvrA), at 500 µg/plate and greater (TA 1535) and at 5000 µg/plate (TA 98).

Because of toxicity in the plate incorporation test dose ranges from 16 to 5000 µg/plate were chosen for the preincubation test and the concentration of 5 µg/plate was included in the treatment series at the tester strain TA 1535.

In the preincubation test toxicity was observed with and without metabolic activation at concentrations of 1600 µg/plate and above in the Salmonella strains.

 

Mutagenicity

In both independent mutation tests Pyrazolsäure 3 TF was tested for mutagenicity with the same concentrations as described. The number of colonies per plate with each strain as well as mean values of 3 plates were given.

The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of S9-mix in either mutation test. No dose-dependent effect was obtained.

All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The results lead to the conclusion that Pyrazolsäure 3 TF is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.

Executive summary:

Pyrazolsäure 3 TF was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA.

Two independent mutagenicity studies were conducted (one plate incorporation test and one preincubation test), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For all studies, the compound was suspended in deionized water, and each bacterial strain was exposed to 5 dose levels, in the preincubation test to 6 (7 in the tester strain TA 1535) dose levels.

The test compound did not precipitate on the plates up to the highest investigated dose of 5000 µg/plate.

The concentrations for the plate incorporation test were 50, 160, 500, 1600 and 5000 µg/plate.

Because of toxicity in the plate incorporation test dose levels from 16 to 5000 µg/plate were chosen for the preincubation test and the concentration of 5 µg/plate was included in the treatment series with the tester strain TA 1535 without S9-mix.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

Toxicity: In the plate incorporation test dose-related toxicity was observed at concentrations of 1600 µg/plate and above in the presence of metabolic activation with the tester strains TA 100, TA 1537 and TA 98 and at 5000 µg/plate with the strain TA 1535. In the absence of metabolic activation, toxicity was observed at concentrations of 1600 µg/plate and greater (TA 100, TA 1537 and WP 2uvrA), at 500 µg/plate and greater (TA 1535) and at 5000 µg/plate (TA 98).

In the preincubation test toxicity was observed with and without metabolic activation at concentrations of 1600 µg/plate and above in the Salmonella strains.

Mutagenicity: In the absence and in the presence of the metabolic activation system Pyrazolsäure 3 TF did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that Pyrazolsäure 3 TF was not mutagenic in this bacterial mutation test at any dose level either in the absence or presence of an exogenous metabolic activation.