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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

268-387-0

EC Name:

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

Cas Number:

68083-38-5

Molecular formula:

C10H11N3O3S

IUPAC Name:

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

Test material form:

solid: particulate/powder

Method

Target gene:

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:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate (Pre-Experiment/Experiment I)
33; 100; 333; 1000; 2500; and 5000 µg/plate (Experiment II)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays):
Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

- OTHER
- Study controls: The solvent (vehicle) control used was dimethyl sulphoxide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.
- Concentrations of positive controls:
Sodium azide (NaN3) - 10µg/plate for TA 1535 and TA 100
4-Nitro-o-phenylene-diamine (4-NOPD) - 10 μg/plate for TA98, 50 μg/plate for TA1537
Methyl methane sulfonate (MMS) - 2 μg/plate for TA102
2-Aminoanthracene (2AA) - 10 μg/plate for TA102, 2.5 μg/plate for TA 98, TA 100, TA1535 and TA1537

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Precipitation: In experiment I the test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with metabolic activation (S9 mix). Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate without S9 mix and from 2500 to 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.
In experiment II no precipitation of the test item was observed in the overlay agar neither in the test tubes nor on the incubated agar plates.

Applicant's summary and conclusion

Conclusions:

The study was conducted under GLP according to OECD guideline 471 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations, the validity criteria are fulfilled, positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce reverse mutations in bacteria. During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471, GLP), Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA 102 were treated with the test item according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II).

The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

In experiment I the test item precipitated in the overlay agar in the test tubes from 1000 to 5000 µg/plate with metabolic activation (S9 mix). Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate without S9 mix and from 2500 to 5000 µg/plate with S9 mix. The undissolved particles had no influence on the data recording.

In experiment II no precipitation of the test item was observed in the overlay agar neither in the test tubes nor on the incubated agar plates.

The plates incubated with the test item showed reduced background growth in strains TA 1535, TA 98 and TA 100 in experiment I and in strain TA 1537 in experiment II.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Iminopyrazolsäure at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, Iminopyrazolsäure  is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.