3-methylpyrazole

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:

2004-04-16 - 2004-08-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

3-methylpyrazole

EC Number:

215-925-7

EC Name:

3-methylpyrazole

Cas Number:

1453-58-3

Molecular formula:

C4H6N2

IUPAC Name:

3-methyl-1H-pyrazole

Test material form:

other: liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

All used bacterial strains are defective in DNA-repair (AuvrB and AuvrA respectively). The Salmonella strains also have a defective lipopolysaccharide barrier on the cell wall (rfa). These properties confer extra sensitivity to DNA damage and also greater permeability to large molecules. The strains TA 98 and TA 100 also contain a plasmid (pKMlOl) which enhances the error prone repair and confers ampicillin resistance.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

All used bacterial strains are defective in DNA-repair (AuvrB and AuvrA respectively). The Salmonella strains also have a defective lipopolysaccharide barrier on the cell wall (rfa). These properties confer extra sensitivity to DNA damage and also greater permeability to large molecules. The strains TA 98 and TA 100 also contain a plasmid (pKMlOl) which enhances the error prone repair and confers ampicillin resistance.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Plate incorporation test without metabolic activation: 10, 50, 100, 500, 1000, 5000 µg/plate
Plate incorporation test with metabolic activation: 50, 100, 500, 1000, 5000 µg/plate
Pre-incubation method without metabolic activation:
TA 1535, TA 98, TA 100, E.coli: 50, 100, 500, 1000, 5000 µg/plate
TA 1537: 10, 50, 100, 500, 1000, 5000 µg/plate
Pre-incubation method with metabolic activation:
TA 1535, TA 98, E. coli: 50, 100, 500, 1000, 5000 µg/plate
TA 1537, TA 100: 10, 50, 100, 500, 1000, 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

3-Methyl-pyrazole is considered to be non-mutagenic in the bacterial reverse mutation test.

Executive summary:

3-MethyJ-pyrazole was tested for a possible potential to induce gene mutation in bacteria. As test organisms the Salmonella lyphimurium strains TA 98, TA 100, TA 1535, TA 1537 and the Escherichia coli strain WP2uvrA were used. The bacteria were exposed to 3-Methyl-pyrazole both, in the presence and absence of rat liver S9 metabolic activation. Two independent experiments were performed according to the standard plate incorporation method (experiment I) or the pre-incubation method (experiment II), respectively.

Only a slight cytotoxicity was observed in the S. typhimurium strain TA 1537 at 5000 µ

/plate in the experiment without metabolic activation in the pre-incubation test. An untreated, a vehicle control and appropriate positive controls were included into the experimental design. Triplicate plates were scored for each experimental point. The revcrtant frequencies of the negative controls were within the expected range and the positive control chemicals induced marked increases in revertant colonies. No biologically relevant increases in revertant numbers were obtained after treatment with 3-Methyl-pyrazole in any bacterial strain and at any concentration tested. This applies to the presence and absence of metabolic activation and as well as the plate incorporation test and the pre-incubation test. Based on the results of the reported study it is concluded that 3-Methyl-pyrazole does not induce gene mutation in Salmonella typhimurium and Escherichia coli under the experimental conditions described. Therefore, 3-Methyl-pyrazole is considered to be non-mutagenic in the bacterial reverse mutation test.