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Genetic toxicity: in vitro

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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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate

Results and discussion

Test resultsopen allclose all
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
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
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
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
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
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.