2-ethyl-4-methylimidazole

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethyl-4-methylimidazole

EC Number:

213-234-5

EC Name:

2-ethyl-4-methylimidazole

Cas Number:

931-36-2

Molecular formula:

C6H10N2

IUPAC Name:

2-ethyl-4-methyl-1H-imidazole

Details on test material:

- Name of test material (as cited in study report): 2-ethyl-4-methylimidazole
- Purity/composition: 2-ethyl-4-methylimidazole: 89.964 %; 4-methylimidazol: 6.559 %; water: 0.076 % (as indicated by the manufacturer)
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Storage stability: guaranteed until 21 jul 2013 by the sponsor. Stored at room temperature, protected against humidity
- Physical state, appearance: Liquid, viscous, yellow, clear

Method

Target gene:

- Salmonella typhimurium: histidine
- Escherichia coli: tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 µg/plate

Vehicle / solvent:

Water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 for all strains

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

without S-9 for TA100 and TA1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

without S-9 for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S-9 for TA1537

Controls 5

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 S-9 for E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

NUMBER OF REPLICATIONS: three plates per dose/control

DURATION
- Exposure duration: 48 -72 hours at 37 °C in the dark

DETERMINATION OF CYTOTOXICITY
- Method: counting of bacterial colonies ( his+ or trp+ revertants)

Evaluation criteria:

Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used. For this approval the titer of viable bacteria has to be ≥ 10^8 colonies per mL.

The test substance is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity: A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was not observed in the standard plate test up to the highest requiered concentration. In the preincubation assay (slight) bacteriotoxicity was observed depending on the strain and test conditions at 5000 µg/plate
Solubility: No test substance precipitation was found with and without S9 mix

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

negative Ames test