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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
testing lab.
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Isopulegol
EC Number:
201-940-6
EC Name:
Isopulegol
Cas Number:
89-79-2
Molecular formula:
C10H18O
IUPAC Name:
isopulegol
Details on test material:
- Test substance : Cyclohexanol, 5-methyl-2-(1-methylethenyl)-, (1R,2S,5R)-
- Name of test material (as cited in study report): L-Isopulegol
- Physical state: liquid, colorless clear
- Analytical purity: 99.4 corr.area-%
- Lot/batch No.: Muster 7 CH
- Expiration date of the lot/batch: 01 Jun 2012
- Storage condition of test material: room temperature, under N2

Method

Target gene:
histidine/tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
liver S-9 mix from phenobarbital / β-naphthoflavone induced rats
Test concentrations with justification for top dose:
33 - 5000 µg/plate (standard plate test)
10 - 2500 µg/plate (preincubation test - Salmonella strains)
33 - 5000µg/plate (preincubation test - E. coli)
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine, 9-aminoacridine and 4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation; preincubation

DETERMINATION OF CYTOTOXICITY
- Method:
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
Evaluation criteria:
Acceptance 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.
• The titer of viable bacteria was ≥ 10^8/mL.

Assessment criteria
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 with or without S9 mix.
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 resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
from about 333 µg/plate onward.
Cytotoxicity / choice of top concentrations:
other:
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: from about 333 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TOXICITY
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 1000 μg/plate onward.
In the preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 333 μg/plate onward.

SOLUBILITY
No test substance precipitation was observed 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

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