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Toxicological information

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:
10 Oct 2011 to 18 Nov 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
22.10.2009
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Golpanol BEO
- Physical state: Liquid, yellow, clear
- Analytical purity: 99.4%
- Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
- Lot/batch No.: 92713124U0
- Storage condition of test material: Room temperature

Method

Target gene:
his operon (S. typhimurium strains) and trp operon (E. coli strains)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days
Test concentrations with justification for top dose:
33; 100; 333; 1000; 2500 and 5000 μg/plate (Standard Plate Test and Preincubation Test)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
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: Standard plate test and preincubation test

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer

OTHER:
Concentration of positive controls
With S9 mix
• 2-aminoanthracene (2-AA)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA

Due to the OECD 471, 2-Aminoanthracene should not be used as the sole indicator of the efficacy of the S9-mix. If 2-aminoanthracene is used, each batch of S9 should also be characterised with a mutagen that requires metabolic activation by microsomal enzymes.
Therefore, in the study the efficacy of the S9 mix aws demonstrated, and the S9 batch was characterized with benzo(a)pyrene.
Evaluation criteria:
The test substance is considered positive 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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test and in the preincubation test either without S9 mix or after the addition of a metabolizing system.
- 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:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.