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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct 2014 - 23 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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

Constituent 1
Chemical structure
Reference substance name:
Etocrilene
EC Number:
226-029-0
EC Name:
Etocrilene
Cas Number:
5232-99-5
Molecular formula:
C18H15NO2
IUPAC Name:
etocrilene
Test material form:
solid
Details on test material:
- Physical state: solid/white

Method

Species / strain
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat S9 mix
Test concentrations with justification for top dose:
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controls
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 2-aminoanthracene (60 µg/plate); without S9: 4-nitroquinoline-N-oxide (5 µg/plate)
Remarks:
with and without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes (PIT)
- Exposure duration: 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS:
Three test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling of the spontaneous mutation rate in the tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for the tester strain were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2 500 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for the tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced trp- background growth, decrease in the number of trp+ revertants) was observed in the standard plate test and in the preincubation assay under all test conditions.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Result

Revertants/plate (mean from three plates)
SPT PIT
Dose (µg/plate) with S9 without S9 with S9 without S9
DMSO 72.0 56.3 62.3 71.3
33 62.3 55.0 60.0 57.0
100 54.3 52.0 62.3 68.3
333 55.3 57.0 63.0 58.7
1000 64.7 54.3 69.0 59.0
2500 59.3 58.7 62.0 67.7
5000 51.0 41.7 63.0 64.7
Positive Control 257.0 860.7 172.7 660.7

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Escherichia coli strain WP2 uvrA in a reverse mutation assay at a dose range of 33 μg - 5000 μg/plate (SPT) and 33 μg - 5000 μg/plate (PIT)

Both Standard plate test (SPT) and preincubation test (PIT) were performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 2500 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. A relevant increase in the number of trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.