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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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
genetic toxicity in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L83-44-2
- Purity: 97,6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator

- Solubility and stability of the test substance in the solvent/vehicle:
The stability of the test substance at room temperature in the vehicle DMSO over a period of
4 hours was verified analytically.
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:
liver S9 mix from induced rats
Test concentrations with justification for top dose:
1st Experiment: 0; 33; 100; 333; 1 000; 2 600 and 5 200 μg/plate
due to the purity of the test substance 5.2 mg/plate was used as top dose in the first experiment (standard plate test)

2nd Experiment (preincubation test): 0; 10; 33; 100; 333; 1 000 and 2 600 μg/plate
A 2nd experiment was performed because no mutagenicity was observed in the standard plate test.
Vehicle / solvent:
DMSO; The substance was not soluble in ultrapure water.
Untreated negative controls:
yes
Remarks:
sterility control; without the addition of tester strains
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA); with S9 mix; for all tester strains
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (NOPD) (w/o S9 mix; for strain TA 98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
1st Experiment: in agar (plate incorporation); 2nd Experiment: preincubation;

DURATION
- Preincubation period: about 20 minutes (Experiment 2)
- Exposure duration: 48 – 72 hours

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

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
was recorded for all test groups both with and without S9 mix in all experiments


- Any supplementary information relevant to cytotoxicity:

Precipitation of the test material was recorded. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and
analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.
Evaluation 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 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.
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 2 600 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Preincubation test
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Preincubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Preincubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Preincubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Preincubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed from 1 000 μg/plate onward with and without S9 mix. Test substance precipitation was found from 2 600 μg/plate onward with and without S9 mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation of the test substance was found from 2 600 μg/plate onward with and without S9 mix.
A bacteriotoxic effect was observed depending on the strain and test conditions from about 1 000 μg/plate onward.

Data source

Materials and methods

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Additional information on results:
negative in all strains with and without metabolic activation

Applicant's summary and conclusion