Registration Dossier

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: Well documented study according to international accepted guidelines and GLP compliant.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Test item: Levodione
CAS number: 21800-83-9
Batch number: 242027N
Physical state: Solid, crystalline powder
Colour: White
Storage: At a temperature of 15 to 30 °C

Method

Target gene:
S. typhimurium TA98: hisD3052; TA100: hisG46; TA1535: hisG46; TA1537: hisC3076
E. coli WP2 uvr A: trpE
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:
S9 mix
Test concentrations with justification for top dose:
Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Ultrapure water (UPW)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, UPW
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Remarks:
Dose: 4 µg/plate; Vehicle: DMSO ; Strain: Salmonella TA98
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
Dose: 2 µg/plate; Vehicle: UPW ; Strain: Salmonella TA100 and TA1535
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
Dose: 50 µg/plate; Vehicle: DMSO ; Strain: Salomella TA1537
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
Dose: 2 µL/plate; Vehicle: UPW ; Strain: E. coli WP2 uvrA
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
Dose: 2 µg/plate; Vehicle: DMSO ; Strain: all of Salmonella strains
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
Dose: 50 µg/plate; Vehicle: DMSO ; Strain: E. coli strain
Details on test system and experimental conditions:
The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test) an Initial Mutation Test (Plate Incorporation Test) and a Confirmatory Mutation Test (Pre-Incubation Test).
In the preliminary Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used.
Evaluation criteria:
A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control;
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the lowest cytotoxic concentration: 500 μg/plate (-S9 mix), 1581 μg/plate (+S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the lowest cytotoxic concentration: 500 μg/plate (-S9 mix), 1581 μg/plate (+S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the lowest cytotoxic concentration: 500 μg/plate (-S9 mix), 1581 μg/plate (+S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the lowest cytotoxic concentration: 500 μg/plate (-S9 mix), 1581 μg/plate (+S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
the lowest cytotoxic concentration: 1581 μg/plate (+/-S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The highest revertant colony number increase over the spontaneous rate of the vehicle control plates was observed in the Confirmatory Mutation Test (Pre-Incubation Test) in Salmonella typhimurium TA1537 at 50 μg/plate, without metabolic activation (-S9 Mix). The mutation rate was: 1.87. This value remained far below the genotoxicological threshold for being positive.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In conclusion, the test item levodione (CAS 21800-83-9) has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.