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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:
18 February 2015 - 01 April 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
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
Specific details on test material used for the study:
Appearance: Blue powder
Purity: ≥85.5%

Method

Target gene:
S.typhimurium TA100- plasmid pKM101
S.typhimurium TA98- plasmid pKM101
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Methods for maintenance in cell culture if applicable: 50 mL of Nutrient Broth


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal glucose agar
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
Examined concentrations in the Initial Mutation Test were 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 μg/plate. Examined concentrations in the Confirmatory Mutation Test were 1250, 625, 312.5, 156.25, 78.13, 39.06, 19.53, 9.77, 4.88 and 2.44 μg/plate.
Concentrations were selected on the basis of the Preliminary Compatibility Test and preliminary Concentration Range Finding Test (Informatory Toxicity Test)*. In the Initial Mutation Test and Confirmatory Mutation Test, different concentrations were used
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Controls
Untreated negative controls:
yes
Remarks:
vehicle
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: 4-nitro-1,2-phenylene-diamine (NPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Minimal glucose agar plates
- Cell density at seeding (if applicable):
DURATION
- Preincubation period: 10-14 hours at 37oC in a Gyrotory Water Bath Shaker
NUMBER OF REPLICATIONS: 3
Rationale for test conditions:
not specified
Evaluation criteria:
The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls
Statistics:
not specified

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range at the non-cytotoxic concentrations (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
Precipitate was detected on the plates in the both tester strains at 5000 and 2500 μg/plate concentrations with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases the appearance of pinpoint colonies were also detected) was observed in both tester strains at 2500 μg/plate concentrations with and without metabolic activation; in Salmonella typhimurium TA98 strain at 1000 μg/plate concentration without metabolic activation and in the Salmonella typhimurium TA100 strain at 1000 μg/plate concentration with and without metabolic activation. Due to the strong precipitate in both strains at 5000 μg/plate concentration, the possible inhibitory effect at this concentration (indicated by the reduced number of revertants) could be clearly detected in the background lawn only in one plate.

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item B331 had no mutagenic activity in the examined bacterial strains under the test conditions of this study.