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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Commission Directive 2000/32/EC, Annex 4D-B13/14 No. L136
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: not reported as such

Method

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:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells.
Controls
Untreated negative controls:
yes
Remarks:
Sterile distilled water
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA98, TA1535, TA1537, TA100, WP2 uvrA +S9), 2-nitrofluorene (TA98 -S9), sodium azide (TA100 and TA1535 -S9), 9-aminoacridine (TA1537 –S9), methyl methane sulphonate (WP2 urvA -S9)
Remarks:
All positive controls were diluted with dimethyl sulfoxide (DMSO) except sodium azide, which was diluted with water.
Details on test system and experimental conditions:
METHOD OF APPLICATION: The plate incorporation method was applied. Treatment without activation was conducted by adding 0.5 mL of overnight culture containing ≥0.3 x10E9 bacteria to top agar supplemented with L-histidine, D-biotin and L-tryptophan. The components were mixed and poured onto a plate containing Vogel-Bonner minimal agar. Treatments with activation were conducted as those without activation except that S9 mix was added to the bacteria/top agar mixture before it was poured onto a Vogel-Bonner minimal agar plate. The plates were incubated at approximately 37±2°C for approximately 48 to 72 hours.

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 2 trials with 3 treatments per concentration
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value. An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. A response is evaluated as negative, if it is neither positive nor equivocal.
Statistics:
Trials were evaluated independently. For each selected tester strain, the average number of revertants and the standard deviation at each concentration with and without S9 activation were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Key result
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

Applicant's summary and conclusion

Conclusions:
Negative when tested in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2 uvr A in the absence and presence of S9 activation.
Executive summary:

The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2 uvr A with and without an exogenous metabolic activation system (S9). The maximum concentration tested was 5000 µg/plate. No evidence of mutagenic activity was detected in either of two independent trials. In this study, the test substance was negative.