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Genetic toxicity: in vitro

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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
Study period:
10.3.2006-21.4.2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Potassium permanganate
- Molecular formula (if other than submission substance): KMnO4
- Molecular weight (if other than submission substance): 158.03
- Substance type: technical product
- Physical state: solid; crystalline powder
- Analytical purity: 99.42 wt.
- Impurities (identity and concentrations): Manganese dioxide cca 0.1 % wt.
- Lot/batch No.: 69
- Stability under test conditions: stable
- Storage condition of test material: at current laboratory conditions, in delivered glass containers

Method

Target gene:
gene for synthesis histidine or tryptphan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine requing strain
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan requing strain
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
1.5, 5, 15, 50, 100, 150 μg in 0.1 ml per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility of the test substance
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: minimal glucose agar plate
DURATION
- Exposure duration: 48 - 72 hours

NUMBER OF REPLICATIONS: 2 series; each with and without metabolic activation, with positive control and solvent control
triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: total growth





Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, its using is comparable with using of statistical methods. After this rule the result is positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc is reached.
Rt number of revertants at tested dose
Rc number of revertants at negative control (water)

Results and discussion

Test resultsopen allclose all
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
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
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500 micrograms See table A.
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in our laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test substance was dissolved in demineralized water till the final concentration 5 000 g per 0.1 mL (plate). This dose is recommended as maximum for mutagenicity testing in the Ames test. With regard to expected toxicity, maximum dose used for toxicity testing was 10 times lower – 500 g/0.1 mL. The toxicity test showed last non-toxic dose of 50 g/0.1 mL (see table A). Since the method accepts (recommends) including of one toxic dose, the presumably toxic dose of 150 g/0.1mL was used as maximum for first mutagenicity test. The other lower doses were diluted according to the guidelines (at maintenance of recommended range among doses) .

Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the above-described experimental design, the test substance Potassium permanganate was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.
Executive summary:

Test substance Potassium permanganate was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria,which is analogous to the OECD Test Guideline No. 471.

Four  indicator Salmonella typhimurium strains TA 98, TA 100,  TA 1535  and

TA 1537 and one indicator Escherichia coli WP2 uvrA strainwere used. The test substance was dissolved in demineralized vater and assayed in doses of 1.5-150mg which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Potassium permanganate was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.