Permanganic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18.07.2020 - 13.08.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21. Jul. 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted 30. May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Permanganic acid

EC Number:

236-695-4

EC Name:

Permanganic acid

Cas Number:

13465-41-3

Molecular formula:

HMnO4

IUPAC Name:

manganesoylol

impurity 1

Reference substance name:

Impurtiy permanganic acid

Molecular formula:

not available

IUPAC Name:

Impurtiy permanganic acid

Test material form:

liquid

Details on test material:

The permanganic acid is only stable in aqueous solution (3-5%) and the solution is placed on the market. Due to the production process only traces of K, P and S ions are to be expected as impurities.

Specific details on test material used for the study:

The permanganic acid is only stable in aqueous solution (3-5%).

Method

Species / strain

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:

Type of metabolic activation system:
S9: produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital-5,6 Benzoflavone

S9-mix
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9 1.0 mL

Test concentrations with justification for top dose:

Experiment 1:
Concentrations to be tested: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate.
Experiments 1b and 1c:
Concentrations to be tested: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate, 0.015 µL/plate and 0.005 µL/plate.
Experiment 2:
For the strains TA98 (+S9), TA100 (-S9 and +S9), TA102 (-S9 and +S9), TA1535 (+S9) and TA1537 (+S9): Concentrations to be tested: 5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate, 0.16 µL/plate, 0.08 µL/plate and 0.04 µL/plate.
For the strains TA98 (-S9) and TA1535 (-S9): Concentrations to be tested: 1.5 µL/plate, 0.75 µL/plate, 0.38 µL/plate, 0.19 µL/plate, 0.09 µL/plate, 0.05 µL/plate and 0.02 µL/plate.

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was determined in demineralized (de-min.) water and dimethyl sulfoxide (DMSO). The test item was soluble in a concentration of 50 mL/L in demin. water.
Based on these results, demin. water was used as solvent in the experiments.

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

Different media and solutions were prepared preliminary (exact production dates are doc-umented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incuba-tion chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9-mix was freshly prepared and stored at 0 °C.

7.2.2 Description of the Method
Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histi-dine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Plate incorporation method:
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

• 100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control)
• 500 µL S9-mix or phosphate buffer (for test without metabolic activation).
• 100 µL bacteria suspension
• 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

Pre-incubation method:
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:

• 100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control)
• 500 µL S9-mix or phos-phate buffer (for test without metabolic activation).
• 100 µL bacteria suspension

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

Five different analysable and non-toxic concentrations should be used for the evaluation of the mutagenic potential of the test item.
The colonies will be counted visually and the numbers will be recorded. A validated spreadsheet software (Microsoft Excel®) will be used to calculate mean values and stand-ard deviations of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tested strain with or without metabolic activation.

A biologically relevant increase is described as follows:
• if in the bacteria strains S. typhimurium TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase fac-tor of at least 2.0)
• if in the bacteria strains S. typhimurium TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A substance is not mutagenic if it does not meet the criteria above.
At least five different analysable and non-toxic concentrations were used for the evalua-tion of the mutagenic potential of the test item in the single experiments. Tables of the values obtained in this test are included in this final report

Statistics:

statistical testing was not done

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Permanganic acid (3-5% in water) is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

Based on the results of the previous experiments,the test item was tested up to concentrations of 5 µL/plate in the bacteria strains TA98 (+S9), TA100 (+S9 and -S9), TA102 (+S9 and -S9), TA1535 (+S9) and TA1537 (+S9 and -S9) and up to 1.5 µL/plate in the strains TA98 (-S9) and TA1535 (-S9) using the pre-incubation method.

The test item showed no precipitates on the plates at any of the test item concentrations.

The bacterial background lawn was reduced at the highest concentration (5 µL/plate) in strain TA102 (-S9) and at the two highest concentrations (5 and 2.5 µL/plate) in strain TA1537 (-S9). At the lower concentrations, no reduction of the bacterial background lawn was seen.

A relevant reduction in colony numbers could be observed in the strains TA100 (-S9) and TA1537 (-S9) at the concentrations 5 and 2.5 µL/plate, in the strains TA102 (-S9) and TA1535 (+S9) at the concentration 5 µL/plate and in strain TA98 (-S9) at the concentration 1.5 µL/plate (highest concentration for this strain). At the lower concentrations no signs of toxicity were observed.

None of the evaluated concentrations showed an increase in the number of revertants in the strains tested in this experiment, in the presence and the absence of metabolic activation.

The results of all experiments performed showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. The test item did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.