Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.5.-21.7.2006
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.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus 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
- Batch No.: 69
- Substance type: technical product
- Physical state: solid crystals
- Analytical purity: 99.42 % wt.
- Impurities (identity and concentrations): Manganese dioxide ca 0.1 % wt.
- Appearance: dark violet-purple crystalline powder with bronze lustre
- pH: 1% solution-6.1

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm BioTest s.r.o., Konárovice, 281 25 Czech Republic, RČH CZ 21760152
- Age at study initiation: 6 – 7 weeks
- Weight at study initiation: males-233,5-275,6 g
females-190,8-210,7 g
- Fasting period before study: no
- Housing: Sterilized shavings of soft wood, Monitored conditions, microbiologically defined background, according to SOP No.40
- Diet: ad libitum, Complete peleted diet for rats
- Water: ad libitum, Drinking tap water
- Acclimation period: At least 5 days
- Stock and health condition: No signs of disease should be observed at clinical check-in.
- Prophylactic arrangement: Cleaning and disinfection of animal room will be regularly performed as it is described in appropriate SOP No.10
- Total number of animals: Pilot experiment: 15 animals for determination of toxicity of the test substance (single administration, acute toxicity test)
Main test: 100 animals
- Identification of animals: Individual labelling of cages and labelling of the animals
- Random selection: According to SOP No. 42

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature 22 +/- 3°C, permanently monitored
- Humidity (%): Relative humidity 30 – 70 %, permanently monitored
- Air changes (per hr): 1/h
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle: 6am-6pm/6pm-6am


Study time schedule
Pilot experiment: 22. 5. – 26. 5. 2006
Main study: 29. 5. – 1. 6. 2006
Examination of microscopic slides: 2. 6. – 21. 7. 2006
Evaluation of results and final report elaboration : 24. 7. – 20. 8. 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s) used: water for injections
- Justification for choice of solvent/vehicle: solubility and relative stability of test substance in water
- Amount of vehicle (if gavage or dermal): Volume of the application form was constant at all dose levels - 1 ml/100g b.w. by adequate adjusting the
concentration.
- Lot/batch no.: 3040105
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
dissolution of test substance in water for injections in appropriate amounts

Duration of treatment / exposure:
1) 1500 mg/kg, exposition – 24 h: 5 males + 5 females
2) 1500 mg/kg, exposition – 48 h: 5 males + 5 females
3) 800 mg/kg, exposition – 24 h: 5 males + 5 females
4) 800 mg/kg, exposition – 48 h: 5 males + 5 females
5) 300 mg/kg, exposition – 24 h: 5 males + 5 females
6) 300 mg/kg, exposition – 48 h: 5 males + 5 females
Frequency of treatment:
The test substance was administered to animals by stomach tube in single dose at 29.5. 2006
Post exposure period:
24 and 48 hours
clinical symptoms of toxicity were observed several times after application
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500 mg/kg b. w.
Basis:
nominal in water
Remarks:
Doses / Concentrations:
800 mg/kg b. w.
Basis:
nominal in water
Remarks:
Doses / Concentrations:
300 mg/kg b. w.
Basis:
nominal in water
No. of animals per sex per dose:
100 animals (50 males and 50 females) were used in the main study
1) 1500 mg/kg, exposition – 24 h: 5 males + 5 females
2) 1500 mg/kg, exposition – 48 h: 5 males + 5 females
3) 800 mg/kg, exposition – 24 h: 5 males + 5 females
4) 800 mg/kg, exposition – 48 h: 5 males + 5 females
5) 300 mg/kg, exposition – 24 h: 5 males + 5 females
6) 300 mg/kg, exposition – 48 h: 5 males + 5 females
- positive control– cyclophosphamide – 20 mg/kg, exposition – 24 h: 5 males + 5 females
- negative control – water for injections, exposition – 24 h: 5 males + 5 females
- negative control – water for injections, exposition – 24 h: 5 males + 5 females
- control without administration: 5 males + 5 females
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide monohydrate
- Batch number: 091K1176
- Justification for choice of positive control(s): Positive control substance was chosen according to the methodology
- Route of administration: intraperitoneal injection
- Doses / concentrations: 20 mg/kg, exposition-24h-5 males+ 5 females

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Doses for main test were selected from results of pilot experiment.
Dose level 1500 mg/kg b.w. was chosen as the highest level (maximal non-lethal dose level)
Dose level 800 mg/kg b.w. was chosen as medium level (low toxicity symptoms)
Dose level 300 mg/kg b.w. was chosen as low level (without clinical toxicity symptoms


DETAILS OF SLIDE PREPARATION:
- Bone marrow harvesting
Bone marrow cells have been obtained from the femora immediately after the sacrifice of animal.
After excising and careful cleansing of the bone both femur ends were be clipped off. Marrow was be gently flushed from the bone by 1 ml of
bovine serum into the tube. Acquired bone marrow was several times mixed by syringe with thin needle.

- Preparation of the bone marrow smears
The bone marrow with serum in tubes was centrifuged (5 min – 1000 rpm). The supernatant was gently removed, one drop of bovine serum was
added to the sediment and this cell suspension is mixed on mixer.
Clean and degreased slides were marked by the number of study, number of animal, sex and dose level. One drop of cell suspension was placed onto the slide and a smear was prepared using a pusher slide. Two slides were prepared per animal.

- Staining of the bone marrow smears
After drying (24 h at laboratory temperature) the smears were fixed by ethanol – 5 minutes. Then they were twice rinsed by distilled water and
stained by 5% solution of Giemsa – 15 minutes.

METHOD OF ANALYSIS:
- Examination of the bone marrow smears
Before examination, the slides were coded.
Stained smears were examined by light microscope. 200 erythrocytes were evaluated per animal for the proportion of immature (polychromatic) and mature (normochromatic) erythrocytes („cytotoxicity index“) in bone marrow.
At least 2000 polychromatic erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

- Criteria for distinguishing the micronuclei
colour – purple
form – generally round or almond shaped, (occasional lightly stained and ring shaped micronuclei may occur)
borders – sharp
size – 5-20% of polychromatic erythrocyte size

- Scoring of micronucleated immature erythrocytes
- number of immature erythrocytes with micronuclei
(number of micronuclei per PE is not a result, occasional more than one micronucleus may appear per PE).

- Data treatment
Individual animal data were summarized to tables. Proportion of immature erythrocytes (cytotoxicity index) and count of micronucleated
immature erythrocytes were determined for each animal. Because the count of evaluated erythrocytes was not the same in each animal (but at
least 2000 erythrocytes were evaluated), the final count of micronucleated immature erythrocytes was adjusted for 2000 erythrocytes per animal.
The Excel software was used for calculation of mean values and standard deviations for each group of animals.
The statistical analysis was performed by the ANOVA test - Analysis of Variance (software QC.Expert 2.5, Trilobyte, Statistical Software, ČR). Each of treatment groups was confronted with negative control group.
Evaluation criteria:
Criteria for distinguishing the micronuclei
colour – purple
form – generally round or almond shaped, (occasional lightly stained and ring shaped
micronuclei may occur)
borders – sharp
size – 5-20% of polychromatic erythrocyte size

Scoring of micronucleated immature erythrocytes
- number of immature erythrocytes with micronuclei
(number of micronuclei per PE is not a result, occasional more than one micronucleus may appear per PE).
Statistics:
ANOVA is used as an aid in evaluating the test results

Results and discussion

Test resultsopen allclose all
Sex:
female
Genotoxicity:
negative
Remarks:
At given experimental conditions the test substance Potassium permanganate, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.
Toxicity:
yes
Remarks:
All of 20 animals of dose level 1500 mg/kg b.w. had mild symptoms of toxicity - piloerection and hunched posture - symptoms faded away until 24 hours after application.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
At given experimental conditions the test substance Potassium permanganate, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.
Toxicity:
yes
Remarks:
All of 20 animals of dose level 1500 mg/kg b.w. had mild symptoms of toxicity - piloerection and hunched posture - symptoms faded away until 24 hours after application.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 300/800/1500 mg/kg b.w. The highest dose level was determined on the basis of result from acute toxicity test. Pilot test was performed in the same laboratory, using the same strain of animals and the same treatment regimen that was used in the main study.
- Solubility: full soluble
- Clinical signs of toxicity in test animals: piloerection, hunched posture
- Evidence of cytotoxicity in tissue analyzed: descrease "cytotoxicity index" was not recorded at any of the three dose levels.
- Harvest times: 24 hours after application for all animals from all three dose levels and 48 hours for animals from dose level 1500 mg/kg b.w.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): in any group of animals administered by test substance statistically significant changes of
count of micronuclated immature erythrocytes were not found out.

Clinical observation
   No animal died during the main experiment.
   All of 20 animals of dose level 1500 mg/kg had mild symptoms of toxicity – piloerection and hunched posture. All symptoms faded away until 24 hours after application. 
    All animals of the dose level 800 mg/kg had low symptoms of toxicity – piloerection.  All symptoms faded away until 24 hours after application. 
    No symptoms of toxicity were observed in all animals of the lowest dose level – 300 mg/kg.
     No symptoms of toxicity were observed in the animals of positive control group and negative control groups. 

Any other information on results incl. tables

Proportion of immature erythrocytes among total erythrocytes  – “cytotoxicity index”

   In any group of animals administered by test substance, statistically significant changes of proportion of immature erythrocytes from total number of erythrocytes were not found out.    

      

Table No:3.    Cytotoxicity index  - group means and standard deviations

 

MALES

FEMALES

Group

Mean

Standard deviation

Mean

Standard deviation

1500 mg/kg

– 24 h

0.380

0.05

0.385

0.04

800 mg/kg

– 24 h

0.386

0.03

0.385

0.04

300 mg/kg

– 24h

0.375

0.04

0.388

0.04

Negative control – 24 h

0.388

0.03

0.387

0.04

1500 mg/kg

– 48 h

0.398

0.04

0.364

0.03

800 mg/kg

– 48 h

0.377

0.04

0.385

0.04

300 mg/kg

– 48 h

0.354

0.06

0.383

0.04

Negative control – 48 h

0.382

0.05

0.383

0.04

Positive control

0.265

0.03

0.239

0.02

Control without treatment

0.378

0.03

0.357

0.04

 

Individual counts of immature and mature erythrocytes from 200 erythrocytes are shown in tables 16 – 35 in Annex I (chapter 7).

 

Count of micronucleated immature erythrocytes

 

In the following tables (4 and 5) the group means and standard deviations of micronucleated IME counts are summarized. In any group of animals administered by test substance, statistically significant changes of count of micronucleated immature erythrocytes were not found out.    

 

Individual counts and percentage expression of micronucleated immature erythrocytes from 2000 immature erythrocytes are presented in tables 16 – 35 in Annex I in attached full-study report.

 

 

Table No: 4.    Micronucleated immature erythrocytes - group means and standard deviations

MALES

Number of micronucleated IME per animal (per 2000 IME)

Percentage expression

       Group

Mean

Standard deviation

Mean

Standard deviation

1500 mg/kg

– 24 h

2.48

0.89

0.124

0.04

800 mg/kg

– 24 h

2.45

0.80

0.123

0.04

300 mg/kg

– 24h

2.70

0.79

0.135

0.04

Negative control – 24 h

2.28

0.55

0.114

0.03

1500 mg/kg

– 48 h

2.66

0.81

0.133

0.04

800 mg/kg

– 48 h

2.66

0.73

0.133

0.04

300 mg/kg

– 48 h

2.10

0.79

0.105

0.04

Negative control – 48 h

2.63

1.04

0.131

0.05

Positive control

- 24 h

15.62

1.85

0.781

0.09

Control without treatment

2.29

0.47

0.114

0.02

IME – immature erythrocytes

 

Table No: 5.    Micronucleated immature erythrocytes - group means and standard deviations

 

FEMALES

Number of micronucleated IME per animal (per 2000 IME)

Percentage expression

        Group

Mean

Standard deviation

     Mean

Standard deviation

1500 mg/kg

– 24 h

2.10

0.78

0.105

0.04

800 mg/kg

– 24 h

2.30

1.08

0.115

0.05

300 mg/kg

– 24h

2.49

0.84

0.125

0.04

Negative control – 24 h

2.50

1.31

0.125

0.07

1500 mg/kg

– 48 h

2.51

0.58

0.126

0.03

800 mg/kg

– 48 h

2.32

0.55

0.116

0.03

300 mg/kg

– 48 h

2.28

0.84

0.114

0.04

Negative control – 48 h

2.49

0.87

0.124

0.04

Positive control

- 24 h

14.09

2.28

0.705

0.11

Control without treatment

2.48

0.53

0.124

0.03

IME – immature erythrocytes

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
The test substance Potassium permanganate, was tested for the assessment of cytogenetic damage using micronucleus test.
Statistically significant increase in the count of micronucleated immature erythrocytes compared to the control was not recorded at any dose level.
Under the given test conditions, the test substance, Potassium permanganate, has negative result in micronucleus test.
Negative results in the micronucleus test indicate that under the test conditions, the test substance Potassium permanganate, does not produce micronuclei in immature erythrocytes in bone marrow of rat.
At given experimental conditions the test substance Potassium permanganate, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.
Executive summary:

The test substance,Potassium permanganate,was tested for the assessment of cytogenetic damage in vivo, using laboratory rat (Wistar). The study is a part of the test substance health hazard evaluation.

 

The test was performed according to the EU Method B.12, Mutagenicity – In vivo Mammalian Erythrocyte Micronucleus Test. The method is analogous to the OECD Test Guideline No. 474, Mammalian Erythrocyte Micronucleus Test.

 

The test substance was administered to animals by stomach tube in single dose. Three dose levels were chosen according to the results of pilot experiment - 300, 800 and 1500 mg/kg of body weight. Two bone marrow sampling intervals were used - 24 and 48 hours after administration. The group of animals without administration and concurrent negative and positive controls were included. Each experimental group consisted of five males and five females.     

The smears obtained from bone marrow were examined by light microscope.

In none of mentioned dose levels the test substance did not cause a significant increase of count of immature erythrocytes with micronuclei in comparison with negative control group.

At given experimental conditions the test substance Potassium permanganate, did not give rise to formation of micronuclei in immature erythrocytes in bone marrow of rat.