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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
20.3.-22.9.2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

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.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

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
- Analytical purity: 99.42
- Impurities (identity and concentrations): Manganese dioxide cca 0.1 %
- Lot/batch No.: 69

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, BioTest, s. r. o., 28125 Konárovice, Czech Republic
- Age at study initiation: 6 - 7 weeks
- Fasting period before study: no
- Housing: in SPF (specific pathogen free) animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood
- Diet: ad libitum, complete pelleted diet for rats and mice in SPF breeding (ST 1 BERGMAN), producer: Mill Kocanda, Jesenice by Prague.
Diet was sterilised before using.

- Water: ad libitum, free access to drinking water (water ad libitum). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law,
Health Ministry. Water was sterilised before using.
- Acclimation period: minimally 6 days
- Identification: Identification of animals was made by colour marks on fur (system 1 – 6), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance and mark of group.

All the study proceeded in SPF (Specifiedc Pathogen Free) animal house of CETA in conditions according to SOP No. 12.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12 hrs light/12 hrs dark

Study time schedule:
Test substance delivery: 3. 3. 2006
Dose-range finding study: 20. 3. – 12. 4. 2006
Main study: 25. 4. – 25. 5. 2006
Date of animal arrival: 18. 4. 2006
Start of administration: 25. 4. 2006
End of administration: 25. 5. 2006
Observation: 25. 4. – 25. 5. 2006 – main groups
25. 4. – 6. 6. 2006 – satellite groups
Urinalysis: 22. – 25. 5. 2006 - main groups
5. – 6. 6. 2006 - satellite groups
Blood taking and necropsies: 23.- 26. 5. 2006 - main groups
6. – 7. 6. 2006 - satellite groups
Histopathological examination: 6. 7. - 22. 9. 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The concentrations of solutions in all three dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form (test substance solution in water for injectione) was prepared daily just before administration. The vehicle control group was administered by water for injectione in the same volume. The application form of the test substance was prepared daily before administration; it
was mixed 10 minutes by magnetic stirrer. The procedure was based on the results of analyses of test substance application form homogeneity
and stability (see ANNEX 2). These results showed that the test substance at defined laboratory conditions (laboratory temperature, mixing of
solutions by defined manner) is homogenously dissolved in water and the solution is stable at last for 120 minutes.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua, the test substance was administered dissolved in water for injectione.
- Manufacturer: Infusia a.s., Hořátev, Czech Republic
- Batch number: 276805
- Attest No.: 2768/05
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of test substance was performed by a spectrophotometry.
Test substance stability and homogeneity were determined by measuring an absorbance of its water solution in visible range of spectrum.
Duration of treatment / exposure:
28 days (4 weeks)
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 - 10.00 a.m.)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg bw/day (m/f)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day (m/f)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
250 mg/kg bw/day (m/f)
Basis:
actual ingested
No. of animals per sex per dose:
Main groups:
1. group - control with water for injectione 6 males + 6 females
2. group - the lowest dose 40 mg/kg/day 6 males + 6 females
3. group - the intermediate dose 100 mg/kg/day 6 males + 6 females
4. group - the highest dose 250 mg/kg/day 6 males + 6 females
Satellite groups:
5. group - satellite control with water for injectione 6 males + 6 females
6. group - satellite the highest dose 258 mg/kg/d 6 males + 6 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose-range finding study with 14-day application period was performed. The dose levels 30, 60, 120 and 240 mg/kg/day (in water for injections) were chosen with respect to the acute toxicity study results. In the dose-range finding study, the dose-dependent decrease of body weight
increments were observed in the males. Clinical observation did not detect the impact of the test substance on the health condition of animals.
Result of haematology examination showed that the test substance does not have an impact to basic blood parameters. Macroscopic changes of
organs (marked structure of liver, changed colour of liver and kidney, change of stomach mucosa) were found in dose levels 60, 120 and 240
mg/kg/day.
- Rationale for animal assignment: Animals were randomly divided into the control and test groups and marked individually.
- Rationale for selecting satellite groups: random
- Post-exposure recovery period in satellite groups: 14 days after the end of application
- Section schedule rationale: all animals were sectioned
Positive control:
no

Examinations

Observations and examinations performed and frequency:
HEALTH CONDITIONS: Yes
- Time shedule: daily

MORTALITY CONTROL: Yes
- Time shedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: performed before the first application and then weekly.
- In the first part of observation, the behaviour of animals in the cage was monitored: posture, position of eyelids, tonic or clonic movements, piloerec tion, stereotypes or bizarre behaviour of animals.
- The second part was the observation during the removal from the cage. These parameters were observed: reaction to handling, elasticity of skin, col our of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
- The body weight of animals was recorded on automatic balances with group average computing module.
First weighing was performed before the first application and then weekly. All animals were weighed immediately before euthanasia too.
Weight increment was computed as an average per group per week (in grams).


FOOD CONSUMPTION AND COMPOUND INTAKE:
-In the given day of every week the remainder of pellets was weighed in each cage, the new food was weighted out and the food consumption for last week was computed.
The average values in groups were calculated for each week of the study.

FOOD EFFICIENCY:
-Food consumption for animal/day was calculated of average values of each group.
From growth increments and food consumption the food conversion was calculated:food conversion (%)=weight increment/food consumption x 100


WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: twice a week
The drinking water requirement consumption was recorded twice a week. The average values in groups (water consumption per animal per day) were calculated for each week of the study.

OPHTHALMOSCOPIC EXAMINATION: Not performed

HAEMATOLOGY: Yes
- Time schedule for collection of blood: : 29th (main groups) and 42nd day of study (satellite groups)
- Anaesthetic used for blood collection: Yes, light diethyl ether narcosis
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 29th and 42nd day of study
- Animals fasted: No
- How many animals: all animals
- Parameters checked in table [No.2] were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: 28th and 41st day of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.3] were examined.


FUNCTIONAL OBSERVATION: Yes
- Time schedule for examinations: fourth week
- Dose groups that were examined: all animals
- Battery of functions tested: the sensory reactivity on auditory, papillary reflex, visual and proprioceptive stimuli were evaluated and motor activity
assessment was conducted.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities was carried out.
Organs for subsequent histopatological examination were taken out and stored in containers with fixative (neutral 9 % formaldehyde).

HISTOPATHOLOGY: Yes (see table No. 4)
- Tissue specimens were fixed in 4% neutral formaldehyde processed by routine paraffin technique and stained by hematoxyline-eosine.
Cryotome sections of liver and kidneys were stained by oil red for neutral lipids.

At the end of study the experimental animals were narcotised and sacrificed by cutting the neck spine and medulla. After the gross necropsy of the cranial, thoracic and abdominal cavities the organs for weighing and further histological examination were collected. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epidydimides or uterus, thymus, spleen, brain, pituitary gland and heart were recorded. Afterwards the somatic indexes (SI) were computed according to the following formula: SI = weight of organ x 100 / body weight (= relative weight of organ).
Statistics:
Data processing
All the primary data (body weight, food consumption, water consumption, health condition control, general observations, clinical observational battery, functional observations, haematological examinations, biochemistry, urinalysis, biometry of organs, necropsy findings, histopathological examinations) were recorded in protocols. These primary data were used for calculations and processed to tables.

Statistical analysis
The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis. This statistical analysis was used for the results of haematology, blood chemistry and biometry of organs. Control group with vehicle was compared with three treated groups and satellite control with vehicle was compared with the satellite highest dose group.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the summary tables.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
In control and treatment animals no clinical changes were observed during the whole study. No mortality occured during the study period.

HEALTH CONDITION CONTROL
Main groups – MALES and FEMALES
The health condition of all animals was controlled daily during the check-in, acclimatisation and application period. No signs of diseases were found out.
Satellite groups – MALES and FEMALES
The health condition of all animals was controlled daily during the check-in, acclimatisation, application period and during the recovery period. No signs of diseases were found out.


BODY WEIGHT AND WEIGHT GAIN
Average body weight:
MALES
Main groups
Since the 1st week the growth curves of all treated groups proceeded slightly under control growth curve. Maximal difference was in dose level 250 mg/kg/day. In this dose level, difference of body weight compared to control increased from 17 grams (in the 1st week application) till 33 grams (in the4th week).
Satellite groups
The growth curves of satellite treated group were slightly below the control one, as in corresponding main group. This weight difference against control group persisted also after the end of application.

FEMALES
Main groups
During the 1st to the 3rd week of study, the growth curves of all treated groups were well-balanced with the control group. In the 4th week, the growth curve of the highest dose level 250 mg/kg/day runs slightly below then in the control group.
Satellite groups
The body weights of females in the dose level 250 mg/kg/day were slightly lower then in the control group. During recovery period after application the growth curve of the treated group had slightly rising trend. In the end of recovery period the body weight of treated animals was slightly higher then in control group.

Body weight increment:
MALES
Main groups
During the 1st week of application, average body increments were lower in all treated males against control group. In the highest dose level marked decrease of increments was recorded and continued during all period of application.
Satellite groups
Average body weight increments of treatment satellite group of males were lower then in control animals. In the 6th week of study (the 2nd week of recovery period) the body weight increments of the highest dose level were higher then in control group.

FEMALES
Main groups
Average body weight increments of females in dose level 250 mg/kg/day were lower then in control group in the 1st week of study. Higher increments of all treated groups were recorded in the 2nd week. During 3rd- 4th week of study increment of animals were lower against control group in the dose levels 100 and 250 mg/kg/day.
Satellite groups
During the 1st – 2nd week of study, average body weight increments were lower in dose level 250 mg/kg/day. In the 3rd week the increment was higher against control group. In the highest dose level decrease of body weight (negative increase) was recorded in the 4th week of study. In the recovery period average body weight increment in treatment group was higher then in control group in 5th week of study and lower in the 6th week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption
MALES
Main groups
Food consumption of all treatment groups of males was lower in comparison the control group, mostly in the dose level 250 mg/kg/day. Consumption was decreased depending on dose level in the1st and 4th week of application.
Satellite groups
Food consumption of treatment group was lower than in control group during the whole study (except the 3rd week).

FEMALES
Main groups
Food consumption of treatment groups of females was analogous with food consumption of control group. Only in the dose level 250 mg/kg/day, food consumption was slightly lower during whole application period.
Satellite groups
Food consumption of treated females was lower than in control during whole application period. The consumption increased during the recovery period.


FOOD EFFICIENCY
Food conversion
MALES
Main groups
In the 1st week of application, food conversion was decreased in the dose level 250 mg/kg/day; in the 2nd week of study conversion was decreased in the dose levels 100 and 250 mg/kg/day; in the 3rd week of study food conversation was decreased only in the highest dose level; in the 4th week food conversion was decreased in all treated groups. In the dose level 250 mg/kg/day the food conversion was decreased against the control group during the whole study.
Satellite groups
In treated group the food conversion was degreased against the control group during whole application period (except the 2nd week). During the 5th week the food conversion of satellite groups were balanced and in the 6th week the conversion in treated group was higher than in the control group.

FEMALES
Main groups
Food conversion of females of all treated groups was analogous with the control group during the 1st week. On the contrary food conversion of all treated groups was higher against the control group in the 2nd week of study. Since the 3rd week the food conversion was lower in dose levels 100 and 250 mg/kg/day, markedly in the highest dose level. In the dose level 40 mg/kg/day the food conversion was lower then in the control group only in the 3rd week of application.
Satellite groups
The food conversion of the dose level 250 mg/kg/day was lower since the 1st week of study; the 3rd week of study the conversion was higher against the control group, but next week was lower again. During 1st week of recovery period (the 5th week of study) the food conversion of treated group was higher and in the 2nd week of study (the 6th week of study) conversion was lower against the control group again.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
MALES
Main groups
During the 1st week of study, the water consumption was decreased depending on dose level. Since 2nd week of study, water consumption was markedly lower only in males of the dose level 250 mg/kg/day. In the dose levels 40 and 100 mg/kg/day the water consumption was analogous to the control group.
Satellite groups
Water consumption of treated group was markedly lower against the control group during whole study (application and recovery period).

FEMALES
Main groups
Water consumption was lower in females of the dose level 250 mg/kg/day. In the dose levels 40 and 100 mg/kg/day the water consumptions were also slightly lower since the 2nd week of application.
Satellite groups
Water consumption of treated group was lower against control group during whole study (application and recovery period).


HAEMATOLOGY
MALES
Main groups
Slight decrease of total erythrocyte count was found in the dose level 100 mg/kg/day. Slight increase of total erythrocyte count and accompanying increase of haemoglobin concentration and haematocrit were recorded in the dose level 40 mg/kg/day. In this dose level, increased value of haemoglobin concentration was statistically significantly different against control group. Value of leucocyte count increased depending on dose levels. APTT was slightly decreased only in the dose level 40 mg/kg/day.
Other measured parameters in the dose level 40 and 100 mg/kg/day and all parameters in the dose level 250 mg/kg/day were similar compared with animals in the control group.

Satellite groups
Statistically significant increases of total erythrocyte count, haematocrite and haemoglobine concentration were recorded in treated group. Value of total leucocyte count was also higher than in the control group. PT was statistically significantly increased against the control group.
Other measured parameters were similar compared with animals in the control group.

FEMALES
Main groups
Decrease of MCV was recorded in the dose level 100 mg/kg/day. Values of leucocyte count and trombocyte count were slightly increased in all treated groups. Decreased counts of monocytes and granulocytes and increased count of lymphocytes were measured in all treated groups; these changes were marked in the dose levels 40 and 100 mg/kg/day.
Other measured parameters were similar compared with animals in the control group.
Satellite groups
Values of total erythrocyte count, leucocyte count ant trombocyte count were slightly higher against the control group in females of treated group. Statistically significantly increased value of monocytes and accompanying decreased value of lymphocytes were recorded in dose level 250 mg/kg/day.PT was also significantly increased against the control group.
Other measured parameters were similar compared with animals in the control group.

BIOCHEMICAL EXAMINATION
MALES
Main groups
Decreased values of total protein and albumine were recorded in the dose levels 100 and 250 mg/kg/day. The changes were statistically significant in the highest dose level. Values of total urea and ALP were increased in dose level 250 mg/kg/day. Increased value of total urea and creatinine were recorded also in the dose level 40 mg/kg/day.
Other measured parameters were similar with the control group.
Satellite groups
Statistically significantly increased value of calcium was recorded in the treated group of animals. Rising trends were detected also in parameters total protein and ALP.
Other measured parameters were similar with the control group.


FEMALES
Main groups
Decreased values of total cholesterol were recorded in all treated groups. Because these decreased values were under of detection limit, statistical evaluation was not possible to perform. Decreased values of total protein and albumine were recorded in the dose levels 100 and 250 mg/kg/day. Value of bilirubine was lower then in the control group in dose level 100 mg/kg/day. Slightly decreased values of total urea were detected in all treated groups.
Other measured parameters were similar with the control group.
Satellite groups
Statistically significant increased value of creatinine was recorded in treated group of females. Decrease was recorded also in value of total urea.


URINALYSIS
MALES
Main groups
Decrease of urine volume; increase of pH, specific weight and content of protein were recorded in all treated group against the control group. These changes were significant in the dose level 250 mg/kg/day. The content of leucocytes, ketones and urobilinogen, in urea were recorded only in the highest dose level.
Satellite groups
Decrease of urine volume, increase of pH and content of protein were recorded in treated satellite group against the control group. The content of leucocytes was also recorded in this dose level.

FEMALES
Main groups
Urine volume was decreased against the control group in the dose levels 40 and 250 mg/kg/day. Increase of pH and specific weight were recorded in all treated groups. The content of protein, ketones and urobilinogen in urea were recorded also in the highest dose level.

Satellite groups
Increase of pH and specific weight were recorded in the satellite treated group.


ORGAN WEIGHTS
MALES
Main groups
In the highest dose level (250mg/kg/day), decreased absolute weight of liver was found out. On the contrary, the relative weights of liver were slightly increased against the control group in all treated groups. Increased relative weight of testes and epididymides were recorded in the dose levels 100 and 250 mg/kg/day. Absolute weights of testes were slightly increased in males of all treated groups. Slightly increased absolute weights of epididymides were found only in dose levels 100 and 250 mg/kg/day. Increased weight (relative and absolute) of the spleen was recorded in exposed groups 250 mg/kg/day.
Satellite groups
Increased weight (relative and absolute) of the spleen was recorded in males of the treated satellite groups. The relative weight of the spleen was statistically significantly increased against the control group. Absolute weight of liver was slightly lower and relative weight was slightly higher then the in control animals.
Absolute and relative weight of testes and epididymides were similar with the control group.

FEMALES
Main groups
Slightly increased weight (relative and absolute) of kidneys and spleen were recorded in the highest dose level 250 mg/kg/day. In the dose level 100 mg/kg/day, absolute and relative weight of spleen were slightly decreased. Increase of absolute and relative weight of uterus was found out in all treated dose levels.
In all treated groups, the weights of next organs were similar with the control group.
Satellite groups
Statistically significantly increased weights (relative and absolute) of uterus were recorded in animals of treated satellite group. In the exposed females, absolute and relative weights of spleen were slightly decreased against control group.
In all treated groups, the weights of other organs were similar with the control group.


GROSS PATHOLOGY
MALES
Main groups
During the macroscopic examination of males no important pathological changes were found out. In 5-4-1-0 males no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity petechiae on thymus in 0-0-2-0 males, and focal changes on lung in 0-0-1-1 males were diagnosed.
In abdominal cavity marked structure of liver in 1-2-0-3 males, irregular ochre colour or discolouration of liver in 1-2-0-2 males was found out. The macroscopic changes were often found in stomach: oedema of mucosa in 0-0-4-0 animals, local changes (erosion) in 0-0-1-2 males, irregular colour 0-0-1-1 and irregular structure of mucosa in 0-0-0-3. Incidental findings included focal change on kidneys in 0-0-0-1 male, atrophy of testes in 1-0-0-0 male, and inflammation of epididymides in 1-0-0-0 male.
In cranial cavity no changes were diagnosed.
Satellite groups
During the macroscopic examination of satellite males no important pathological changes were found out. In 5-3 males no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity focal changes on lung in 1-1 males were only found out.
In abdominal cavity irregular colour of liver in 0-1 males and atrophy of testes in 0-1 male were recorded.
In cranial cavity no changes were diagnosed.

FEMALES
Main groups
During the macroscopic examination of females no important pathological changes were found out. In 6-4-0-1 females no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity petechiae and irregular colour of thymus in 0-0-1-0 females, focal changes on lung in 0-0-1-1 females, and irregular colour of lung in 0-0-1-0 female were recorded.
In abdominal cavity marked structure of liver in 0-1-3-1 females, irregular ochre colour or discolouration of liver in 0-2-5-3 females was found out. Atrophy of spleen in 0-0-2-1 females and irregular colour of spleen in 0-0-4-1 females were diagnosed. The macroscopic changes were often found in stomach and forestomach: oedema of mucosa in 0-0-2-3 animals, local changes (erosion) in 0-0-1-4 males, mucosa with mucin in 0-0-1-1 females and irregular structure of mucosa in 0-0-0-3 females. Irregular colour of forestomach was only found out in 0-0-0-1 female. Dilatation of uterus with fluid was diagnosed in 0-0-2-1 females.
In cranial cavity no changes were diagnosed.


Satellite groups
During the macroscopic examination of satellite females no important pathological changes were found out. In 4-3 females no macroscopic changes were observed. Examination of the external surface of body revealed no change.
In thoracic cavity petechiae on thymus in 1-0 females were only found out.
In abdominal cavity marked structure and irregular ochre colour of liver in 0-1 female, in stomach: oedema of mucosa in 0-1 female, mucosa with mucin 0-1 female and swelling of Peyer´s patches in 1-0 female were recorded. Dilatation of uterus with fluid was diagnosed in 0-2 females.
In cranial cavity no changes were diagnosed.


HISTOPATHOLOGY: NON-NEOPLASTIC
MALES
Main groups
The affections were often diagnosed in stomach and liver. Focal atrophy of phundal glands was observed in stomach of 1-3-1-0 males and eosinophile infiltration was recorded in stomach of 0-0-1-2 animals. Various alterations were registered in liver (in all groups). Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 2-4-4-3 males. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 4-2-2-3 males. Biliary proliferation was found out in 0-1-3-0 males and oval cells proliferation was recorded in 0-1-1-0 males. Extramedullary haemopoiesis was found out in liver of 0-0-0-1 males. Focal inflammation of liver was detected in 4-3-3-0 males. The changes of intestine were found sporadically: hyperplasia of MALT (MALT – mucosa associated lymphoid tissue) in large intestine of 1-0-0-0 males, eosinophiles infiltration in small intestine of 0-1-0-0 males, and hyperplasia of MALT in small intestine of 1-0-0-0 males. Areactive necrosis of the mucosa was diagnosed in rectum of 0-0-0-3 males.
Steatosis of kidneys was found in 5-4-6-6 males. Next sporadic changes were recorded in kidneys: tubular atrophy in 0-0-1-0 males, hydronephrosis in 0-0-0-1 males, intersticial nephritis in 0-1-0-0 males, proteinuria in 0-0-2-0 animals.
In lymph nodes the following pathological changes were diagnosed: hyperplasia in 2-0-0-0 males, reactive histiocytosis in 0-2-2-0 males, activation in 1-0-0-0 males. Extramedullary haemopoiesis was found in spleen of all males (6-6-6-6). Intensity of haematopoiesis was similar in all groups of males.
Nodular hyperplasia of adrenal gland cortex was detected in 1-0-0-0 males. Active haematopoiesis were kept in bone marrow of all males (6-6-6-6).
Inflammation of larynx was recorded in 0-2-1-2 males. Sporadic affections were also diagnosed in next organs: inflammation in treachea of 0-1-0-0 males, inflammation of lung in 0-0-1-1 males, focal proliferation of glial cells of brain in 0-0-0-1 males.
Incidence of pathological affections in male genital tract was sporadic. Atrophy of germinal epithelium of testes was found in 1-0-0-0 males and inflammation of epididymis in 1-0-0-0 males. Histopathological findings in prostate gland were also sporadic: focal intersticial inflammation in 3-0-0-0 males. The affections in mammary gland were more often: lactating mammary gland in 3-2-1-0 males and involution in 0-2-0-0 males.
Satellite groups
The affections in stomach and intestine were only sporadic. Focal atrophy of phundal glands was observed in stomach of 0-1 males. Focal inflammation of small intestine was recorded in 0-1 males. More often alterations were registered in liver. Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 1-1 males. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 5-5 males. Extramedullary haemopoiesis was found in liver of 0-1 males. Focal inflammation of liver was detected in 4-1 males.
Steatosis of kidneys was found in 6-5 males. Extramedullary haemopoiesis was found in spleen of all satellite males 6-6 males. Intensity of haematopoiesis was similar in both groups of males. Active haematopoiesis were kept in bone marrow of all satellite males (6-6).
Sporadic affections were also diagnosed in next organs: nodular hyperplasia of adrenal gland cortex in 2-0 males, focal inflammation of the myocardium of heart in 2-0 males, and focal oedema of medulla spinalis in 1-0 males.
Inflammation of larynx was recorded in 4-2 males. The affections were also diagnosed in lungs: inflammation in 2-3 males, dystelectasis in 1-0 males.
Focal interstitial inflammation in prostate gland was recorded in 4-1 males. Proliferation of epithelium in prostate gland was found out in 1-0 males. Histopathological findings in mammary gland were sporadic: lactating mammary gland in 2-0 males.

FEMALES
Main groups
The affections were often diagnosed in stomach and liver. Focal atrophy of phundal glands of stomach was diagnosed in 3-2-0-0 females. Eosinophile infiltration was recorded in stomach of 0-0-0-6 animals and oedema of mucosa of stomach in 0-0-0-6 animals. Various alterations were registered in liver (in all groups). Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 3-3-2-3 females. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 3-3-4-3 males. Biliary proliferation was found in 0-0-3-0 females and oval cells proliferation in 0-0-3-0 females. Extramedullary haemopoiesis was found in liver of 0-0-1-0 females. Focal inflammation of liver was detected in 1-1-1-2 females. The changes were found in small intestine: hyperplasia of MALT (MALT – mucosa associated lymphoid tissue) in 1-0-0-0 animals, and eosinophile infiltration in 1-3-0-0 females.
Steatosis of kidneys was found in 5-5-6-6 females. Metastatic mineralization was recorded only in 0-0-1-0 females.
Sporadic affections were also diagnosed in next organs: reactive histiocytosis in lymph node of 1-0-0-0 females, cyst in thyroid gland of 1-0-0-0 male and focal proliferation of ependymal cells in brain of 0-0-0-1 females.
Extramedullary haemopoiesis was found in spleen of all females (6-6-6-6). Intensity of haematopoiesis was similar in all groups of females.
Also active haematopoiesis were kept in bone marrow of all females (6-6-6-6).
Inflammation of lung was recorded in 1-1-0-4 females and dystelectasis in lung of 1-0-0-0 female. Sporadic affections were also diagnosed in larynx: inflammation in 0-0-2-0 females
Female genital tract: Hydrometra of uterus in 0-0-1-0 females. The affections in mammary gland were more often: involution in 5-5-3-3 females.
Satellite groups
The affections were sporadically diagnosed in stomach. Eosinophile infiltration was recorded in stomach of 1-0 animals, oedema of mucosa in stomach of 1-0 females and muscular dystrophy of stomach of 1-0 animals. Various alterations were registered in liver (in all satellite groups). Irregular steatosis (increased amount of lipids in hepatocytes irregularly disseminated in liver parenchyma) was observed in 1-2 females. Peripheral steatosis (increased amount of lipids in hepatocytes of periportal area) was detected in 5-4 females. Focal inflammation of liver was detected in 3-4 females.
Steatosis of kidneys was found in 6-5 females. Metastatic mineralization was recorded only in 0-1females.
Sporadic affections were also diagnosed in next organs: inflammation of larynx in 1-3 females, inflammation of trachea in 0-1 females, histiocytosis in lung of 0-1 female, inflammation of lung in 0-2 female, oedema of brain in 0-1 animal, and focal proliferation of ependymal cells in brain of 0-1 female.
Extramedullary haemopoiesis was found in spleen of all females (6-6). Intensity of haematopoiesis was similar in both groups of females.
Also active haematopoiesis were kept in bone marrow of all females (6-6).

Hydrometra of uterus was diagnosed in 0-2 females and fibrosis of endometrium was recorded in uterus of 0-4 females. The affections in mammary gland were more often: involution in 3-4 females.




Effect levels

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no toxic effect observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION OF RESULTS

   From the results of laboratory examination of rats strain Wistar, the negative effect of 28-day oral application of the test substance Potassium permanganate is apparent in animals of both sexes.

   Stress for organism was manifested by lower food consumption and food conversion, what caused lower growth increment of treated animals in comparison with control animals. This effect was recorded since 1st week of application and was markedly in males.

   Negative effect of the test substance on digestive tract was confirmed by macroscopic examination. In the middle and highest dose level, numerous changes of stomach mucosa, changes of structure and irregular colour of liver were recorded. By histological examination the inflammation changes of stomach mucosa (eosinophile infiltration) and oedema of mucosa were diagnosed in females of the highest dose level. In males these changes were recorded only sporadically.         

  Increased relative weight of liver was measured in males of all treated groups. Biliary proliferations were diagnosed in liver of males and females in the middle dose level. In both sexes (markedly in females) of the middle dose level the proliferation of oval cells in liver was found out. These cells are considered to be liver stem cells. Peripheral or irregular steatosis was diagnosed with similar frequency in all animals of treated and control groups. The changes in hepatic function were confirmed by biochemical examination of blood. In the highest dose level, increased value of ALP (alkaline phosphatase) and decreased values of total protein and albumin were recorded in males. Also the presence of urobilinogen in urea might have been influenced by dysfunction of liver. Biochemical examination of blood showed markedly decreased concentration of total cholesterol in all treated females. In males the decrease of this value lead to the levels under limits of measurability.

   The histopathological changes of the other organs in gastro-intestinal tract were found out only sporadically. Only in males of the highest dose level presence of areactive necrosis of the mucosa in rectum was recorded in half of animals. This change was not recorded in any other group of treated males and females or control animals. Dysfunction of digestion might have been a cause of impairment of the physical condition of animals. The grip strength (one of parameters of functional observation battery) decreased in both sexes of the highest dose level

The damage of gastro-intestinal tract was reversible. After 14-day period without application the food consumption, growth increments and food conversion were comparable with control animals. Macroscopic changes of liver were still recorded in satellite animals, but their frequency was lower in comparison with corresponding main group. Accompanying biochemical parameters were similar to the control groups or even lower.

  The haematology parameters of red blood cells were the second sphere of serious damage. Decreased value of total erythrocyte count and accompanying decrease of haematocrite and haemoglobine concentration in peripheral blood were recorded in males of the highest and middle dose level and females of the highest dose level. At the end of period without test substance application the contrary effect was recorded, which bears evidence of compensation process. Increased values of total erythrocyte count, haematocrite and haemoglobine concentration were measured in both sexes. The effect was statistically significant only in males.

  The changes in blood coagulation were the third sphere of damage. Increased value of trombocyte count was recorded in all treated groups of females. After 14-day observation period this increase was significant. Slightly increased value of trombocyte count was recorded also in satellite treated males. Also prolongation of protrombine time detected in all treated groups might have been a symptom of disbalance among components of haemocoagulation. The effect of the test substance on this blood component was protracted – the effect manifested itself even after period without application.

 The application of the test substance had also effect on urine excretion system. Urinalysis showed decrease of urine volume depending on increasing dose level. On the contrary specific weight of urine and pH urine were increased depending of dose level. Presence of protein in urine was recorded in all groups of males (also in control males), but the protein content was increased depending on dose level. In females the presence of protein in urine was recorded only in the highest dose level. The presence of leucocytes in urine was recorded only in males of the highest dose level. These changes in urine were irreversible, but expected damage of kidney was not approved histologicaly. Only sporadic findings of proteinuria and hydronephrosis in kidney could be associated with above-mentioned findings. Statistically significantly increased value of creatinine in blood in satellite treated females also could signalise the kidney damage.

   Accompanying symptom of the above-mentioned changes could be the changes in proportion of white blood cells - increased value of total leucocyte count and increased portion of monocytes. These changes might have been caused by defensive action of organism against toxicity of the test substance. Alternatively they could by attributed to inflammation changes in respiratory air-ways. Histopathological examination showed these inflammation changes in organs of males and females of all treated main groups and also in satellite treated groups.

       Histopathological changes in brain (focal proliferation of glial cells, proliferation of ependymal cells and oedema) were not numerous, but were unusual. These changes were recorded only in animals of the highest dose level (both males and females). The 28-day study is not long enough for determination, if these changes are really caused by application of the test substance.   

       Histopathological findings in genital organs showed rather changes caused by senility of organism. Only hydrometra of uterus in females of treated groups (also in satellite animals) might have been caused by activation of estral cycle by application of the test substance. In males slightly increased weights of testes and epididymis were recorded in treated animals.

Applicant's summary and conclusion

Conclusions:
The dose level 40 mg/kg/day could be regarded as the NOAEL (no adverse effect level). Sporadically finded changes at this dose level are considered to be a result of normal variation for rats of the strain and age used and they has no toxicological importance.
Executive summary:

 Following effects were detected after repeated 28-day repeated oral application of the test substance,Potassium permanganate

 

Dose level 250 mg/kg/day

    reversible changes:

-          decrease of body weight increment in both sexes

-          decrease of food consumption in both sexes, markedly in males

-          decrease of food conversion in both sexes

-          decreased values of total protein and albumine in both sexes, statistically significant in males

-          increased content of protein in urea – in both sexes, markedly in males

-          occurrence of urobiline and ketones in urea – in both sexes

-          slightly increased weight of testes and epididymides

 irreversible changes:

-         decrease of water consumption – in both sexes

-         increased value of total leucocyte count – in both sexes, markedly in males

-         slightly increased value of thrombocyte count - in females

-         slightly increased value of ALP – in males

-         decrease of urine volume – in both sexes

-         increased value of pHand specific weight of urine – in both sexes

-         increased weight of spleen – statistically significant in males (this change was reversible in females)

-         increased weight of uterus – statistically significant in females

protractedchanges:

-         increased value of total erythrocyte count, haematocrite and concentration of haemoglobine – in both sexes, statistical significant in males

-         increased value of monocyte fraction – statistically significant in females

-         prolongation of PT - protrombine time - statistically significant in both sexes

-         increased value of calcium in blood – statistically significant in males

-         increased value of creatinine in blood – statistically significant in females

 

 

 

Dose level 100 mg/kg/day

-         increased value of pHand specific weight of urine – in both sexes (not so marked as in the dose level 250 mg/kg/day)

-         slightly increased weight of testes and epididymides

 

Dose level 40 mg/kg/day

 -   no toxic effect observed

 

    Overall assessment showed, that the test substance Potassium permanganate after 28-day oral application cased decrease of growth increments, food consumption and food conversion. Slightly damage of gastro- intestinal tract was confirmed by the other examination.

    Haematological examination showed negative effect on red blood component. Increased values of total erythrocytes, concentration of haemoglobin and haematocrit were recorded after end of observation period. These changes have been accompanied by increased weight of spleen. Prolongation of protrombine time was pronounced disbalance among components of haematocoagulation.

    Increased specific weight and pH urine (together with decreased urine volume) was connected with decreased water consumption. Also decreasedvalue of total protein and albumin in blood showed negative effect on urinary tract. But histopathological changes of kidney were recorded only sporadically.

  Accompanying symptoms of changes was increase value of leucocyte count and increased portion of monocytes. These changes might have been caused by defensive of organism against toxicity of the test substance but also by inflammation changes in respiratory air-ways.

The findings of genital organs showed rather changes caused by senility of organism. Only hydmometra of uterus in females of treated groups (also in satellite animals) and fibrosis in endometrium (only in satellite treated animals) might have been caused by activation of oestral cycle after application of the test substance.