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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.10. 2008-28.1.2009
Reliability:
2 (reliable with restrictions)
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:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
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
- 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:
other: Wistar Han
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 9 weeks
- Weight at study initiation:
- Fasting period before study: no
- Housing: Animals were housed in SPF animal room in plastic cages containing sterilised clean shavings of soft wood. Before mating 2 rats of the same sex in one cage, during mating period – one male and two females in one cage were housed. During pregnancy 1 female was housed in one cage.
- Diet (e.g. ad libitum): Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used, manufacturer: Ing. Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using.
Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosphorus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted
groats, Wheat sprouts, Dehydrated yeast, Calcium carbonate, Vitamin and Mineral complex.
- Water (e.g. 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: 5 days
- Identification: Identification of females was made by colour marks on fur (system 1 – 10), each cage was marked with the number of study, number of animals, sex, number of cage, name and dose of the test substance, mark of group and date of planned euthanasia.
- Additional information: The standard pelleted laboratory animal diet was analysed for nutrients (once a year) and bacteriological contaminants (each batch) on a regular basis. Results were retained in the CETA archives. Certificates of drinking water analysis (performed twice a year) were retained in the CETA archives. Bedding (sterilised clean shavings of soft wood) was examined for bacteriological contaminants once a year and results were also retained in the CETA archives.
Analysis of diet, water and bedding, did not reveal any findings that could affect study integrity.


ENVIRONMENTAL CONDITIONS
Animals were housed in a controlled environment
- Temperature (°C): 22+/-3°C
- Humidity (%): 30-70%
- Air changes (per hr): in which optimal conditions were considered to be approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12 hour dark cycle.



Study time schedule
Main study
Date of animal arrival: 22. 10. 2008
Mating: 29. 10. - 6. 11. 2008
Start of administration: 4. 11. 2008
End of administration: 25. 11. 2008
Clinical observation: 4. – 25. 11. 2008
Necropsies: 19. – 26. 11. 2008
Microscopical examination: 6. – 28. 1. 2009
Evaluation of results and final report elaboration: 10. 1. – 20. 4. 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The application form (test substance solution in water for injection) was prepared daily just before administration.
The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.
The application form of the test substance was prepared daily before administration; these solutions were mixed for 10 minutes by magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): Complete peleted diet for rats and mice in SPF breeding (ST 1 BERGMAN) was used, manufacturer: Ing. Mir oslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda, Kocanda No. 19, 252 42 Jesenice u Prahy. Diet was sterilised before using.
- Nutrient content of the diet: Crude protein – min. 21%, Drip – max. 14%, Fat – min. 3%, Fiber – max. 4.1%, Ash – max. 7%, Calcium – min. 1%, Phosp horus – min. 0.8%, Magnesium – min. 0.2%, Sodium – max. 0.25%.
- Composition of food: Wheat, Oats, Fish meal powder, Dried Snail-clover, Soya extracted groats, Wheat sprouts, Dehydrated yeast, Calcium carbo nate, Vitamin and Mineral complex.
- Storage temperature of food: laboratory temperature, bacteriological contaminants are checked once per two month


VEHICLE
- Justification for use and choice of vehicle (if other than water): Aqua pro injectione
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): 0101210308
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.
The procedure was based on the results of analyses of test substance application form homogeneity and stability (Annex 2 of the Study No. 15/06/7: Potassium permanganate - Repeated Dose (28 days) Toxicity (Oral), VUOS-CETA Report No. 0680, 2006).
Details on mating procedure:
- Impregnation procedure: After acclimatisation females were mated with males (1 male and 2 females).
Vaginal smears were carried out daily in the morning to control fertilization (first time: 24 hours after the first removing to male).
Presence of sperms was examined.
Day 0 of pregnancy was the day on which sperms in vaginal smears were observed.
Pregnant females were randomly distributed to experimental groups – 24 or 25 probably gravid females were at each group (2 females from beginning total number 100 were not used for study).
- If cohoused:
- M/F ratio per cage: 1 male and 2 females
- Mating: 29. 10.- 6. 11. 2008
- Proof of pregnancy: sperm in vaginal smear referred to as day 0
- Vaginal smears: daily in mating period
Duration of treatment / exposure:
4.11.-25.11.2008
Frequency of treatment:
The treated and control females were administered daily by gavage – from the 5th to the 19th day of pregnancy. The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Duration of test:
22.10.2008-28.1.2009
No. of animals per sex per dose:
1. Control: 25 probably pregnant females No. 101 - 125
2. 20 mg/kg: 24 probably pregnant females No. 126 - 149
3.100 mg/kg: 24 probably pregnant females No. 151 - 174
4. 500 mg/kg: 25 probably pregnant females No. 176 - 200

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were determined on the basis of results of Study No. 15/06/7: Potassium permanganate – Repeated Dose (28 days) Toxicity (Oral); VUOS-CETA Report No. 0680, 2006 and Study No. 15/06/15: Potassium permanganate – One-Generation Reproduction Toxicity Test; VUOS-CETA Report No. 08142, 2008.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the 1st, 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: on the 5th, 8th, 11th, 14th, 17th and 20th day of pregnancy
- In the given day the remainder of pellets of each cage was weighed, the new food was weighed out and the food consumption for one female and one day was computed (average value for each cage).

MORTALITY CONTROL: Yes
- Time schedule: daily-during the acclimatization, mating and pregnancy

HEALTH CONDITION CONTROL
- Time schedule: daily-during the acclimatization, mating and pregnancy

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on the 20th day of pregnancy
- Organs examined: biometry of organs, microscopical examination,


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes half per litter
Statistics:
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 body weight of pregnant females at the end of pregnancy, absolute and relative weight of uterus, number of foetuses, number of male foetuses, number of female foetuses, weight of foetuses,
weight of male foetuses and weight of female foetuses. Control group with vehicle was compared with three treated groups.
The results statistically significant on probability level 0.05 are indicated by figures with asterisk in the tables of averages.
Indices:
Preimplantation loss, postimplatation loss were calculated from number of implantations, corpora lutea and resorptions.
Historical control data:
none

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Preimplantation loss, postimplatation loss.
Decreased average body weight of foetuses with dependence on the dose level was recorded at all dose levels

Effect levels (fetuses)

Dose descriptor:
LOAEC
Effect level:
20 mg/kg bw/day
Basis for effect level:
other: overall effects litter size and weights; number viable (number alive and number dead); sex ratio; postnatal growth; postnatal survival; grossly visible abnormalities; external, soft tissue and skeletal abnormalities.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Administration of the test substance Potassium permanganate affected growth, clinical status and macroscopic structure of organs in treated maternal animals. These effects were significantly manifested in the results of health condition controls and clinical observations (cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy). During maternal animal necropsies the pathologic changes in stomach and uterus and slightly lower relative weight of pregnant uterus were found. Above mentioned changes were recorded markedly at the highest dose level and sporadically at the middle dose. Important effect on the significant decreased maternal weight was detected at the highest dose level.
In individual foetuses examination the effect to the weight of foetuses was found especially at the highest dose level. Increased number of aborted females and related negative changes of reproduction parameters (postimplantation losses) was also recorded in treated groups with higher incidence at highest and middle dose levels. The increased incidence of some of skeletal variations was found out in foetuses of all treated groups and could be an adverse effect of treatment.
Developmental effect of the test substance together with toxicity to the maternal animals was detected at the highest and middle dose levels. At the lowest dose level 20 mg/kg/day developmental effect (the decreased weight of foetuses, the increased incidence of delayed ossification of vertebrae) occurred in the absence of toxicity to the maternal animals.
Executive summary:

Introduction

   The test substance,Potassium permanganatewastested for prenatal developmental toxicity using the EUMethod B.31, Prenatal Developmental Toxicity Study, Council Regulation (EC) No. 440/2008, Published in O.J. L142, 2008.

 

Study performance

     Wistar rat females of SPF quality were used for testing. After acclimatization the females were mated with males. The test substance was then administered to pregnant females - daily from the 5thto the 19thday of pregnancy. Thestudyincluded four groups of females – 3 treated groups and 1 control group (vehicle only).The test substance was administered dissolved in water by stomach tube and the concentrations of solutions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. 

 

    The dose levels for study – 20, 100 and 500 mg/kg/day were chosen on the basis of results of Study No. 15/06/7: Potassium permanganate – Repeated Dose (28 days) Toxicity (Oral); VUOS-CETA Report No. 0680, 2006 and Study No. 15/06/15: Potassium permanganate – One-Generation Reproduction Toxicity Test; VUOS-CETA Report No. 08142, 2008.

       

   The health condition, clinical status after application, body weight and food consumption of maternal animals were monitored during developmental toxicity study. On the 20thday of pregnancy the maternal animals were killed, the uterine contents were examined and the foetuses were evaluated for soft tissue and skeletal changes.

Results

    At the dose level of 20 mg/kg/day the weight increments, mortality, health condition, clinical status after application, macroscopical structure of organsof pregnant females, values of reproduction parameters (number of live and dead foetuses, intra uterine death early), development of foetuses (foetal weight, evaluation of external and visceral affections)were unaffected by treatment of the test substance.    

    At the dose level of 100 mg/kg/day the mortalityof pregnant females and development of foetuses (evaluation of external and visceral affections)were unaffected by treatment of the test substance.    

    At the dose level of 500 mg/kg/day the mortality,values of some reproduction parameters (number of dead foetuses, intra uterine death early) of pregnant femalesand foetal development judged according to the external and visceral observationwere unaffected by treatment of the test substance.    

    

    The following effects could be attributed to the administration of the test substance:

    At the dose level of 20 mg/kg/day food consumption (decrease) anduterus biometry (decrease of relative weight of uterus) was very slightly influenced by administration of the test substance. Effect of the test substance on the weight of foetuses (slightly decrease) and the increased incidence of delayed ossification of vertebrae (skeletal variations) was recorded.

    At the dose level of 100 mg/kg/day body weight increment (slight decease at the end of application), food consumption (decrease), health condition, clinical status after application (sporadic occurrence of clinical symptoms of toxicity), biometry of uterus (decrease of relative weight of uterus), macroscopical structure of organsofmaternal animals were influenced by administration of the test substance. Some of reproduction parameters (intra uterine death earlyand late, decreased number of live foetuses), weight of foetuses (decrease) and increased incidence of skeletal variation (delayed ossification of vertebrae) were also affected.

   At the dose level 500 mg/kg/day body weight increment (significantly decreased), food consumption (markedly decreased), health condition, clinical status after application (occurrence of clinical symptoms of toxicity), biometry of uterus (decrease of relative weight) and macroscopical structure of organs of maternal animals (findings in digestive system) were influenced by administration of the test substance. Important effect on reproduction parameters (high incidence of aborted females, hight incidence of resorptions and accompanying high postimplantation losses), number of live foetuses (slight decrease of number), weight of male and female foetuses (significantly decrease in female foetuses) and increased incidence of skeletal variation (delayed ossification of vertebrae) were also recorded.

Administration of the test substance Potassium permanganateaffectedgrowth,clinical status and macroscopic structure of organs in treated maternal animals. These effects were significantly manifested in the results of health condition controls and clinical observations (cachexia, anemia, cough or hoarse breath, secretion from nostrils and eyes, piloerrection, apathy). During maternal animalnecropsies the pathologic changesin stomach and uterus and slightly lower relative weight of pregnant uterus were found. Above mentioned changes were recorded markedly at the highest dose level and sporadically at the middle dose. Important effect represented by the significantly decreased maternal weight was detected at the highest dose level. 

    In individual foetuses examination the effect to the weight of foetuses was found especially at the highest dose level. Increased number of aborted females and related negative changes of reproduction parameters (postimplantation losses) was also recorded in treated groups with higher incidence at highest and middle dose levels. The increased incidence of some of skeletal variations was found out in foetuses of all treated groups and could be attributed to an adverse effect of treatment.

  Developmental effect of the test substance together with toxicity to the maternal animals was detected at the highest and middle dose levels. At the lowest dose level 20 mg/kg/day developmental effect(the decreased weight of foetuses, the increased incidence of delayed ossification of vertebrae)occurred in the absence of toxicity to the maternal animals.