Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9.9.2009 – 25.3.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1995
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Semi Dry Absorption (SDA) Product
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available

Composition of test substance:
Calcium sulphate 6.49 %
Calcium sulphite 41.40 %
Calcium carbonate 29.50 %
Calcium hydroxide 2.27 %
Calcium chloride 17.98 %
Calcium fluoride 0.57 %
Oxides (SiO2 + Al2O3 + Fe2O3 + TiO2) sum 0.97 %
Sum of toxic metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum < 0.1 %

Batch No.: SDA/0609/Sk
Appearance: White solid powder
Stability / Expiration date: 15 years (06/2024)
- Stability under test conditions: stable
- Storage condition of test material: the substance was stored in PE container at room temperature.
- Other: pH 11 approx. (by contact of application form with universal indicator pH strip moistened with water, strip producer Lach-Ner, s.r.o. Neratovice)

Test animals

Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic
- sex: males and virgin females
- Age at study initiation: 9 weeks
- Fasting period before study: no

- Housing:
Animals were housed in SPF animal room, 2 rats of the same sex in one plastic cage (40x25x20 cm) containing sterilised clean shavings of soft wood. During mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother.

- Diet:
Complete peleted diet for rats and mice in SPF breeding - ST 1 BERGMAN. Diet was sterilised before using.

- Water:
Free access to drinking water (ad libitum). Water was sterilised before using.

- Acclimation period: 14 days

- Identification:
Identification of animals 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 and mark of group.

- Additional Information: The standard pelleted laboratory animal diet are analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis. Results are retained in the CETA archives. Reports of analysis of water (twice a year) are retained in the CETA archives. Results of sterilizer effectivity control (performed once a year) are retained in the CETA archives.
Analysis of diet and water and steriliser control, did not reveal any findings that could affect study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): a relative humidity of 30-70%
- Air changes (per hr): Animals were housed in controlled environment, 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
Date of animal arrival: 9.9.2009
Start of administration: 23.9. 2009
End of administration: males 20.10.2009, females 8.11.2009
Clinical examination: 9.9. – 9.11.2009
Necropsy and biometry of organs: males 21.10.2009, females 2.11. – 9.11.2009
Histopathological examination: 9.11.2009 – 24.2.2010

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
not specified
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The application form (test substance suspension in olive oil) was prepared daily just before administration.The concentrations of suspensions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight.

VEHICLE
olive oil (pharmaceutical quality), manufacturer: Dr.Kulich Pharma s.r.o.
- Lot/batch no. (if required): 4683401
Details on mating procedure:
- M/F ratio per cage: Mating 1 : 1 (one male to one female) was used in this study.
- Length of cohabitation: until the presence of spermatozoa
- Proof of pregnancy: Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found.

- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance consists of various oxides insoluble in the application form (olive oil). A suitable analytical method cannot be found for homogeneity and stability testing. Since undissolved particles of the test substance are easily visible in the application form, homogeneity was checked by eye (suspension were mixed for 10 minutes by magnetic stirrer). Stability of the test substance in the application form cannot be verified but there is no indication that a mixture of rigid oxides would be unstable in its solution (in olive oil) for that short time period (1 hour).
Duration of treatment / exposure:
The treated groups were administered daily for the following period:
males and females – 2 weeks prior to the mating period and then during the mating period
pregnant females - during pregnancy and till the 3rd day of lactation
males were then administered after mating period – totally for 28 days
non-pregnant females (mated females without parturition) were administered 26 days after the confirmed mating.
Frequency of treatment:
The animals were treated 7 days per week at the same time (8.00 – 10.00 am).
Doses / concentrationsopen allclose all
Dose / conc.:
160 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3 treated groups (dose 160, 400, 1000 mg/kg body weight/day) and one control group (vehicle only). Each group consisted of 10 males and 10 females.

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for study – 160, 400 and 1000 mg/kg bw/day were chosen on the basis of the results of the Study No. 67/09/8: Semi Dry Absorption (SDA) Product – Repeated Dose 90-day Oral Toxicity Study (dose-range finding experiment with 28-day application period).

Examinations

Parental animals: Observations and examinations:
MORTALITY CONTROL: Yes
daily during the treatment periods for vitality or mortality changes

HEALTH CONDITION CONTROL: Yes
Time schedule: daily during the acclimatization and the experimental part.

CLINICAL OBSERVATIONS: Yes
Clinical Observation of Males and Females daily during the administration period (in their cages) in order to record possible clinical effects after application and all changes in behaviour of animals.

BODY WEIGHT: Yes
- Time schedule for examinations: on specified days, all animals were weighed immediately before euthanasia too
Weight increment was computed as an average per group per time interval (in grams). Nonpregnant females were not included in calculation of averages in pregnancy and lactation period.
males - weekly
females - weekly in premating and mating period
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 0. or 1st and 4th day

FOOD CONSUMPTION:
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males average values were calculated for each week of the study (except of mating period). Food consumption for animal/day was calculated from average values of each group.
The same way of calculation of average food consumption was used for females in premating period. In pregnancy and lactation period average individual values (grams/animal/day) were calculated for each week of the study. Average food consumption for each group was calculated from individual values. Nonpregnant and aborted females (females without parturition) were not included in calculation of average food of pregnant females.

males - weekly
females - weekly during premating period and after mating period
during pregnancy: 0., 7th, 14th, 20th day
during lactation: 0. or 1st and 4th day


EXAMINATION OF VAGINAL SMEARS: Yes
The pregnancy was determined by the presence of spermatozoa in vaginal smear. The vaginal smears were carried out daily in the morning during mating period. The smears were stained and the presence of sperm was evaluated. Day 0 of pregnancy was defined as the day when sperms were found.
Oestrous cyclicity (parental animals):
It was not monitored.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
In all males of all groups surviving to scheduled necropsy the sperm parameters were examined: sperm motility and sperm morphology.

Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension. The result of observation was evaluated subjectively according to following grades: 1 – fast progressive motility, 2 - slow progressive motility, 3 – no progressive motility, 4 – non-motile sperm.

Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology were assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.
All deviations – e.g. broken tail, abnormal form of tail, double head, amorphous head, no head, abnormal form of neck – were recorded.
Litter observations:
PARAMETERS EXAMINED
BODY WEIGHT: Yes
pups (litters) – 0. or 1st and 4th day

CLINICAL OBSERVATION: Yes
All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.








Postmortem examinations (parental animals):
SACRIFICE and PATHOLOGICAL EXAMINATION:
Parental males - at the end of the administration period – after 28 days of administration.
Parental females were killed on the 4th day of lactation. Mated females without delivery were killed 27th day after confirmed mating.
Then they were macroscopically examined for any pathological changes with special attention to the organs of the reproductive systems. All macroscopic abnormalities were recorded.

BIOMETRIC OF REPRODUCTIVE ORGANS: Yes
The absolute weights of testes, epididymis, prostate gland and pituitary gland were recorded in males and absolute weight of ovaries, uterus (incl. uterine tube and cervix) and pituitary gland were recorded in females. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

HISTOLOGICAL TECHNIQUE
The following tissue and organs were collected from all killed males and females at necropsy and fixed in neutral 4% formaldehyde suspension (v/v) for further histopathological evaluation:
relevant gross lesions, pituitary gland, coagulation gland, prostate gland, seminal vesicles, epididymis and testes (fixed in Davidson´s suspension), cervix of uterus, ovaries, uterus and vagina.
For histopathological processing the routine histopathological paraffin technique with haematoxylin-eosin staining was used.

Detailed histological examination was performed on testes (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Spermatogenesis and spermatogenic cycle were evaluated according to criteria mentioned in the publication: Hess, R.A.; Quantitative and qualitative Characteristics of the Stages and Transitions in the Cycle of the Rat Seminiferous Epithelium: Light Microscopic Observation of Perfusion-Fixed and Plastic-Embedded Testes (Biology of Reproduction 43, 525-542, 1990). Pathological changes were evaluated according to criteria mentioned in the publication: Creasy, D.M.; Evaluation of Testicular Toxicity in Safety Evaluation Studies: The Appropriate Use of Spermatogenic Staging (Toxicologic Pathology 25, 119-131, 1997).
Postmortem examinations (offspring):
SACRIFICE AND GROSS EXAMINATION OF DEAD PUPS
- on the 4th day of lactation
- these animals were subjected to external examination of the cranium, and to macroscopic examination of the thoracic and abdominal tissues and organs. All macroscopic changes were recorded.

- Dead pups were sexed and externally examined; the stomach was examined for the presence of milk.
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 and biometry of organs and number of pups. 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 summary tables.
Reproductive indices:
For each of parental females the following parameters were counted:
Pre-implantation loss, Post implantation loss, Post-natal loss
For each dose group reproduction parameters will be counted:
Percentage mating, Fertility index, Conception index, Gestation index
Offspring viability indices:
Percentage of postnatal loss days 0-4 post partum, Viability index

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
M: No statistically significant differences were detected.
F: see box Details on results (P0)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
M/F: see box Details on results (P0)
The most of histopathological findings recorded in parental males and females responded to common findings in animals in reproductive period.
Histopathological findings: neoplastic:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
Negative influence of the test substance on sperm motility and sperm morphology was observed in males of the highest dose level (limit) - see box Details on results (P0) but this affection had no negative effect on mating and reproduction parameters of males at the highest dose level.
Reproductive performance:
no effects observed
Description (incidence and severity):
see box Details on results (P0)

Details on results (P0)

MORTALITY (PARENTAL ANIMALS)
There were no unscheduled deaths at any dose levels and at the control group during whole study.

CLINICAL SIGNS (PARENTAL ANIMALS)
Males and females: no signs of diseases were found out during the check-in, acclimatisation and application period. No treatment-related effects were detected during the health condition control. No clinical changes were observed in males of all dose levels after application of the test substance.

FOOD CONSUMPTION
Males - food consumption of males at all treated dose levels was lower than in the control group during the whole study. In comparison with the control group, statistically significant decrease of average food consumption was recorded at the middle and highest dose level in the 2nd week and at all treated groups in the 4th week of study.
Females
Pre-mating period - during pre-mating period, average food consumption was well-balanced in the control group and in the lowest treated group. In the groups 400 and 1000 mg/kg/day, average food consumption was decreased in comparison with the control group. This decrease was statistically significant in the 1st week of study.
Pregnancy - females without parturition (non pregnant or aborted females) were not included in evaluation of food consumption during pregnancy.
In the second and third week of pregnancy, average food consumption at all treated groups was relatively well-balanced but lower compared to the control group. In the 3rd week of pregnancy, this decrease was statistically significant in all treated group.
Lactation - only mothers (females with live pups) were included in evaluation of food consumption during lactation period. Average food consumption of treated mothers was well-balanced, but lower than in mothers of control group. Decrease of average food consumption of the highest dose level was statistically significant.

BODY WEIGHT
Males: no statistically significant differences were detected.
The animal body weights at all dose levels were relatively well-balanced with the control group during whole application period.
Body weight increments at all treated animals were well-balanced, but were a little bit lower in comparison with control group.

Females
Pre-mating period - average body weight increments of all dose levels were relatively well-balanced, but a little bit lower in comparison with the control group. Only in the 2nd week, average body weight increment of females in the lowest dose level (160 mg/kg/day) was higher than in the control group. No statistically significant changes during the whole pre-mating application period were found out.
Pregnancy - females without parturition (non pregnant or aborted females) were not included in evaluation of the body weight increments during pregnancy.
Only in the 1st week of pregnancy, average body weight increment of females at the dose level 160 mg/kg/day persisted higher compared to the control group. The second and third week of pregnancy, average body weight increment of all treated groups was well balanced but lower than in control the group.
Lactation - only mothers (females with live pups) were included in evaluation of body weight increment during lactation period.
Average weight increments of treated mothers were markedly lower in comparison with mothers of control group.

SPERM QUALITY
The test substance had negative effect on sperm quality of treated males. Sperm motility was affected in males of all treated groups (0-40-30-60% impaired motility); mostly in males of the highest dose level 1000 mg/kg. Microscopical examination of sperms revealed increased percentage portion of morphologically changed sperms in males of all dose levels (5-6.1-5.8-6.9% affected sperms), mostly in males of the highest dose level 1000 mg/kg.
These findings were not accompanied by damage of spermiogenesis in testicular tubules and had no effect on reproduction.

BIOMETRY OF REPRODUCTIVE ORGANS
Males
Absolute and relative weights of all observed organs were similar in control males and in males of the highest dose level.
Decreased weight of testes was recorded in males of the dose level 160 mg/kg/day; decreased weight of epididymis was recorded in males of the dose levels 160 and 400 mg/kg/day. On the contrary, increased weight of prostate gland was recorded at the dose level 400 mg/kg/day.
No statistically significant differences were detected.
Females
Nonpregnant females and females with abortion were not used for calculation of means and evaluation of organs weight.
Absolute weight of uterus at the dose level 160 mg/kg/day was lower in comparison with control group; at the dose level 400 mg/kg/day and the dose level 1000 mg/kg/day, weight of uterus was higher than at the control group. At these dose levels, also relative weight of uterus was higher.
Absolute and relative weight of ovaries was similar at the control group and the highest dose level. Absolute weight of ovaries at the lowest and middle dose levels was lower in comparison with the control group.
Absolute and relative weight of pituitary gland of all treated groups was lower compared to weight of pituitary gland at the control group. This difference was not dependent on dose level.
No statistically significant differences of relative and absolute weights of reproductive organs were detected.

REPRODUCTION DATA
Number of females achieving pregnancy and accompanying conception index was well balanced at the control group and at the lowest and middle dose levels. At the highest dose level, these parameters were higher than in other groups. Duration of mating and pregnancy of treated groups was similar to control group.
Number of females bearing live pups was higher at the dose level 1000 mg/kg/day, in the rest of the treated group and control group, this number was similar.
Number of pregnant females was similar at the control group and lowest and the middle dose level; the highest number of pregnant females was recorded at the highest dose level. At the lowest dose level and at the middle dose level 2 females aborted. None abortion was observed at the control and the highest dose level.
Pre-implantation losses were relative well balanced among treated groups. At the lowest dose level abortion in two females together with increase of post-implantation losses were recorded. There were no gross or microscopic findings that could be related to treatment and theirs abortion was considered spontaneous without toxicological importance. At the middle and the highest dose level post-implantation loss were well-balanced with the control group. Slight disbalance of other fertility parameters recorded in parental females was without toxicological importance.

PATHOLOGY - MACROSCOPIC AND MICROSCOPIC FINDINGS
Males
Histopathological examination of reproductive system of parental males showed increased incidence of tubules with destructed epithelium in testes of males in the dose levels 400 and 1000 mg/kg/day; increased incidence of lymphocyte infiltration in prostate gland in males of the dose levels 160 and 400 mg/kg/day. Increased incidence of vacuolation of spermatogoium in testes was recorded in males at all treated groups.

Females
Macroscopic structure of reproductive organs of treated parental females was not markedly affected by treatment of the test substance.
Histopathological examination of reproductive system of parental females revealed increased incidence of microscopic affections in ovaries – follicular cysts in females of the dose level 1000 mg/kg/day, but this affection was recorded also sporadically at the control group and at the middle dose level. Atrophy of epithelium of vagina was recorded in two females of the highest dose level. Affections (atrophy, hypertrophy, hyperplasia) in cervix were also observed in females of treated groups, especially in the highest dose level. These findings had no negative effect on parental females.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The test substance did not affect fertility of treated males and females and development of pups

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
see box Details on results (F1)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

BODY WEIGHT
In comparison with the control group, the average weights of litter at all treated groups on 0/1 day after parturition were lower. Average body weight of pup per litter was relatively well-balanced.
On the 4th day of lactation, average weights of litter were still lower in comparison with the control group, but average body weight of pup per litter was slightly higher at all treated groups compared to average body weight of pup at the control group.

DEVELOPMENT AND ABNORMAL
No pups died at the control group and at the dose levels 160 mg/kg/day during lactation period. At the dose level 400 and 1000 mg/kg/day one pup of each dose level died during lactation period.
No differences in development of pups were observed at the control group and at all treated groups.

VIABILITY (OFFSPRING)
The test substance did not affect number of pups. The total number of live pups was the highest at the highest dose level but average number of pups per litter in comparison with the control group was lower.
Marked decrease of total number of live pups and average number of pups per litter (in the day of parturition/1st day after parturition and the day 4th after parturition) in comparison with control group were recorded at the lowest and middle dose levels. This decrease is not influenced by the test substance, but by number of pregnant females (average number was well-balanced).

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. No pathologic findings were recorded at control group and at the all dose levels.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: The test substance did not affect fertility of treated males and females and development of pups

Results: F2 generation

Effect levels (F2)

Based on:
not specified
Sex:
not specified
Remarks on result:
not determinable

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Effect on average food consumption (decrease) was observed in males of the middle and the highest dose level. The same effect on average food consumption (decrease) was recorded in females of the highest dose level. Marked decrease of average body weight increment was recorded in all treated mothers on 4th day of lactation.
Negative influence of the test substance on sperm motility and sperm morphology was observed in males of the highest dose level (limit); but this affection had no negative effect on mating and reproduction parameters of males at the highest dose level.
The most of histopathological findings recorded in parental males and females responded to findings of animals in reproductive period.
The test substance did not affect number of pups. Different total number of pups was not influenced by the test substance, but by number of pregnant females (average number was well-balanced).
Number of females achieving pregnancy, durations of mating and pregnancy, number of pups, sex ratio, average weight of litter, average body weight and postnatal development of pups were unaffected by the test substance treatment.
The test substance did not affect fertility of treated males and females and development of pups.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of males was established as 1000 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for REPRODUCTION of females and pups DEVELOPMENT was established as 1000 mg/kg body weight/day.
Executive summary:

Introduction

The test substance, Semi Dry Absorption (SDA) Product, was tested for reproduction toxicity using the OECD Test Guideline No. 421 Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on July 27th 1995.

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered dissolved in olive oil using a stomach tube; oral application of rats was made daily. The concentrations of suspensions at all dose levels were adjusted to ensure the administered volume of 1 mL per 100 g of body weight. Four groups of animals were included in the study - 3 treated groups (doses 160, 400, 1000 mg/kg of body weight/day) and one control group (vehicle only). Each group consisted of 10 males and 10 females. The dose levels for study – 160, 400 and 1000 mg/kg/day were chosen on the basis of the results of the Study No. 67/09/8: Semi Dry Absorption (SDA) Product – Repeated Dose 90-day Oral Toxicity Study, Dose-range finding experiment with 28-day application period.

The treated groups were administered daily for the following periods:

males and females – 2 weeks prior to the mating period and during the mating period,

pregnant females – during pregnancy and till the 3rd day of lactation,

males  after mating period – totally for 28 days,

non-pregnant females (mated females without parturition) – for 26 days after the confirmed mating.

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in specified time intervals. Vaginal smears were prepared daily during mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters: sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.