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Repeated dose toxicity: oral

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Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24.03.2016 to 22.01.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 24.03.2016 to 22.01.2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 93%
- Expiry date: 06.03.2018
Species:
rat
Strain:
Wistar
Remarks:
Cmdb:WI; outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from the husbandry of laboratory animals of the Experimental Medicine Centre at the
Medical University in Białystok,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks old.
- Weight at study initiation: average body weights:
- group 0: males 403.50 g; females 243.75 g
- group 1: males 403.50 g females 243.92 g
- group 2: males 403.40 g; females 243.75 g
- group 3: males 403.50 g; females 243.92 g
- group 0 SAT: males 404.00 g; females 244.90 g
- group 3 SAT: males 404.10 g; females 245.10 g.
- Fasting period before study:
- Housing: Air-conditioned rooms. The animals were kept in plastic cages covered with wire bar
lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). During the main e
xperiment, there were 2 or 3 animals/cage. Each sex was kept separately.
- Diet (e.g. ad libitum): ad libitum access,
- Water (e.g. ad libitum): ad libitum access.
- Acclimation period: All animals were quarantined and observed for at least 5 days.

DETAILS OF FOOD AND WATER QUALITY: Food: Altromin 1324 P TPF (Phytoestrogen Deficient,
Total Pathogen Free) standard granulated fodder produced by Altromin Spezialfutter GmbH & Co.
KG, Lage, Germany, batch number: 1/17 -200418/0453. A certificate of analysis is included in the
report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25ºC
- Humidity (%): 43 - 81%
- Air changes (per hr): 13-18 times/h
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: Experimental starting date 13.06.2017 To: Experimental completion date 22.08.2017
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
During the main study solutions of the test item in a vehicle (corn oil) were prepared once a week.
Prepared solutions were stored in a fridge (temperature 4-8#C). During administration, solutions were
kept at room temperature and they were thoroughly mixed.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable vehicle, as the test item hydrolyses in water.
- Concentration in vehicle: 6.66 mg VL3/1 mL solution, 20 mg VL3/1 mL solution and 60 mg VL3/1mL solution
- Amount of vehicle (if gavage): the total volume administered was 0.5mL/100 g b.w. (including testitem)
- Lot/batch no. (if required): L5294 (Supplier BASSO FEDELE & FIGLI srl, ITALY)
- Purity: 100 % corn oil
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: [vaginal plug referred to as day 0 of pregnancy
- Further mattings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Pregnant females were housed individually. After delivery, females were housed with their offspring. A label was attached to each cage. It provided information of the study code, the cage number, sex and numbers of the animals, the dose of the test item, the starting and ending dates of the study, day
0 of pregnancy (when appropriate) and the date of delivery (when appropriate)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was conducted by BLIRT S.A. Laboratory, Trzy Lipy 3/1.38, 80-172 Gdańsk, Poland, which holds the Good Laboratory Practice Certificate GLP. Solutions of the test item in a vehicle (corn oil) were prepared once a week. Two samples (2 x 2 ml) were collected from each batch of prepared solutions. One of them was frozen and sent in a pack of dry ice to BLIRT S.A. (27 samples in 9 batches) for analysis and the other one (spare sample) was archived in the freezer (-15 - -20°C).

TECHNIQUE AND TEST METHOD: Gas chromatography coupled with flame ionization detector (FID).

TEST PARAMETERS: Chromatographic column: ZB-5MS, 30 m x 0.25 mm x 0.25 μm, catalog no.:7HG-G010-11, serial no.: 221879, Phenomenex.
Temperature gradient: 175ºC (Injection port), 280ºC (detector), 25ºC/min column rate., Column flow:0.69 mL/min, Carrier gas: helium, Injection volume: 1 μL
Split ratio: 33.3 mg/kg b. w. (6.66 mg/mL of VL3 in corn oil) and 100 mg/kg b. w. (20 mg/mL of VL3 in corn oil): 1:50, 300 mg/kg b. w. (60 mg/mL of VL3 in corn oil): 1:100.

ACCEPTANCE CRITERIA: The relative standard deviation, RSD [%], of the VL3 content for 3 results for 33.3 mg/kg b. w. (6.66 mg/mL of VL3 in corn oil) dose is 6.67 %, so it is ≤ 10.0 %. The relative standard deviation, RSD [%], of the VL3 content for 3 results for 100 mg/kg b. w. (20 mg/mL of VL3 in corn oil) dose is 9.91 %, so it is ≤ 10.0 %. The relative standard deviation, RSD [%], of the VL3 content for 3 results for 300 mg/kg b. w. (60 mg/mL of VL3 in corn oil) dose is 0.48 %, so it is ≤ 10.0 %. The acceptance criteria are fulfilled.

QUANTITATIVE AND QUALITATIVE DETERMINATION: All average VL3 content in study samples were at the expected level.
Duration of treatment / exposure:
The test item/vehicle control was administered to males for 28 days. Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (51 – 59 days). Males from groups 0 SAT and 3 SAT were treated for 28 days, females from groups 0 SAT and 3 SAT were treated for 53 days. Post treatment, the satellite groups were observed for 14 days.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
33.3 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group 0, 1, 2, 3: 10 males, 12 females
Satellite groups: 10 males, 10 females
Control animals:
yes, concurrent vehicle
other: other: two satellite groups one receiving vehicle control and a second one receiving the highest dose level 300 mg/kg bw/d
Details on study design:
Two Dose range-finding studies were conducted.
First Dose range-finding study: administered 250, 500 and 1000 mg/kg bw/d (n= 5animals/sex/group) during 7 days. In haematological examinations, statistically significant decrease in the number of leukocytes in males from group 1 and females from group 1 and 3 were stated. Biochemical examinations revealed following statistically significant changes between the treated and the control groups: the decreased total protein concentration in males from group 1, the decreased albumin concentration in males from group 1, the decreased globulin concentration in males from group 1, the decreased calcium concentration in males from group 1, the decreased creatinine concentration in females from group 3.

Based on these results, a second Dose range-finding study was conducted: administered 31.3, 65.5,125 mg/kg b.w./day for 7 days.It was not observed treatment-related changes.
Based on both dose range finding studies, the doses of 33.3, 100 and 300 mg/kg b.w./day were selected for the main study.

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: control and highest dose group in order to evaluate the reversibility, stability, or delay in the onset of possible harmful effects.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not conducted.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day or once a day (on days off)
- Cage side observations checked: General condition of the animals, i.e. the observation of all animals for morbidity and mortality was conducted.

DETAILED CLINICAL OBSERVATIONS: Yes, evaluation of skin, fur, eye and mucosa changes, the respiratory, circulatory, and autonomous and central nervous systems, somatic activity, and behaviour .
- Time schedule: before the experiment and then once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week during the entire experiment. Body weights of females were measured twice a week before gestation, on days 0, 7, 14, and 20 of gestation, after delivery (day 0), and day 4, 7, 10 and 13 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, food intake by parental males was measured once a week during the pre-mating period. Food intake
by parental females was measured once a week during the pre-mating period, on days 0, 7, 14, and 20 of gestation, on day 0, 4, 7 and 13 post-partum. Food intake by females and males from satellite groups was measured once a week during the entire experiment.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (g/100 g b.w./day)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: the day before the introduction of the animals to the experiment and one day before euthanasia.
- Dose groups that were examined: all adult animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the experiment, blood was collected for clinical-chemical examinations.
- Anaesthetic used for blood collection: Yes, anesthetized with a mixture of xylazine and ketamine at the doses of 10 mg xylazine/kg b.w. and 100 mg ketamine/kg b.w.
- Animals fasted: Yes, prior to the clinical-chemical examinations, the animals were fasted for about 18 – 19 hours. Blood samples were taken from the heart as part of the procedure for euthanasia of the animals.
- How many animals: 5 adult males and 5 adult females from each group (0, 1, 2, 3, 0 SAT, and 3 SAT). Additionally 5 other males (from all groups) and 7 other females (from group 0, 1, 2, 3) or 5 females (from 0 SAT and 3 SAT group) were subjected to hematological (without bone marrow) biochemical and enzymatic examinations.. The level of thyroid hormone, i.e. total T4 (TT4) in serum was determined in all adult males after termination.
- Parameters examined: numbers of leukocytes, erythrocytes, and thrombocytes, the concentration of hemoglobin, hematocrit, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin weight), and MCHC (mean corpuscular hemoglobin concentration), number of reticulocytes, circulatory blood and bone marrow microscopic examinations, Prothrombin time (PT) AND Partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the experiment, blood was collected for clinical-chemical examinations.
- Animals fasted: Yes, prior to the clinical-chemical examinations, the animals were fasted for about
18 – 19 hours.
- How many animals: 5 adult males and 5 adult females from each group (0, 1, 2, 3, 0 SAT, and 3 SAT). Additionally 5 other males (from all groups) and 7 other females (from group 0, 1, 2, 3) or 5 females (from 0 SAT and 3 SAT group) were subjected to hematological (without bone marrow) biochemical and enzymatic examinations.. The level of thyroid hormone, i.e. total T4 (TT4) in serum was determined in all adult males after termination.
- Parameters examined: - total protein, albumin, globulin (albumin subtracted from total protein, total cholesterol, urea nitrogen, creatinine, glucose, bilirubin, bile
acids, sodium, potassium, chlorides and calcium. Also aspartate aminotransferase (AST), alanine a minotransferase (ALT) and alkaline phosphatase (AP).

URINALYSIS: Yes
- Time schedule for collection of urine: On the last day of the experiment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined.: colour, volume (mL), specific gravity, pH, protein (g/L), glucose (mmol/L), ketone bodies (mmol/L), bilirubin (qualitative test), blood (Ery/μL), urobilinogen (μmol/L), leukocytes (leu/μL), urine sediment, squamous and columnar epithelium, leukocytes, erythrocytes, and crystals were determined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males from group 0, 1, 2 and 3 were examined at the end of the administration period (1-2 days before euthanasia). Females from group 0, 1, 2 and 3 were examined during the last week of lactation (1 day before euthanasia). The behavioural studies on the males and females from satellite groups were performed at the end of the administration period (measurement 1) and then before the end of the additional observation period (measurement 2).
- Dose groups that were examined: 5 adult males and 5 adult females from each group were subjected to the studies.
- Battery of functions tested: observations of animal behaviour in an open field, responses to stimuli, locomotor activity, and forehand hindlimb grip strength.

IMMUNOLOGY: Yes
- Time schedule for examinations:
- How many animals: 5 adult males and 5 adult females
- Dose groups that were examined: from each group (0, 1, 2, 3, 0 SAT, and 3 SAT).
- Parameters examined.: The evaluation of the immune system was based on the results of clinical-chemical examinations and post-mortem examinations (gross and histopathological examinations) of the spleen, thymus, and lymph nodes, as well as the statistical analysis of the absolute and relative weights of the spleen and thymus. The clinical-chemical parameters included blood morphology with a picture of peripheral blood and bone-marrow smears, the concentrations of albumin, total protein, the albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase.

OTHER - HORMONE TESTING:
The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined. The Mouse/Rat Thyroxine (T4) ELISA kit produced by Calbiotech were used.
Blood samples were collected from:
- all dams,
- all adult males after termination.
Blood samples from adult males were measured for TT4. There was no statistically significant changes in the hormone level, absolute/relative weight of the thyroid and histopathological changes of the thyroid connected with the test item, therefore further measurements of TT4 (dams) were not made.
Oestrous cyclicity (parental animals):
Females were screened for normal ostreus cycles in a 2-week pre-treatment period. Ostreus cycles were monitored before treatment starts to select for the study females with regular cyclicity. Females that fail to exhibit typical 4-5-day cycles were not included in the study. Vaginal smears were monitored daily during the pre-treatment period and then from the beginning of the treatment period until evidence of mating. Vaginal smears were examined on the day of necropsy to determine the stage of the ostreus cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
Spermatogonia, spermatocytes and spermatides were evaluated as well as the accuracy of the particular layers of the sex epithelium structure. The lumen of the seminiferous tubules was examined to exclude the presence of the immature forms of spermatogenetic cells or giant cells. In epididymides the presence of sperm content was evaluated.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Day 0: clinical observations, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, physical or behavioural abnormalities,
Day 4: AGD (anogenital distance) of each pup, body weight
Day 13: presence of nipples/areolae in male pups

Body weights of pups in litter were measured on day 0, 4, 7 and 13 post-partum

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Dead pups and the ones euthanized on day 4 and 13 post-partum were carefully examined for gross abnormalities. The examinations involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with all organs and their content. Particular attention in pups was paid to the external reproductive genitals which were examined for signs of altered development.

Additionally the thyroid gland was collected from 1 male and 1 female from each litter in the control and treated groups for histopathological examination, there was an exception, because of insufficient number of live males and females per litter thyroid gland was collected from 2 females (litter from female no.13 of group 0) .

OTHER - HORMONE TESTING:
The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined. The Mouse/Rat Thyroxine (T4) ELISA kit produced by Calbiotech were used.
Blood samples were collected from:
- two pups per litter on day 4 after birth,
- two pups per litter on day 13 after birth,
Blood samples from 13-day pups were measured for TT4. There was no statistically significant changes in the hormone level, absolute/relative weight of the thyroid and histopathological changes of the thyroid connected with the test item, therefore further measurements of TT4 (4 – days pups) were not made.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after at least 28 days of administration including pre-mating and mating periods
- Maternal animals: All surviving animals after about 51- 59 days of administration including pre-mating, mating, gestation, and 13 days of lactation.
For post-mortem examinations all animals were euthanized with a mixture of xylazine and ketamine at the doses of 20 mg xylazine/kg b.w. and 200 mg ketamine/kg b.w.

GROSS NECROPSY
- Gross necropsy consisted of detailed observations of external body surface, all natural apertures, the cranial, thoracic and abdominal cavities with all organs and their contents. During the necropsy in all parental females the numbers of corpora lutea (separately in the left and the right ovary) and implantation sites (separately in the left and the right horn of the uterus) were determined. Special attention was paid to the organs of the reproductive system in all adult animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively.
Weights of organs of the animals used in the study were measured. Absolute and relative weights of brain, pituitary gland, thymus, thyroid gland, heart, liver, spleen, kidneys and adrenals from all adult rats from each group were recorded. Additionally, absolute and relative weights of testes, epididymides, levator ani, bulbocavernosus muscle complex, Cowper’s glands and glans penis, prostate with seminal vesicles and coagulating glands of all adult males as well as ovaries and uterus with cervix of all adult females were determined. Relative weights of internal organs were calculated on the basis of absolute weights, with reference to body weights of living animals.

The following organs and tissues of animals were collected for histopathological examinations from all adult males and females (groups 0, 1, 2 ,3, 0 SAT and 3 SAT): all gross lesions, brain (cerebrum, cerebellum and pons), spinal cord, eye with optic nerve, pituitary gland, thyroid with parathyroids, stomach, small and large intestines including Peyer’s patches (duodenum, jejunum, ileum, cecum, colon), pancreas, liver, kidneys, ureters, adrenals, spleen, heart, aorta, thymus, trachea, lungs (preserved by inflation with fixative and then immersion), testes, ovaries with oviducts, uterus with cervix, epididymides, accessory sex organs (prostate, seminal vesicles and coagulating glands), vagina, skin, mammary gland, urinary bladder, lymph nodes, skeletal muscle with tibial nerve and femur with knee joint. Organs with gross lesions i.e. lesion in liver (7mm long, yellowish, grainy, on the visceral side of the left lobe) of the female no. 46, group 0 SAT; lesion in subcutaneous tissue of cervical area above the salivary gland (yellowish, elastic, in the size of 5 x 2 mm) of the male no. 31, group 3 SAT; lesion in fat tissue in left ovary area (yellowish, 5 mm long) of the female no. 41, group 3 SAT, lesion located in fat tissue in pelvis cavity inlet area (yellowish, 5 mm long) of the female no. 46, group 3 SAT were also collected for evaluation.
As for the gross lesions, thyroid, testicles, epididymides and prostate with accessory sex organs i.e. vesicular and coagulating glands as well as the ovaries with oviducts, they were examined in adult animals of all experimental groups (0, 0 SAT, 1, 2, 3, 3 SAT).
As for the organs and tissues of the reproductive system, despite of routinely proceeded evaluation, detailed histopathological evaluation of the testicles and ovaries for the prospective gametogenesis disorders were carried out. In the testicles spermatogonia, spermatocytes and spermatides were evaluated as well as the accuracy of the particular layers of the sex epithelium structure. The lumen of the seminiferous tubules was examined to exclude the presence of the immature forms of spermatogenetic cells or giant cells. In epididymides the presence of sperm content was evaluated. In the ovaries there were ovarian follicles at various stages of maturation and corpora lutea organisation evaluated. During the histopathological examination special attention was paid to the tissues and organs which could have been the indicators of endocrine disruption influenced by the test item (thyroid, pituitary, adrenals, mammary gland and organs of the reproductive system of tested animals).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring was subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Dead pups and the ones euthanized on day 4 and 13 post-partum were carefully examined for gross abnormalities.

GROSS NECROPSY
- Gross necropsy consisted of the examinations involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with all organs and their content. Particular attention in pups was paid to the external reproductive genitals which were examined for signs of altered development.


HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid gland was collected from 1 male and 1 female from each litter in the control and treated groups for histopathological examination, there was an exception, because of insufficient number of live males and females per litter thyroid gland was collected from 2 females (litter from female no.13 of group 0)
Statistics:
The treated groups, i.e. groups 1, 2, and 3 were compared to the control group. The treated satellite group, i.e. 3 SAT was compared to group 0 SAT. Each sex was analyzed separately.
The clinical results were statistically analyzed using the one-way analysis of variance and the Dunnet’s test (group 0, 1, 2 and 3) and Student’s t-test (group 0 SAT, 3 SAT), (p ≤ 0.05).

Clinical-chemical examinations results: The results obtained in the groups 0, 1, 2 and 3 were statistically analysed using the one-way analysis of variance or Kruskal-Wallis test (in the absence of normal distribution or heterogeneous variances) and then Dunnet’s test (p ≤ 0.05). The results of the clinical-chemical examinations in satellite group were statistically analyzed using Student’s t test (p ≤ 0.05) or U Mann-Whitney test (in the absence of normal distribution).
Some results of the urinalysis such as bilirubin and urine sediment were statistically analyzed using nonparametric statistics test - Kruskal-Wallis and then Dunnet’s test (p ≤ 0.05) (group 0, 1, 2, 3) and U Mann-Whitney test (group 0 SAT, 3 SAT), (p ≤ 0.05).

Post-mortem examinations results
The results, i.e. absolute and relative weights of internal organs as well as the numbers of implantation sites and corpora lutea were statistically evaluated using Dunnett’s test for groups 0, 1, 2, 3 and student’s T-test for satellite groups 0 SAT and 3 SAT (at p≤0.05).

Statistical analyses were conducted using the Microsoft Excel 2010 and the Statistica 10 programmes.
Reproductive indices:
Mating index for males, mating index for females, fertility index for males, fertility index for females, pregnancy index, the average number of days required for copulation, the length of gestation.
Offspring viability indices:
Index of live births, index of 4-day survival, index for 13-day survival,
Clinical signs:
no effects observed
Description (incidence and severity):
There were no differences in physical appearance and behaviour between the treated and the control groups. The animals didn’t show any clinical signs during the entire experiment. The only exception was the thinning of hair all over the body in one female from group 1 but this change is not considered to be treatment-related.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
During the experiment, no mortality of adults animal was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in body weights between parental animals from treated and control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences in food intake by males and also by females from the treated (1, 2, 3, 3 SAT) and the control groups (0, 0 SAT).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmic examinations did not reveal any pathological changes.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological examinations: In case of peripheral blood examinations of animals from groups 1, 2 and 3, statistically significant changes in females of group 1 were stated (statistically significant: decrease in the hemoglobin concentration, decrease in the hematocrit value and decrease in the number of erythrocytes). Mentioned above changes were considered as accidental due to no confirmation in higher dosage i.e. group 2 and 3. There were no changes in males of group 1, 2, and 3. In case of peripheral blood examinations of males and females from group 3 SAT, statistically significant increase in the number of thrombocytes was stated compared to the control group (0 SAT). However, number of thrombocytes in group 3 SAT compared to the group 0 was at a similar level, so this change should not be connected with the test item administration. Statistically significant increase in the MCHC value in females of group 3 SAT was considered as accidental because this change was isolated, and no other deviations in red blood cells parameters were stated. In bone marrow examinnations, statistically significant decrease in the number of proerythroblasts was stated in males of group 3 SAT. This change was regarded as random because there were no changes on the next stages of erythroblast development. Statistically significant decrease in the number of filamented eosinophils (bone marrow examination) in males of group 3SAT was also considered as accidental.

Coaglulological examinations: The treatment with the test item at three different dose levels did not affected coagulological parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical examinations: In case of biochemical parameters, statistically significant increase in the chloride concentration in males of group 1, 2 and 3 were observed. Increase in chloride concentration was small, by about 2% compared to the control group and did not increase with increasing dose. For this reason statistically significant change which was observed may be considered as not treatmentrelated. These change was not observed in the satellite group, after 14-days post-treatment. In males of group 3 stattistically significant increase in the calcium concentration was stated, it can be also considered as random due to low intensity. Statistically significant increase in the creatinine concentration was stated in females of group 3. This change was considered as accidental because the histopathological examination of the kidneys revealed no changes considered as treatment related. Statistically significant increase in the total protein concentration and decrease in the glucose concentration was stated in males of group 3 SAT compared to the control group (0 SAT) and those changes could be random. In case of females of group 3 SAT, statistically significant decrease in the urea nitrogen concentration and increase in the sodium concentration were stated and those changes were considered as accidental due to change in sodium was small (increase by about 0.9% compared to the group 0 SAT) and change in urea nitrogen is in the range of variability of the control group.

Enzymatic examinations: All changes observed in the males and females of group 1 and 2 (statistically significant decrease in the AP activity in males of group 1 and 2 and statistically significant decrease in the AST activity in females of group 2) were considered as random due to no confirmation in the higher dosage group. Statistically significant decrease in AST activity in males of group 3 SAT was consider as accidental, because in case of toxic impact on the liver function, an increase in activity of AST is observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in the protein level was stated in females of group 1, 2 and 3 but this is not an adverse change so it will not be taken into account. Statistically significant increase in the level of urine protein in males of group 3SAT was stated and this change was considered as accidental.
Statistically significant decrease in the level of urine bacteria in males of group 3SAT is not an adverse change so it will not be taken into account.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The behavioural studies showed that the nervous system functioned properly at dose levels of 33.3, 100, and 300 mg/kg b.w./day.
The detailed clinical observations made during and after the treatment, and the open field observations did not reveal any muscarinic symptoms (weakness, lacrimation, salivation, and anxiety), nicotinic symptoms (diarrhea and motor coordination difficulties), or central symptoms caused by the test item. Delayed neurotoxicity, i.e. of paralysis of the hindlimbs, clumsiness, sensory disturbances, and general paralysis were not observed. During the open field observations, a few cases of increased and decreased arousal were found. Changes in arousal was observed both in treated and control groups so it should not be connected with the administered material.
A statistically significant increase of horizontal locomotor activity was noticed in males and females from group 2 compared to the control group. This change may be considered as accidental because there was not appear in other treated groups. There were statistically significant higher average of the number of urine pools left by males from group 3 SAT compared to control group (0 SAT) at the end of the 14-day period of additional observation (measurement 2). However, no similar changes were found in males from other treated groups and also in males from group 3 SAT at the end of the treatment (measurement 1), so it can be assumed as fortuitous change and not related to the administration of the test item. It was also discovered that the test item did not affect responses to stimuli. The latency of pain responses in females in the satellite group 3 SAT at the end of the treatment (measurement 1) was significantly shorter compared to the group 0 SAT. However, similar changes were not observed in females in group 3 (measurements of females from group 3 and 3 SAT was conducted at the same stage of the experiment - at the end of the treatment). Hence, it can be concluded that this change was accidental.

The administration of the test item did not influence fore- and hindlimb grip strength in the treated groups. Only exception was statistically significant increase of forelimb grip strength observed in females from group 3 SAT at the end of the additional observation period (measurement 2) compared to the group 0
SAT. It can be assumed that this is an accidental change.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant changes considered as test item related were found in the results of clinical-chamical examinations therefore it can be stated that the test item did not affect the immune system. The gross and histopathological examination did not reveal any harmful effects of the test item on the immune system. Although cases of histopathological lesions in the immune system were observed, they should not be linked as the effect of the action of the test item since they were con sider accidental and / or occurred in both, the treated and the control animals. There were also stated no incidence of overt infections, and occurrence of significant tumors in the treated rats. It was concluded that the test item is not immunotoxic for rats. The observed changes are the following: Statistically significant change was stated in the creatinine concentration in females of group 3. The macroscopic examination of the thymus, spleen, and lymph nodes did not reveal any pathological changes in any group with the exception of ecchymosis of thymus, petechiae on thymus and decrea sed of thymus. This changes occurred in a few animals both in control and treated groups. Hence, it was defined as accidental and should not be associated with the action of the test item. Statistically significant differences, i.e. decrease in absolute weights of spleen in males from group 3 and decrease in relative weights of spleen in males from groups 1, 2 and 3. There were no other statistically significant changes in absolute and relative weights of thymus, spleen, and lymph nodes of males and females in all treated groups.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The statistical analysis of absolute and relative weights of organs showed statistically significant changes in weight of the pituitary of males (absolute and relative in the group 3) and females (absolute in the groups: 2 and 3), the thyroid of males (relative in the group 3), the spleen of males (absolute in the group 3 and relative in the groups: 1, 2 and 3), the kidneys of females (relative in the group 3 SAT). In connection with the absence of compatibility of occurrence of these changes between both sexes and / or the lack of occurrence of significant histopathological lesions in these organs the events of the organ weight disorders seem to be accidental.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Regarding the gross lesions stated in the treated rats, the majority of them was localized in the lungs. Macroscopically, they were particularly noticeable as the differences of a size (thymus, lungs, heart, uterus, kidneys, spleen), color (thymus, lungs, heart, uterus, ovaries, mandibular lymph nodes, intestines), consistence (lungs, uterus), and content (cranial cavity, trachea, uterus).
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
On the basis of the results of the histopathological examination of organs and tissues it may be concluded that all of the revealed lesions were not associated with the test item.
Histopathological, there were observed cases of congestion (thymus, heart, lungs, liver, spleen, mandibular and mesenteric lymph nodes, glomeruli of kidneys, islets of pancreas, prostate, uterus, ovaries), erythrocytorrhagia (heart, lungs, thymus, kidneys, mandibular lymph nodes), edema (lungs), effusion (thymus, liver, mandibular lymph nodes, uterus), inflammatory lesions such as lymphocyte infiltration (lungs, colon, kidneys, prostate, urinary bladder), foamy cells infiltrations (lungs), leukocyte infiltrations (heart), lymphatic follicle reactive hyperplasia (cecum, colon), regressive lesions such as lymphocytic tissue depletion (thymus), emphysema (lungs), foci of fibrosis (heart), foci of atrophy of lobules with fibrosis (pancreas), left gland atrophy with medulla microcyst (adrenals), glomeruli atrophy, Bowman’s capsule extension and retention microcysts (kidneys), hemosiderin deposits in spleen and progressive lesions such as mesenteric lymph nodes hyperplasia, left ventricular myocardium hypertrophy (heart), islets of Langerhans hypertrophy and ducts proliferation (pancreas), glomeruli hypertrophy (kidneys).
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were also observed individual cases of small benign tumors such as lipomas of the adipose tissue and fibroids of the uterus. In the subcutaneous tissue of region of cervical area above the salivary gland (male in the group 3 SAT) and region of the abdominopelvic cavity (two females in the group 3 SAT) the small lipomas were occurred. As for the uterus (two females in the group 1 and one female in the group 2) the fibroids were observed. The occurrence of them was clearly incidental to the treatment and should not be linked with the test item.
Both, the lipomas and fibroids were considered as spontaneous lesions, consistent with background findings commonly observed in rats. The occurrence of the left adrenal gland atrophy with medulla microcyst and right adrenal gland hyperplasia in one of the females in the group 3 is also accidental. It seems to be a kind of a malformation.
Other effects:
no effects observed
Description (incidence and severity):
Hormonal examinations: Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in all males were not observed, so the determination of TT4 levels were not extended to females.
There were found no adverse effects of the test item on an endocrine system. There were no significant histopathological lesions in the thyroid of adults in all experimental group of animals.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The detailed histopathological examination of the ovaries has proven that the test item did not affect the gametogenesis process in animals exposed at all dose levels. No lesions or disorders in the gonads (ovaries) were observed. One of the females from the group 2 was non-pregnant (no corpora lutea in ovaries and no implantation sites in uterus were found)
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The detailed histopathological examination of testicles and epididymides has proven that the test item did not affect the gametogenesis process in animals exposed at all dose levels. No lesions or disorders in the gonads (ovaries) were observed. As for the testicles particular layers of the sex epithelium were correct. In the epididymides a sperm reserve content was preserved. There were also no pathological lesions in the seminal vesicles and coagulating glands. Cases of lymphocyte infiltration in the prostate in males exposed at all dose levels and the control were observed. These changes can be considered as the background lesions, not associated with the test item.
Reproductive performance:
no effects observed
Description (incidence and severity):
The indices relating to fertility of the parental animals, including mating indices for males and females, fertility indices for males and females, and pregnancy indices in the treated and the control groups were similar, which suggests that the test item at the dose levels of 33.3, 100, and 300 mg/kg b.w./day did not affect fertility of the parental rats.
However, one female (no. 11 from group 2) was not pregnant, although sperm was found. Histopatological examination revealed no corpora lutea in ovaries and no implantation sites. Pathomorphological (gross and histopathological examinations) results of the male which mounted the non-pregnant female were correct, therefore it is difficult to identify the reason for the female not being pregnant. Two females in control group were also not pregnant (although sperm was found), so the lack of pregnancy in one treated female from group 2 can not be associated with the test item.
The test item had no impact on numbers of corpora lutea in the ovaries and implantation sites in the uterus.
As for the adrenals, pituitary, mammary gland, gonads (ovaries and testes), accessory sex organs (uterus with the cervix, vagina, epididymides, seminal vesicles with coagulating glands, and prostate) there were also found no adverse effects of the test item.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item adverse effects observed at the highest dose tested.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hematoma on the snout in one pup from group 0 and hematoma on the head in one pup from group 2 were transient. In a few pups thinning of hair all over the body, thinning of hair on the back and smaller body were also noticed. These changes occurred both in treated and control groups. Hence, they should not be associated with the administered test item. Hematomas in pups after birth were also observed in pups, including control ones in other combined repeated dose toxicity studies with the reproduction/developmental toxicity screening test performed at the same laboratory [OECD 422; unpublished reports].
The analysis of the AGD and number of nipples/areolae showed that the test item did not cause any significant changes in development of pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
There were 541 pups in the study, whereof: 34 pups were born dead, 29 animals were eaten by mother, 4 pups was found dead at the first day of life. The remaining animals survived the 13-day observation period. The test item did not influence the total numbers of pups/litters, the numbers of live births and the percentages of males and females in litters in group 1, 2 and 3. number of stillbirths in group 1 (2.8% of total number of pups in group), group 2 (11.8% of total number of pups in group) and group 3 (8.8% of total number of pups in group) were higher compared to the control group (0.9% of total number of pups in group). Moreover, mortality (number of death pups during the study period) was also higher in treated groups compared to the control group. In group 1 it was 2.8%, in group 2 - 15.0% and in group 3 - 5.6% of total number of pups in group, while in control group it was 0.9% of total number of pups in group. Based on this results, it may be concluded that the test item in group 2 and 3 affected pups survival.
The analysis of the indices relating to pups survival from the treated parental animals (indices of live births, 4-day survival and 13-day survival of offspring) also confirmed a negative influence of the test item in group 2 and 3.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight determinations: It may be concluded that the test item at the dose levels of 33.3, 100 and 300 mg/kg b.w./day (males and females) had no adverse effects.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food intake measurements: It may be concluded that the test item at the dose levels of 33.3, 100 and 300 mg/kg b.w./day (males and females) had no adverse effects.
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:
no effects observed
Description (incidence and severity):
Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in 13 days pups were not observed, so the determination of TT4 levels were not extended to 4-days pups.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
no effects observed
Description (incidence and severity):
Histopathological examination of the thyroid of the 13-day pups revealed no lesions.
Other effects:
no effects observed
Description (incidence and severity):
Hormonal examinations: Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in 13 days pups were not observed, so the determination of TT4 levels were not extended to 4-days pups. Histopathological examination of the thyroid of the 13-day pups revealed no lesions.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 33.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Table 5: Main study. Clinical signs - adult animals

Clinical signs

Group / sex / number of animals / computer numbers of animals

0

1

2

3

males

females

males

females

males

females

males

females

Test animals

10

12

10

12

10

12

10

12

Animals with clinical signs

0

0

0

1

0

0

0

0

Animals with ophthalmic

changes

0

0

0

0

0

0

0

0

Death of animals

-

-

-

-

-

-

-

-

SKIN AND COAT

Thinning of hair all over the body

-

-

-

1 [16]

-

-

-

-

[ ] computer numbers of animals

Table 6- VL3. Main study. Clinical signs - offsring

Clinical signs

Group / sex / number of animals / computer numbers of animals

0

1

2

3

males

females

males

females

males

females

males

females

Number of live births pups

52

57

64

76

56

56

88

58

Number of pups with clinical

signs

9

10

0

0

5

7

1

0

Hematoma on the snout

1 [13]1

-

-

-

-

-

-

-

Hematoma on the head

-

-

-

-

1 [20]1

-

-

-

Thinning of hair all over the

body

2 [15]

9 [15]

-

-

4 [15]

4 [15]

-

-

Thinning of hair on the back

6 [21]

1 [21]

-

-

-

-

-

-

Smaller body

-

-

-

-

-

1 [15]

1 [20]

1 [21]1

1 [21]

 

-

Stillbirth

-

1 [13]

-

1 [11]

3 [20]

1 [12]

3 [17]

2 [19]

1 [17]

4 [19]

1 [20]

4 [22]

1 [13]

1 [19]

4 [22]

Death of animal

1 [13]2

-

2 [20]3

2 [20]3

4 [16]3

3 [17]3

1 [18]3

1 [19]3

1 [16]2

2 [16]3

6 [17]3

1 [18]3

1 [12]3

2 [18]3

1 [19]3

1 [22]3

1 [14]3

2 [22]2

1 [22]3

Undefined sex stillbirth*

-

-

3 [19]

1[13]

3[22]

[ ] – computer numbers of mothers of animals in which clinical signs were observed

1 – temporary change 2 – animal died at the first day of life 3 – pups eaten by mother * - pups sex could not be identified because pup were bitten by mother.

Table 7. VL3. Main study. Open field observations – females

Parameter

Group

0

n = 5

1

n = 5

2

n = 5

3

n = 5

involuntary clonic

movement

no changes

no changes

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

no changes

no changes

gait

no changes

no changes

no changes

no changes

arousal

no changes

no changes

[12]4, [15]4, [16]4

[11]1, [12]4, [14]4

stereotypies

no changes

no changes

no changes

no changes

number of defecations

2.80±2.17

1.80±2.49

2.20±2.05

1.00±1.73

number of

urinations

2.60±1.52

4.20±0.84

4.00±1.73

2.20±1.64

distance 0-3 min [m]

109.44±18.48

106.48±12.37

162.06±40.87*

125.40±39.00

number of rearings

0-3 min

13.60±4.22

12.80±2.77

18.60±8.20

19.20±7.09

x± SD

nnumber of test animals

*-statistically significant difference at p ≤ 0.05-Dunnett’s test

[ ] - computer numbers of animals with changes

Classification of changes:

1 - slight decrease (a rat sometimes does not move, but exploratory activity is observed)

2 - moderate decrease (a rat is slightly numb; only the head moves)

3 - strong decrease (paralysis, coma)

4 - slight increase (a rat is slightly excited or tense; it suddenly moves forward and stays without motion)

5 - strong increase (a rat is very vigilant; it runs and suddenly moves from its place

Table 8. VL3. Main study. Mating of parental animals – list of results

Parameter

Group

0

1

2

3

Number of males to

mating

10

10

10

10

Number of females to

mating

12

12

12

12

Number of males which

copulated

10

10

10

10

Number of females

which were mated

12

12

12

12

Average number of

days to copulation

1.0

1.2

1.0

1.1

Number of fertilized

females

12

12

12

12

Number of pregnant

females

10

12

11

12

Number of females

which delivered

10

12

11

12

Number of not

pregnant females

2

0

1

0

Number of females

which delivered

offspring

10

12

11

12

Number of females

which delivered live

offspring

10

12

11

12

Table 9. VL3. Main study. Indices concerning fertility of parental animals

Parameter

Group

0

1

2

3

Mating index for males

100.0

100.0

100.0

100.0

Mating index for females

100.0

100.0

100.0

100.0

Fertility index for males

90.0

100.0

100.0

100.0

Fertility index for females

83.3

100.0

91.7

100.0

Pregnancy index

100.0

100.0

100.0

100.0

Length of gestation

22.5 ± 0.5

22.5 ± 0.5

22.5 ± 0.7

22.5 ± 0.7

Table 10. VL3. Main study. Average number of pups in litter

Parameter

Group

0

1

2

3

Total number of pups

in litter

11.00 ± 2.75

12.00 ± 3.05

11.55 ± 2.50

13.33 ± 2.90

Number of live births

in litter

10.90 ± 3.03

11.67 ± 3.03

10.18 ± 3.22

12.17 ± 4.22

Number of pups in

litter, min.max.

4 - 13

4 - 17

7 - 15

7 - 16

Percentage of males

in litter

47.27

44.44

50.00

58.97

Percentage of

females in litter

52.73

55.56

50.00

41.03

Table 11. VL3. Main study. Indices connected with survival of offspring

Parameter

Group

0

1

2

3

Index of live

births

99.09

97.22

88.19

91.25

Index of 4-day

Survival

99.08

97.14

83.04

93.84

Index of 13-day

survival

98.90

96.61

80.21

92.74

Mortality [%]

0.9

2.8

15.0

5.6

Table 12. VL3. Main study. Average AGD [mm] in male pups in litter on 4th day of life 

Day 4

Group

0

n= 10

1

n = 12

2

 n = 11

3

n = 12

Male

5.93±0.38

5.90±0.30

5.78±0.29

5.83±0.51

Female

3.92±0.32

3.90±0.30

3.76±0.93

3.74±0.22

x± SD

n - number of litters in group

 

Table 13. VL3. Main study. Average number of nipples / areolae in male pups in litter on 13th day of

Life

Day

Group

0

n= 10

1

n = 12

2

 n = 11

3

n = 12

13

0.0±0.0

0.0±0.0

0.0±0.0

0.0±0.0

Table 14. VL3. Main study. Results of hormonal examination - pups 13 day after birth

Parameter

MALES

GROUP

0

1

 

 

2

3

n=9

n=12

 

 

n=9

n=11

TT4

5.31 ± 0.73

5.54 ± 0.91

5.36 ±0.79

5.200.93

 

FEMALES

GROUP

0

1

 

2

3

n=11

n=12

 

n=9

n=10

TT4

5.40 ±0.90

5.74 ± 0.87

5.37 ±0.72

5.030.51

Table15. VL3. Main study. Offspring subjected to pathomorphological examination.

Group

Sex / number of animals / computer numbers of parental females

Males

Females

Undefined sex

Number of animals per group

Dead

animals

Stillbirth

Euthanized animals at day 4

Euthanized animals at day 13

Number of animals per group

Dead

animals

Stillbirth

Euthanized animals at day 4

Euthanized animals at day 13

Number

of

animals

Dead

animals

Stillbirth

0

52

1

 

8

43

58

 

1

10

47

 

 

 

 

 

[13]

 

 

 

 

 

[13]

 

 

 

 

 

 

 

 

 

 

 

 

 

1[11]

 

 

 

 

 

1

62

-

-

11

51

78

-

3[20]

11

63

-

-

-

 

 

 

1[12]

 

 

 

 

1[17]

 

 

 

 

 

2

53

-

3[17]

8

39

53

1[16]

4[19]

8

38

3

-

3[19]

 

 

 

2[19]

 

 

 

 

1[20]

 

 

 

 

 

 

 

 

 

 

 

 

 

1[13]

 

 

 

 

 

3

87

-

4[22]

10

73

62

2[22]

1[19]

12

42

4

-

1[13]

 

 

 

 

 

 

 

 

4[22]

 

 

 

 

3[22]

[]-computer numbers of parental females

 

Table 16. VL3. Main study. Gross lesions: euthanized, dead and stillborn pups.

Gross lesions

Group / number of animals / computer numbers of parental females

0

1

2

3

Animals euthanized at day 4

18

22

16

22

Animals with gross lesions

-

-

-

-

Animals euthanized at day 13

90

114

77

115

Animals with gross lesions

-

-

-

-

Dead animals

1

-

1

2

Partial autolysis of internal organs

-

-

-

2 [22]

Autolysis of internal organs

-

-

1[16]

-

Partially eaten by mother

1[13]

-

-

-

Stillbirth

1

4

15

14

Partial autolysis of internal organs

1[13]

3[20]

4[17]

8[22]

Autolysis of internal organs

-

-

6[19]

1[13]

Partially eaten by mother

-

-

3[19]

1[13]

Partial autolysis of internal organs, partially eaten by mother

-

-

-

3[22]

No lesions

-

1[11]

1[12], 1[20]

1[19]

 

Table 17. VL3. Main study. Number of corpora lutea and implantation sites

 

Group

Number of

0

1

2

3

 

n=10*

n=12

n=11*

n=12

Corpa lutea

15.200          2.530

16.500          2.541

20.000          7.266

17.334                  3.576

Implantation sites

13.200          2.936

13.417          2.644

14.273                 1.348

14.667                  1.723

xSD

n - number of tested animal

*number of tested animals excluding non-pregnant female

Table 18. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Pituitary

microcyst

2

[1,4]

-

-

-

-

-

-

Thyroid

congestion

-

-

-

-

-

-

1

[9]

parathyroid

hypertrophy

-

1

P2]

-

-

-

-

-

 

congestion

1

[2]

-

-

1

[18]

-

-

2

[3,9]

Thymus

erythrocytorrhagia

-

-

-

-

-

-

1

[3]

effusion

-

1

[22]

-

1

[17]

 

 

-

 

lymphocytic tissue depletion

-

1

[17]

-

-

-

1

[13]

-

 

erythrocytorrhagia

3

[1,2,10]

-

-

1

[18]

-

-

1

[8]

 

leukocyte infiltration

1

[10]

-

-

 

-

-

1

[10]

 

Heart

foci of fibrosis

1

[10]

-

-

-

-

-

-

 

foci of fatty degeneration

1

[10]

-

-

 

-

-

-

 

left ventricular myocardium hypertrophy

-

-

-

1

[18]

-

1

[18]

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Heart

left and right ventricular myocardium hypertrophy

1

[10]

-

-

-

-

-

-

 

congestion

1

[6]

-

3

[3,6,9]

2

[19,20]

1

[2]

2

[14,22]

2

[1,5]

2

[11,13]

 

erythrocytorrhagia

5

P6,8,9,10]

9

[12,13,14,

15,16,18,

20,21,22]

4

[3,6,7,9]

10

[11,14,15,

16,17,18,

19,20,21,

22]

3

P,3,4]

9

[13,14,15,

16,17,19,

20,21,22]

6

[1,2,4,5,7,

10]

8

[11,12,13,

14,15,17,

18,22]

 

edema

3

P6,10]

4

[13,14,18,

21]

3

[3,6,9]

2

[11,17]

-

2

[17,22]

2

[5,10]

1

[18]

Lungs

emphysema

4

[2,4,8,10]

9

[11,13,14,

15,17,19,

20,21,22]

-

10

[11,12,14,

15,16,18,

19,20,21,

22]

1

[2]

3

[13,14,21]

5

[1,4,5,6,10]

3

[11,18,22]

 

foamy cell infiltration

2

[7,8]

7

[11,15,17,

18,20,21,

22]

-

3

[11,16,17]

-

4

[13,19,20,

21]

1

[6]

1

[18]

 

lymphocyte

infiltration

1

[8]

-

-

-

-

-

1

[10]

 

leukocyte infiltration

1

[10]

-

-

-

-

-

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

 

congestion

1

[10]

2

[15,17]

-

-

-

-

1

[9]

 

effusion

1

[10]

-

-

-

-

-

-

Liver

bile ducts proliferation

1

[10]

-

-

-

-

-

-

 

Browicz-Kupffer cell proliferation

1

[10]

-

-

-

-

-

-

 

hepatocyte fine- droplet fatty degeneration

1

[3]

-

-

-

-

-

-

Spleen

congestion

-

1

[18]

-

-

-

-

-

depletion of the white pulp

-

2

[17,18]

-

-

-

-

-

Pancreas

islets of Langerhans hypertrophy

1

1

P1]

-

-

-

-

3

[4,6,8]

ducts proliferation

-

1

P1]

-

-

-

-

-

Jejunum

lymphatic follicle reactive hyperplasia

1

[5]

-

-

-

-

-

-

Ileum

lymphocyte

infiltration

-

1

[11]

-

-

-

-

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Cecum

lymphatic follicle reactive hyperplasia

1

[7]

-

-

-

-

-

-

1

[14]

Colon

lymphatic follicle reactive hyperplasia

1

[4]

-

-

-

-

-

2

P,4]

1

[14]

 

erythrocytorrhagia

1

[8]

1

[15]

-

-

-

-

1

[2]

 

glomeruli congestion

6

[1,2,3,5a

7]

4

[12,17,20,

22]

-

-

-

-

10

[1,2,3,4,5,6,7

8,9,10]

6

[13,14,15,

17,20,22]

 

lymphocyte

infiltration

-

2

[12,21]

-

-

-

-

1

[8]

Kidneys

glomeruli

hypertrophy

9

[1,2,3,5,6,7,8

,9,10]

7

[12,15,17,

19,20,21,

22]

-

-

-

-

9

[1,3,4,5,6,7,8

9,10]

9

[12,13,14,

15,17,19,

20,21,22]

glomeruli

atrophy

4

[1,8,9,10]

5

[15,17,18,

20,21]

-

-

-

-

10

[1,2,3,4,5,6,7

8,9,10]

8

[12,13,14,

15,19,20,

21,22]

 

Bowman's capsule extension

1

[9]

2

[18,21]

-

-

-

-

6

[2,3,5,6,8,9]

 

retention microcysts

-

2

[12,15]

-

-

-

-

1

[8]

2

[14,21]

 

hyaline casts

-

1

[12]

-

-

-

-

-

 

 Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0f

1m

2m

3f

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Adrenals

left adrenal gland atrophy with medulla microcyst; right adrenal gland hyperplasia

-

-

-

-

-

-

-

1

[11]

Urinary bladder

lymphocyte

infiltration

-

-

-

-

-

-

5

[1,3,5,6,7]

Prostate

congestion

2

[1,9]

 

-

-

-

-

2

[2,8]

 

edema

2

[1,9]

-

-

-

-

-

-

lymphocyte

infiltration

5

[1,3,7,9,10]

-

3

P,4,9]

-

6

[2,4,5,7,8,9]

-

6

[2,3,5,8,9,

10]

Uterus

congestion

-

-

-

1

[16]

-

2

[16,20]

-

effusion

-

-

-

2

[12,16]

-

2

[20,21]

-

fibroid

-

-

-

2

[12,16]

-

1

[20]

-

Mandibular lymph nodes

congestion

1

[6]

-

-

-

-

-

-

hyperplasia

1

[6]

-

-

-

-

-

-

lymphocytic tissue depletion

-

1

[17]

-

-

-

-

-

 

Table 18 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0f

1m

2m

3f

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Mesenteric lymph nodes

congestion

-

-

-

-

-

-

1

[6]

hyperplasia

1

[6]

-

-

-

-

-

1

[6]

lymphocytic tissue depletion

-

1

[17]

-

-

-

-

-

[]-computer numbers of animals in which the histopathological lesions were found n - number of tested animals f - full histopathology

m - histopathology of thyroid, testicles, ovaries with oviducts, epididymides, prostate, seminal vesicles, coagulating glands and gross lesions were conducted

Table 19. VL3. Main study. Histopathological lesions: thyroid of 13-day offspring.

Histopathological examinations of thyroid

Group / sex / number of animals / computer numbers of parental females

0

1

2

3

males

females

males

females

males

females

males

females

13-day offspring

9

11

12

12

9

9

11

11

Animals with histopathological lesions

-

-

-

-

-

-

-

-

 

Table 20. VL3. List of effects on reproduction/development

OBSERVATIONS

VALUES

Dosage (mg/kg b.w./day)

0

33.3

100

300

Pairs started (N)

12

12

12

12

Ostreus cycle (at least mean length and frequency of irregular cycles)

4.1

0/12

(0%)

4.0

0/12

(0%)

4.0

0/12

(0%)

4.0

0/12

(0%)

Females showing evidence of copulation (N)

12

12

12

12

Females achieving pregnancy (N)

10

12

11

12

Conceiving days 1-5 (N)

10

12

11

12

Conceiving days 6 -N)

0

0

0

0

Pregnancy < 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

5

6

6

7

Pregnancy > 23 days (N)

5

6

5

5

Dams with live young born (N)

9 + 1*

10 + 2*

7 + 4*

9 + 3*

Dams with live young at day 4 pp (N)

9 + 1**

11+1**

9 + 2**

7 + 5**

Implants/dam (mean)

13.2

13.4

14.3

14.7

Live pups/dam at birth (mean)

10.9

11.7

10.2

12.2

Live pups/dam at day 4 (mean)

10.8

11.3

8.5

11.4

Sex ratio (m/f) at birth (mean)

1.1

0.9

1.1

1.5

Sex ratio (m/f) at day 4 (mean)

1.1

1.1

1.0

1.5

Litter weight at birth (mean) [g]

66.27

70.63

60.65

69.90

Litter weight at day 4 (mean) [g]

116.02

118.93

102.61

127.49

Pup weight at birth (mean) [g]

6.14

6.15

5.96

5.70

Pup weight at the time of AGD measurement (mean males, mean females, PND 4)

(M) 10.93

(F) 10.54

(M) 10.86

(F) 10.65

(M) 10.28

(F) 9.86

(M) 10.81

(F) 10.35

Pup AGD on the same postnatal day, birth­day 4 (mean males, mean females, PND

4)

(M) 5.93

(F) 3.92

(M) 5.90

(F) 3.90

(M) 5.78

(F) 3.76

(M) 5.83

(F) 3.74

Pup weight at day 4 (mean) [g]

10.74

10.75

10.07

10.58

Pup weight at day 13 (mean) [g]

28.51

28.61

28.57

28.20

Male pup nipple retention at day 13 (mean)

0.00

0.00

0.00

0.00

ABNORMAL PUPS

Dams with 0

7

12

7

11

Dams with 1

1

0

1

1

Dams with >2

2

0

3

0

LOSS OF OFFSPRING

 

Pre-natal (implantations minus births)

 

Females with 0

2

2

1

5

Females with 1

3

3

2

2

Females with 2

0

4

3

0

Females with > 3

5

3

5

5

Post-natal (live births minus alive at postnatal day13)***

 

 

 

 

Females with 0

9

11

7

7

Females with 1

1

0

1

3

Females with 2

0

0

1

1

Females with > 3

0

1

2

1

(1) last day of the mating period * number of dams with at least 1 dead young born ** number of dams with at least 1 dead young to day 4

*** pups taken at day 4 after birth to blood examination, were not taken into account

(M) male (F) female

Conclusions:
Based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.
Executive summary:

A Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats according to the OECD Guideline No. 422 (2016) with GLPs. The study involved the determination of the effects of the test item on body weights, food intake, behaviour, clinical, haematological, coagulation, biochemical, enzymatic, urine, and hormonal parameters and the evaluation of gross and histopathological lesions in tissues and internal organs. Two dose range finding tests and the main study were conducted. The first Dose range finding stated that 7-day exposure of rats to VL3 at the doses of 250, 500 and 1000 mg/kg b.w./ day cause one treatment-related change, i.e. decrease in number of leukocytes (males from group 1 and females from group 1 and 3).The second Dose-range finding test revealed that 7-day exposure of rats to VL3 at the doses of 31.3, 62.5 and 125.0 mg/kg b.w./day did not cause treatment-related changes. The main study was performed on 60 males and 68 females of Wistar rats treated with vehicle control or with 33.3, 100, 300 mg/kg b.w./day of test item. The animals received the test item/vehicle control by gavage once a day (7 days/week). After two weeks of initial treatment, the animals were mated. The test item/vehicle control was administered to males for 28 days (2 weeks prior to mating, during the mating and post-mating period). Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (about 51-59 days, depending on the duration of the mating and pregnancy period). Furthermore, two satellite groups were used;: a control group and one treated group which was given VL3 at the dose of 300 mg/kg b.w/day. The satellite groups were not be mated. Males from groups 0 SAT and 3 SAT were treated for 28 days. Females from groups 0 SAT and 3 SAT were treated for 53 days (on average as long as females from groups 0, 1, 2 and 3). After that, males and females from satellite groups were observed for 14 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects. Offspring was examined for survival, body weights, clinical signs and hormonal parameters. Reproduction parameters such as oestrous cycle, sperm parameters and reproductive organs were also examined on parental animals. During the experiment, no mortality of the adults was observed. There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. No treatment-related clinical signs, ophthalmic alterations, T4 levels altered, or significant differences in body weights and food intake were observed. Statistical analysis of the clinical-chemical examinations results obtained in males and females of group 1, 2 and 3 and satellite groups revealed some statistically significant changes however all of them were considered as not related with the administration of the test item. Fertility parameters on parental animals did not reveal significant changes. There were gross changes observed in control group 0, treated groups 1, 2 and 3 and satellite groups 0 SAT and 3 SAT. However, these changes occurred regardless of the degree of exposure to the test item (this changes were observed both in control and treated groups). Statistical analysis of absolute and relative weights of internal organs revealed statistically significant changes but these organ weight disorders seem to be accidental. The macroscopic and the histological examinations of the rats treated with the test item at the doses of 33.3, 100, and 300 mg/kg b.w./day did not reveal any toxic effects. Additionally, based on results, it was concluded that the test item is not immunotoxic for rats. As for offspring, 34 pups were born dead, the remaining offspring survived the 13-day observation period, except for 4 pups which died at the first day of life. Number of stillbirths in group 2 and 3 were higher compared to the control group. Moreover, mortality was also higher in group 2 and 3 with comparison to the control group. Based on this results, it may be concluded that the test item at dose 100 and 300 mg/kg b.w./day affected pups survival. Nor significant clinical signs were observed among pups neither body weight changes. In conclusion, the treatment of adult (male and female) rats with the test item at the dose levels of 33.3, 100 and 300 mg/kg bw/day by the repeated oral administration, revealed no adverse effects. Based on the results, the no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day. As for pups, based on number of stillbirths and mortality in group 2 and 3, it may be concluded that the test item affected pups survival. Therefore, on the grounds of the used doses, the no observed adverse effect level (NOAEL) for reproduction/developmental toxicity for pups can be determined on 33.3 mg/kg b.w./day.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Stabilisation: in water undergoes hydrolysis
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Analytical purity: 93%
- Expiry date: 06.03.2018

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Cmdb:WI; outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: from the husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok,
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 13-14 weeks old.
- Weight at study initiation: average body weights:
- group 0: males 403.50 g; females 243.75 g
- group 1: males 403.50 g females 243.92 g
- group 2: males 403.40 g; females 243.75 g
- group 3: males 403.50 g; females 243.92 g
- group 0 SAT: males 404.00 g; females 244.90 g
- group 3 SAT: males 404.10 g; females 245.10 g.

- Fasting period before study:
- Housing: Air-conditioned rooms. The animals were kept in plastic cages covered with wire bar lids. The dimensions of the cages were 58 x 37 x 21 cm (length x width x height). During the main experiment, there were 2 or 3 animals/cage. Each sex was kept separately.
- Diet (e.g. ad libitum): ad libitum access,
- Water (e.g. ad libitum): ad libitum access.
- Acclimation period: All animals were quarantined and observed for at least 5 days.

DETAILS OF FOOD AND WATER QUALITY: Food: Altromin 1324 P TPF (Phytoestrogen Deficient, Total Pathogen Free) standard granulated fodder produced by Altromin Spezialfutter GmbH & Co. KG, Lage, Germany, batch number: 1/17 -200418/0453. A certificate of analysis is included in the report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25ºC
- Humidity (%): 43 - 81%
- Air changes (per hr): 13-18 times/h
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: Experimental starting date 13.06.2017 To: Experimental completion date 22.08.2017

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item and the vehicle control were administered to animals once a day for seven days by gavage, in a constant volume of 0.5 mL/100 g b.w. The amounts of the solution of the test item were adjusted to animals’ body weights determined on body weight measurement days.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
During the main study solutions of the test item in a vehicle (corn oil) were prepared once a week. Prepared solutions were stored in a fridge (temperature 4-8 ºC). During administration, solutions were kept at room temperature and they were thoroughly mixed.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): most suitable vehicle, as the test item hydrolyses in water.
- Concentration in vehicle: 6.66 mg VL3/1 mL solution, 20 mg VL3/1 mL solution and 60 mg VL3/1 mL solution
- Amount of vehicle (if gavage): the total volume administered was 0.5mL/100 g b.w. (including test item)
- Lot/batch no. (if required): L5294 (Supplier BASSO FEDELE & FIGLI srl, ITALY)
- Purity: 100 % corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
It was conducted by BLIRT S.A. Laboratory, Trzy Lipy 3/1.38, 80-172 Gdańsk, Poland, which holds the Good Laboratory Practice Certificate GLP. Solutions of the test item in a vehicle (corn oil) were prepared once a week. Two samples (2 x 2 ml) were collected from each batch of prepared solutions. One of them was frozen and sent in a pack of dry ice to BLIRT S.A. (27 samples in 9 batches) for analysis and the other one (spare sample) was archived in the freezer (-15 - -20°C).

TECHNIQUE AND TEST METHOD: Gas chromatography coupled with flame ionization detector (FID).

TEST PARAMETERS: Chromatographic column: ZB-5MS, 30 m x 0.25 mm x 0.25 μm, catalog no.: 7HG-G010-11, serial no.: 221879, Phenomenex.
Temperature gradient: 175ºC (Injection port), 280ºC (detector), 25ºC/min column rate., Column flow: 0.69 mL/min, Carrier gas: helium, Injection volume: 1 μL
Split ratio: 33.3 mg/kg b. w. (6.66 mg/mL of VL3 in corn oil) and 100 mg/kg b. w. (20 mg/mL of VL3 in corn oil): 1:50, 300 mg/kg b. w. (60 mg/mL of VL3 in corn oil): 1:100.

ACCEPTANCE CRITERIA: The relative standard deviation, RSD [%], of the VL3 content for 3 results for 33.3 mg/kg b. w. (6.66 mg/mL of VL3 in corn oil) dose is 6.67 %, so it is ≤ 10.0 %. The relative standard deviation, RSD [%], of the VL3 content for 3 results for 100 mg/kg b. w. (20 mg/mL of VL3 in corn oil) dose is 9.91 %, so it is ≤ 10.0 %. The relative standard deviation, RSD [%], of the VL3 content for 3 results for 300 mg/kg b. w. (60 mg/mL of VL3 in corn oil) dose is 0.48 %, so it is ≤ 10.0 %. The acceptance criteria are fulfilled.

QUANTITATIVE AND QUALITATIVE DETERMINATION: All average VL3 content in study samples were at the expected level.
Duration of treatment / exposure:
The test item/vehicle control was administered to males for 28 days.
Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (51 – 59 days).
Males from groups 0 SAT and 3 SAT were treated for 28 days, females from groups 0 SAT and 3 SAT were treated for 53 days. Post treatment, the satellite groups were observed for 14 days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Only receiving corn oil (vehicle control)
Dose / conc.:
33.3 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Group 0, 1, 2, 3: 10 males, 12 females
Satellite groups: 10 males, 10 females
Control animals:
yes, concurrent vehicle
other: two satellite groups
Details on study design:
- Dose selection rationale:
Two Dose range-finding studies were conducted.
First Dose range-finding study: administered 250, 500 and 1000 mg/kg bw/d (n= 5animals/sex/group) during 7 days. In haematological examinations, statistically significant decrease in the number of leukocytes in males from group 1 and females from group 1 and 3 were stated. Biochemical examinations revealed following statistically significant changes between the treated and the control groups: the decreased total protein concentration in males from group 1, the decreased albumin concentration in males from group 1, the decreased globulin concentration in males from group 1, the decreased calcium concentration in males from group 1, the decreased creatinine concentration in females from group 3.

Based on these results, a second Dose range-finding study was conducted: administered 31.3, 65.5, 125 mg/kg b.w./day for 7 days.It was not observed treatment-related changes.
Based on both dose range finding studies, the doses of 33.3, 100 and 300 mg/kg b.w./day were selected for the main study.

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: control and highest dose group in order to evaluate the reversibility, stability, or delay in the onset of possible harmful effects.
- Post-exposure recovery period in satellite groups: 14 days
Positive control:
Not conducted

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day or once a day (on days off)
- Cage side observations were: General condition of the animals, i.e. the observation of all animals for morbidity and mortality was conducted.

DETAILED CLINICAL OBSERVATIONS: Yes, evaluation of skin, fur, eye and mucosa changes, the respiratory, circulatory, and autonomous and central nervous systems, somatic activity, and behaviour .
- Time schedule: before the experiment and then once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week during the entire experiment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, once a week
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (g/100 g b.w./day)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: the day before the introduction of the animals to the experiment and one day before euthanasia.
- Dose groups that were examined: all adult animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the experiment, blood was collected for clinical-chemical examinations.
- Anaesthetic used for blood collection: Yes, anesthetized with a mixture of xylazine and ketamine at the doses of 10 mg xylazine/kg b.w. and 100 mg ketamine/kg b.w.
- Animals fasted: Yes, prior to the clinical-chemical examinations, the animals were fasted for about 18 – 19 hours. Blood samples were taken from the heart as part of the procedure for euthanasia of the animals.
- How many animals: 5 adult males and 5 adult females from each group (0, 1, 2, 3, 0 SAT, and 3 SAT). Additionally 5 other males (from all groups) and 7 other females (from group 0, 1, 2, 3) or 5 females (from 0 SAT and 3 SAT group) were subjected to hematological (without bone marrow) biochemical and enzymatic examinations.. The level of thyroid hormone, i.e. total T4 (TT4) in serum was determined in all adult males after termination.
- Parameters examined: numbers of leukocytes, erythrocytes, and thrombocytes, the concentration of hemoglobin, hematocrit, MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin weight), and MCHC (mean corpuscular hemoglobin concentration), number of reticulocytes, circulatory blood and bone marrow microscopic examinations, Prothrombin time (PT) AND Partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the experiment, blood was collected for clinical-chemical examinations.
- Animals fasted: Yes, prior to the clinical-chemical examinations, the animals were fasted for about 18 – 19 hours.
- How many animals: 5 adult males and 5 adult females from each group (0, 1, 2, 3, 0 SAT, and 3 SAT). Additionally 5 other males (from all groups) and 7 other females (from group 0, 1, 2, 3) or 5 females (from 0 SAT and 3 SAT group) were subjected to hematological (without bone marrow) biochemical and enzymatic examinations.. The level of thyroid hormone, i.e. total T4 (TT4) in serum was determined in all adult males after termination.
- Parameters examined: - total protein, albumin, globulin (albumin subtracted from total protein, total cholesterol, urea nitrogen, creatinine, glucose, bilirubin, bile acids, sodium, potassium, chlorides and calcium. Also aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (AP).

URINALYSIS: Yes
- Time schedule for collection of urine: On the last day of the experiment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined.: colour, volume (mL), specific gravity, pH, protein (g/L), glucose (mmol/L), ketone bodies (mmol/L), bilirubin (qualitative test), blood (Ery/μL), urobilinogen (μmol/L), leukocytes (leu/μL), urine sediment, squamous and columnar epithelium, leukocytes, erythrocytes, and crystals were determined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males from group 0, 1, 2 and 3 were examined at the end of the administration period (1-2 days before euthanasia). Females from group 0, 1, 2 and 3 were examined during the last week of lactation (1 day before euthanasia). The behavioural studies on the males and females from satellite groups were performed at the end of the administration period (measurement 1) and then before the end of the additional observation period (measurement 2).
- Dose groups that were examined: 5 adult males and 5 adult females from each group were subjected to the studies.
- Battery of functions tested: observations of animal behaviour in an open field, responses to stimuli, locomotor activity, and forehand hindlimb grip strength.

IMMUNOLOGY: Yes
- Time schedule for examinations:
- How many animals: 5 adult males and 5 adult females
- Dose groups that were examined: from each group (0, 1, 2, 3, 0 SAT, and 3 SAT).
- Parameters examined.: The evaluation of the immune system was based on the results of clinical-chemical examinations and
post-mortem examinations (gross and histopathological examinations) of the spleen, thymus, and lymph nodes, as well as the statistical analysis of the absolute and relative weights of the spleen and thymus. The clinical-chemical parameters included blood morphology with a picture of peripheral blood and bone-marrow smears, the concentrations of albumin, total protein, the albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, and the activity of aspartate aminotransferase,
alanine aminotransferase, and alkaline phosphatase.

OTHER - HORMONE TESTING:
The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined. The Mouse/Rat Thyroxine (T4) ELISA kit produced by Calbiotech were used.
Blood samples were collected from:
- all dams,
- all adult males after termination.
Blood samples from adult males were measured for TT4. There was no statistically significant changes in the hormone level, absolute/relative weight of the thyroid and histopathological changes of the thyroid connected with the test item, therefore further measurements of TT4 (dams) were not made
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see corresponding table)
A pathological examination involved macroscopic examinations of tissues and organs and a statistical analysis of absolute and relative weights of selected organs.
All adult animals (128 animals: 60 males and 68 females) used in the experiment survived the experiment and after being euthanized were examined pathologically.
The animals used in the study were subjected to the detailed gross examination which involved observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with all organs and their content.
Weights of organs of the animals used in the study were measured. Absolute and relative weights of brain, pituitary gland, thymus, thyroid gland, heart, liver, spleen, kidneys and adrenals from all adult rats from each group were recorded.

HISTOPATHOLOGY: Yes (see corresponding table)
The aim of the histopathological examination was to provide information on influence of the test item on tissues and organs of tested rats in following contexts:
determination of potential significant lesions in the brain (cerebrum, cerebellum, pons), spinal cord, eye with the optic nerve, pituitary gland, thyroid with parathyroids, stomach, small and large intestines including Peyer’s patches (duodenum, jejunum, ileum, cecum, colon), pancreas, liver, kidneys, ureters, adrenals, spleen, heart, aorta, thymus, trachea, lungs with the bronchi, urinary bladder, gonads: testes and ovaries with oviducts, uterus with the cervix, epididymides, accessory sex organs (prostate, seminal vesicles and coagulating glands), vagina, skin, mammary gland, lymph nodes, skeletal muscle with the peripheral nerve and femur with knee joint.
It has been conducted full histopathological examination of organs and tissues of adult animals of the control groups (group 0 and group 0 SAT) and the ones treated with the test item at the highest dose (group 3 and group 3 SAT).
Statistics:
The results (average values and standard deviation) are presented in tables. The treated groups, i.e. groups 1, 2, and 3 were compared to the control group. The treated satellite group, i.e. 3 SAT was compared to group 0 SAT. Each sex was analyzed separately.
Clinical studies results: The clinical results were statistically analyzed using the one-way analysis of variance and the Dunnet’s test (group 0, 1, 2 and 3) and Student’s t-test (group 0 SAT, 3 SAT), (p ≤ 0.05).
Clinical-chemical examinations results: The results obtained in the groups 0, 1, 2 and 3 were statistically analysed using the one-way analysis of variance or Kruskal-Wallis test (in the absence of normal distribution or heterogeneous variances) and then Dunnet’s test (p ≤ 0.05). The results of the clinical-chemical examinations in satellite group were statistically analyzed using Student’s t test (p ≤ 0.05) or U Mann-Whitney test (in the absence of normal distribution). Some results of the urinalysis such as bilirubin and urine sediment were statistically analyzed using nonparametric statistics test - Kruskal-Wallis and then Dunnet’s test (p ≤ 0.05) (group 0, 1, 2, 3) and U Mann-Whitney test (group 0 SAT, 3 SAT), (p ≤ 0.05).
Post-mortem examinations results: The results, i.e. absolute and relative weights of internal organs as well as the numbers of implantation sites and corpora lutea were statistically evaluated using Dunnett’s test for groups 0, 1, 2, 3 and student’s T-test for satellite groups 0 SAT and 3 SAT (at p≤0.05).
Statistical analyses were conducted using the Microsoft Excel 2010 and the Statistica 10 programmes.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no differences in physical appearance and behaviour between the treated and the control groups. The animals didn’t show any clinical signs during the entire experiment. The only exception was the thinning of hair all over the body in one female from group 1 but this change is not considered to be treatment-related.
Mortality:
no mortality observed
Description (incidence):
No mortality and morbidity of the adult animals was observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in body weights between parental animals from treated and control groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no statistically significant differences in food intake by males and also by females from the treated (1, 2, 3, 3 SAT) and the control groups (0, 0 SAT).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmic examinations did not reveal any pathological changes.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological examinations: In case of peripheral blood examinations of animals from groups 1, 2 and 3, statistically significant changes in females of group 1 were stated (statistically significant: decrease in the hemoglobin concentration, decrease in the hematocrit value and decrease in the number of erythrocytes). Mentioned above changes were considered as accidental due to no confirmation in higher dosage i.e. group 2 and 3. There were no changes in males of group 1, 2, and 3. In case of peripheral blood examinations of males and females from group 3 SAT, statistically significant increase in the number of thrombocytes was stated compared to the control group (0 SAT). However, number of thrombocytes in group 3 SAT compared to the group 0 was at a similar level, so this change should not be connected with the test item administration. Statistically significant increase in the MCHC value in females of group 3 SAT was considered as accidental because this change was isolated, and no other deviations in red blood cells parameters were stated. In bone marrow examinations, statistically significant decrease in the number of proerythroblasts was stated in males of group 3 SAT. This change was regarded as random because there were no changes on the next stages of erythroblast development. Statistically significant decrease in the number of filamented eosinophils (bone marrow examination) in males of group 3SAT was also considered as accidental.

Coaglulological examinations: The treatment with the test item at three different dose levels did not affected coagulological parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Biochemical examinations: In case of biochemical parameters, statistically significant increase in the chloride concentration in males of group 1, 2 and 3 were observed. Increase in chloride concentration was small, by about 2% compared to the control group and did not increase with increasing dose. For this reason statistically significant change which was observed may be considered as not treatment-related. These change was not observed in the satellite group, after 14-days post-treatment. In males of group 3 statistically significant increase in the calcium concentration was stated, it can be also considered as random due to low intensity. Statistically significant increase in the creatinine concentration was stated in females of group 3. This change was considered as accidental because the histopathological examination of the kidneys revealed no changes considered as treatment related. Statistically significant increase in the total protein concentration and decrease in the glucose concentration was stated in males of group 3 SAT compared to the control group (0 SAT) and those changes could be random. In case of females of group 3 SAT, statistically significant decrease in the urea nitrogen concentration and increase in the sodium concentration were stated and those changes were considered as accidental due to change in sodium was small (increase by about 0.9% compared to the group 0 SAT) and change in urea nitrogen is in the range of variability of the control group.

Enzymatic examinations: All changes observed in the males and females of group 1 and 2 (statistically significant decrease in the AP activity in males of group 1 and 2 and statistically significant decrease in the AST activity in females of group 2) were considered as random due to no confirmation in the higher dosage group. Statistically significant decrease in AST activity in males of group 3 SAT was consider as accidental, because in case of toxic impact on the liver function, an increase in activity of AST is observed.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant decrease in the protein level was stated in females of group 1, 2 and 3 but this is not an adverse change so it will not be taken into account. Statistically significant increase in the level of urine protein in males of group 3SAT was stated and this change was considered as accidental.
Statistically significant decrease in the level of urine bacteria in males of group 3SAT is not an adverse change so it will not be taken into account.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The behavioural studies showed that the nervous system functioned properly at dose levels of 33.3, 100, and 300 mg/kg b.w./day.

Open field observations: In case of males and females from groups 1, 2, 3 and control group, no involuntary clonic and tonic movements, changes in gait, or stereotypical behaviour were observed. A slight decrease in arousal was noticed in two males from group 0 (no. 4 and no. 5) and one female (no. 11) from group 3. A slight increase in arousal was noticed in one male (no. 3) from group 1, one male (no. 5) and three females (no. 12, no. 15 and no. 16) from group 2 and two females (no. 12 and no. 14) from group 3. A statistically significant increase of horizontal locomotor activity was noticed in males and females from group 2 compared to the control group.
A slight increase in arousal was noticed in one male (no. 34) and one female (no. 44) from group 0 SAT. A slight decrease in arousal was observed in one male (no. 34) and one female (no. 42) from group 0 SAT and in two males (no. 32 and no. 33) and two females (no. 43 and no. 45) from group 3 SAT. A moderate decrease in arousal was observed in two males (no. 31 and no. 32) and one female (no.44) from group 0 SAT and in three males (no. 31, no. 34 and no. 35) from group 3 SAT. A slight increase in arousal was observed in one female (no. 42) from group 3 SAT. There were statistically significant increase in the numbers of urine pools left by males from group 3 SAT compared to the group 0 SAT.
Evaluation of sensorimotor responses to stimuli: No differences between groups.
Measurement of the fore- and hindlimb grip strength: A statistically significant increase of the forelimb grip strength was observed in females from group 3
SAT compared to the control group (0 SAT). The hindlimb grip strength in females from group 3 SAT and 0 SAT was similar.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant changes considered as test item related were found in the results of clinicalchamical examinations therefore it can be stated that the test item did not affect the immune system. The gross and histopathological examination did not reveal any harmful effects of the test item on the immune system. Although cases of histopathological lesions in the immune system were observed, they should not be linked as the effect of the action of the test item since they were consider accidental and / or occurred in both, the treated and the control animals. There were also stated no incidence of overt infections, and occurrence of significant tumors in the treated rats. It was concluded that the test item is not immunotoxic for rats.
The observed changes are the following:
Statistically significant change was stated in the creatinine concentration in females of group 3. The macroscopic examination of the thymus, spleen, and lymph nodes did not reveal any pathological changes in any group with the exception of ecchymosis of thymus, petechiae on thymus and decreased of thymus. This changes occurred in a few animals both in control and treated groups. Hence, it was defined as accidental and should not be associated with the action of the test item.
Statistically significant differences, i.e. decrease in absolute weights of spleen in males from group 3 and decrease in relative weights of spleen in males from groups 1, 2 and 3. There were no other statistically significant changes in absolute and relative weights of thymus, spleen, and lymph nodes of males and females in all treated groups.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The analysis of absolute and relative weights of internal organs of the males from groups 0, 1, 2, and 3 showed statistically significant decreases in absolute weights of pituitary gland and spleen in males from group 3 and in relative weights of pituitary gland and thyroid in males from group 3, as well as in relative weights of spleen in males from groups 1, 2 and 3.
The analysis of absolute weights of internal organs of the females from groups 0, 1, 2, and 3 showed statistically significant decreases in absolute weight of pituitary gland in females from group 2 and 3. There were no statistically significant changes in relative weights of organs of females in all treated groups.
There were no statistically significant changes in absolute and relative weights of organs in both satellite groups (0 SAT and 3 SAT) in males. Similarly there were no statistically significant changes in absolute weights of organs in both satellite groups (0SAT and 3SAT) in females. But there was statistically significant increase in relative weight of kidneys in females of group 3 SAT.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant lesions in the treated rats stated. The lesions (gross as well as histopathological) which were occurred in some organs and tissues, were mostly scattered among the treated animals and the control or occurred individually (all differences between treated and control groups were considered accidental, since they were of small magnitude, sporadic, not dose-related, and/or not consistent between sexes). On the basis of the results of the histopathological examination of organs and tissues it may be concluded that all of the revealed lesions were not associated with the test item.

The post-mortem examination revealed:
Group 0: Trachea filled with foamy liquid in 2 rats (females), Thymus petechiae in 1 rat (female), decreased in 1 rat (female), Lungs increased in 3 rats (2 males and 1 female), bright in 1 rat (female), bright foci above the surface in 3 rats (females), marbled in 5 rats (2 males and 3 females), dark red in 1 rat (male), dark red foci in 3 rats (1 male and 2 females), dark red area in 7 rats (1 male and 6 females), Heart slightly increased in 1 rat (male), Uterus congestion in 1 rat(female).

Group 1: Trachea filled with foamy liquid in 2 rats (females), Thymus ecchymosis of 1/3 organ in 1 rat( female), petechiae in 1 rat (female), decreased in 2 rat (females), Lungs increased in 8 rats (3 males and 5 females), bright in 1 rat (female), bright foci above the surface in 5 rats (females), foci collapsed under the surface in 1 rat (female), red in 1 rat (female), marbled in 2 rats (females), dark red in 3 rats (males), dark red foci in 1 rat (female), dark red area in 3 rats (1 male and 2 females), Heart slightly increased in 1 rat (female), small intestine congestion in 1 rat (female), Cecum congestion in 1 rat (female), Uterus congestion in 1 rat (female), cyanosis of the horns in 1 rat (female).

Group 2: Trachea filled with foamy liquid in 1 rat (female), Thymus petechiae in 1 rat (female) decreased in 1 rat (female), Lungs increased in 2 rats (females), bright in 1 rat (female), bright foci above the surface in 1 rat (female), marbled in 5 rats (females), dark red foci in 1 rat (female), dark red area in 4 rats (2 males and 2 females), Heart slightly increased in 1 rat (female), Ovaries congestion in 1 rat (female), Uterus congestion in 2 rats (females), partial cyanosis in 1 rat (female), expanded in 1 rat (female), cyanosis of the horns in 3 rats (female).

Group 3: Thymus petechiae in 1 rat (female), Lungs increased in 3 rats (2 males and 1 female), bright in 1 rat (female), bright foci above the surface in 1 rat (female), marbled in 5 rats (1 male and 4 females), dark red in 1 rat (female), dark red area in 4 rats (female), Uterus congestion in 1 rat (female), expanded in 1 rat (female), thickened wall in 1 rat (female), filled with clear liquid in 1 rat (female).

Group 0 SAT: Trachea filled with foamy liquid in 2 rats (1 male and 1 female), Thymus petechiae in 8 rats (3 males and 5 females), decreased in 2 rats (1 male and 1 female), Lungs bright red in 5 rats (4 males and 1 female), bright red foci in 2 rats (1 male and 1 female), bright red foci above the surface in 4 rats (2 males and 2 females), increased in 3 rats (2 males and 1 female), marbled in 7 rats (6 males and 1 female), dark red foci in 9 rats (3 males and 6 females), Heart increased in 4 rats (males), Liver lesion 7mm long, yellowish, grainy on the visceral side of the left lobe in 1 rat (female), Spleen increased in 1 rat (male)
Cecum, congestion in 1 rat (male), Colon petechiae in 1 rat (male), Kidneys increased in 2 rats (males), Ovaries congestion in 1 rat (female), Uterus congestion in 1 rat (female), slightly expanded in 4 rats (females), filled with clear liquid in 4 rats (females).

Group 3 SAT:
Trachea filled with foamy liquid in 4 rats (3 males and 1 female), Thymus petechiae in 9 rats (5 males and 4 females), decreased in 1 rat (male), Lungs bright red in 3 rats (males), bright red foci above the surface in 5 rats (3 males and 2 females), increased in 3 rats (2 males and 1 female), marbled in 8 rats (7 males and 1 female), dark red in 1 rat (male), dark red foci in 9 rats (1 male and 8 females), dark red foci under the surface in 2 rats (males), doughy consistency in 1 rat (female),Heart increased in 2 rats (males), congestion in 2 rats (males),mandibular lymph nodes petechiae in 3 rats (1 male and 2 females), Ovaries congestion in 1 rat (female), Uterus congestion in 1 rat (female), slightly expanded in 2 rats (females), filled with clear liquid in 2 rats (females), Other lesions yellowish, elastic, size 5x2mm in subcutaneous tissue of cervical area above the salivary gland in 1 rat (male), yellowish, 5mm long located in fat tissue in left ovary area in 1 rat (female), yellowish 5mm long located in fat tissue in pelvis cavity inlet area in 1 rat (female), Cranial cavity filled with blood in 1 rat (male)
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant histopathological lesions observed in the animals treated with the highest dose of the test item (groups: 3 and 3 SAT) therefore the full histopathological examination was not extended to organs and tissues of the animals exposed to the mid and the low dose of the test item. As for the animals of the satellite group (group 3 SAT) there were no delayed significant changes in organs and tissues for 14-days additional observation after the treatment.
Changes observed were:
Group 0 - pituitary: microcyst in 2 males, thyroid: parathyroid hypertrophy in 1 female, thymus: congestion in 1 male, effusion in 1 female, lymphocytic tissue depletion in 1 female, heart: erythrocytorrhagia in 3 males, leukocyte infiltration in 1 male, foci of fibrosis in 1 male, foci of fatty degeneration in 1 male, left and right ventricular myocardium hypertrophy in 1 male, lungs: congestion in 1 male, erythrocytorrhagia in 5 males and 9 females, edema in 3 males and 4 females, emphysema in 4 males and 9 females, foamy cell infiltration in 2 males and 7 females, lymphocyte infiltration in 1 male, leukocyte infiltration in 1 male, liver: congestion in 1 male and 2 females, - effusion in 1 male, bile ducts proliferation in 1 male, Browicz-Kupffer cell proliferation in 1 male, hepatocyte fine-droplet fatty degeneration in 1 male, spleen: congestion in 1 female, depletion of the white pulp in 2 females, pancreas: islets of Langerhans hypertrophy in 1 male and 1 female, ducts proliferation in 1 female, jejunum: lymphatic follicle reactive hyperplasia in 1 male, ileum: lymphocyte infiltration in 1 female, cecum: lymphatic follicle reactive hyperplasia in 1 male, colon: lymphatic follicle reactive hyperplasia in 1 male, kidneys: erythrocytorrhagia in 1 male and 1 female, glomeruli congestion in 6 males and 4 females, lymphocyte infiltration in 2 females, glomeruli hypertrophy in 9 males and 7 females, glomeruli atrophy in 4 males and 5 females, Bowman's capsule extension in 1 male and 2 females, retention microcysts in 2 females, hyaline casts in 1 female, prostate: congestion in 2 males, edema in 2 males, lymphocyte infiltration in 5 males, mandibular lymph nodes: congestion in 1 male, hyperplasia in 1 male, lymphocytic tissue depletion in 1 female, mesenteric lymph nodes: hyperplasia in 1 male, lymphocytic tissue depletion in 1 female.

Group 1 - thymus: congestion in 1 female, effusion in 1 female, heart: erythrocytorrhagia in 1 female, left ventricular myocardium hypertrophy in 1 female, lungs: congestion in 3 males and 2 females, erythrocytorrhagia in 4 males and 10 females, edema in 3 males and 2 females, emphysema in 10 females, foamy cell infiltration in 3 females, prostate: lymphocyte infiltration in 3 males, uterus: congestion in 1 female, effusion in 2 females, fibroid in 2 females.

Group 2: thymus: lymphocytic tissue depletion in 1 female, heart: left ventricular myocardium hypertrophy in 1 female, lungs: congestion in 1 male and 2 females, erythrocytorrhagia in 3 males and 9 females, edema in 2 females, emphysema in 1 male and 3 females, foamy cell infiltration in 4 females, prostate: lymphocyte infiltration in 6 males, uterus: congestion in 2 females, effusion in 2 females, fibroid in 1 female.

Group 3: thyroid: congestion in 1 male, thymus: congestion in 2 males, erythrocytorrhagia in 1 male, heart: erythrocytorrhagia in 1 male, leukocyte infiltration in 1 male, lungs: congestion in 2 males and 2 females, erythrocytorrhagia in 6 males and 8 females, edema in 2 males and 1 female, emphysema in 5 males and 3 females, foamy cell infiltration in 1 male and 1 female, lymphocyte infiltration in 1 male, liver: congestion in 1 male, pancreas: islets of Langerhans hypertrophy in 3 males, cecum: lymphatic follicle reactive hyperplasia in 1 female, colon: lymphatic follicle reactive hyperplasia in 2 males and 1 female, kidneys: erythrocytorrhagia in 1 male, glomeruli congestion in 10 males and 6 females, lymphocyte infiltration in 1 male, glomeruli hypertrophy in 9 males and 9 females, glomeruli atrophy in 10 males and 8 females, Bowman's capsule extension in 6 males, retention microcysts in 1 male and 2 females, adrenals: left adrenal gland atrophy with medulla microcyst; right adrenal gland hyperplasia in 1 female, urinary bladder: lymphocyte infiltration in 5 males, prostate: congestion in 2 males, lymphocyte infiltration in 6 males, mesenteric lymph nodes: congestion in 1 male, hyperplasia in 1 male.

Group 0 SAT - pituitary: hypertrophy in 1 female, microcyst in 2 males, thymus: congestion in 2 males, effusion in 1 male and 3 females, heart: congestion in 1 male, erythrocytorrhagia in 1 male, foci of fibrosis in 2 males, myocardium of the left ventricular hypertrophy in 3 males, lungs: congestion in 4 males, erythrocytorrhagia in 8 male and 9 females, edema in 3 males, emphysema in 5 male and 4 females, foamy cell infiltration in 1 female, lymphocyte infiltration in 1 male, inflammatory infiltration in 1 male, foci of fibrosis in 1 male, liver: congestion in 1 male, Browicz-Kupffer cell proliferation in 1 male and 1 female, focus of fibrosis in 1 female, spleen: congestion in 1 male, pancreas: islets of Langerhans congestion in 2 males, islets of Langerhans hypertrophy in 7 males and 2 females
- lymphocyte infiltration in 2 males and 1 female, jejunum: lymphatic follicle reactive hyperplasia in 1 female, cecum: lymphatic follicle reactive hyperplasia in 1 male, colon: lymphatic follicle reactive hyperplasia in 1 female, lymphocyte infiltration in 1 female, kidneys: glomeruli congestion in 5 males and 4 females, glomeruli hypertrophy in 6 males and 4 females, glomeruli atrophy in 3 males and 2 females, Bowman's capsule extension in 1 male, retention microcysts in 1 male and 2 females, prostate: congestion in 1 male, lymphocyte infiltration in 3 males, ovaries: congestion in 1 female, mandibular lymph nodes: lymphocytic tissue reactive hyperplasia in 1 female.

Group 3 SAT - thymus: congestion in 2 males and 1 female, effusion in 2 males and 4 females, heart: congestion in 3 males, erythrocytorrhagia in 4 males and 1 female, foci of fibrosis in 3 males, myocardium of the left ventricular hypertrophy in 2 males, lungs: congestion in 4 males and 1 female, erythrocytorrhagia in 9 males and 9 females, edema in 4 males and 1 female, emphysema in 4 males and 4 females, foamy cell infiltration in 1 male and 1 female, liver: congestion in 1 male, effusion in 1 male, spleen: congestion in 1 male, hemosiderin deposits in 1 male and 2 females, pancreas: islets of Langerhans congestion in 2 males and 2 females, islets of Langerhans hypertrophy in 3 males and 3 females, foci of atrophy of lobules with fibrosis in 1 male, ducts proliferation in 1 male, cecum: lymphatic follicle reactive hyperplasia in 2 males and 3 females, colon: lymphocyte infiltration in 1 female, kidneys: glomeruli congestion in 5 males and 6 females, glomeruli hypertrophy in 6 males and 5 females, glomeruli atrophy in 3 males and 3 females, Bowman's capsule extension in 1 male, urinary bladder: lymphocyte infiltration in 1 male, prostate: lymphocyte infiltration in 2 males, mandibular lymph nodes: congestion in 1 male and 1 female, erythrocytorrhagia in 1 female, effusion in 1 male and 1 female, gross lesion isolated from subcutaneous tissue of cervical area above the salivary gland (nodule): lipoma in 1 male gross lesion isolated from the fat tissue of the left ovary area (nodule): lipoma in 1 female, gross lesion isolated from the fat tissue of the pelvis cavity inlet area (nodule): lipoma in 1 female.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were also observed individual cases of small benign tumors such as lipomas of the adipose tissue and fibroids of the uterus. In the subcutaneous tissue of region of cervical area above the salivary gland (male in the group 3 SAT) and region of the abdominopelvic cavity (two females in the group 3 SAT) the small lipomas were occurred. As for the uterus (two females in the group 1 and one female in the group 2) the fibroids were observed. The occurrence of them was clearly incidental to the treatment and should not be linked with the test item.
Both, the lipomas and fibroids were considered as spontaneous lesions, consistent with background findings commonly observed in rats. The occurrence of the left adrenal gland atrophy with medulla microcyst and right adrenal gland hyperplasia in one of the females in the group 3 is also accidental. It seems to be a kind of a malformation.
Other effects:
no effects observed
Description (incidence and severity):
Hormonal examinations: Statistically significant changes in the TT4 level, absolute/relative weight of the thyroid and histopathological changes of the thyroid related with the test item in all males were not observed, so the determination of TT4 levels were not extended to females.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effects observed at the highest dose tested.

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 5: Main study. Clinical signs - adult animals

Clinical signs

Group / sex / number of animals / computer numbers of animals

0

1

2

3

males

females

males

females

males

females

males

females

Test animals

10

12

10

12

10

12

10

12

Animals with clinical signs

0

0

0

1

0

0

0

0

Animals with ophthalmic

changes

0

0

0

0

0

0

0

0

Death of animals

-

-

-

-

-

-

-

-

SKIN AND COAT

Thinning of hair all over the body

-

-

-

1 [16]

-

-

-

-

[ ] computer numbers of animals

Table 6. Main study. Clinical signs - satellite groups

Clinical signs

Group / sex / number of animals / computer numbers of animals

0 SAT

3 SAT

males

females

males

females

Test animals

10

10

10

10

Animals with clinical signs

0

0

0

0

Animals with ophthalmic

changes

0

0

0

0

Death of animals

-

-

-

-

Table 7. VL3. Main study. Open field observations – males

Parameter

Group

0

n = 5

1

n = 5

2

n = 5

3

n = 5

involuntary clonic

movement

no changes

no changes

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

no changes

no changes

gait

no changes

no changes

no changes

no changes

arousal

[4]1, [5]1

[3]4

[5]4

no changes

stereotypies

no changes

no changes

no changes

no changes

number of defecations

2.20±1.30

2.40±2.30

1.00±1.73

2.80±1.92

number of

urinations

2.00±0.71

1.40±0.89

2.40±0.55

1.80±1.10

distance 0-3 min [m]

69.26±33.14

100.28±38.03

157.78±52.14*

119.38±43.20

number of rearings

0-3 min

5.60±5.13

10.00±11.40

13.60±8.20

7.40±3.51

x± SD

nnumber of test animals

*-statistically significant difference at p ≤ 0.05-Dunnett’s test

[ ] - computer numbers of animals with changes

Classification of changes:

1 - slight decrease (a rat sometimes does not move, but exploratory activity is observed)

2 - moderate decrease (a rat is slightly numb; only the head moves)

3 - strong decrease (paralysis, coma)

4 - slight increase (a rat is slightly excited or tense; it suddenly moves forward and stays without motion)

5 - strong increase (a rat is very vigilant; it runs and suddenly moves from its place

Table 8. VL3. Main study. Open field observations – females

Parameter

Group

0

n = 5

1

n = 5

2

n = 5

3

n = 5

involuntary clonic

movement

no changes

no changes

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

no changes

no changes

gait

no changes

no changes

no changes

no changes

arousal

no changes

no changes

[12]4, [15]4, [16]4

[11]1, [12]4, [14]4

stereotypies

no changes

no changes

no changes

no changes

number of defecations

2.80±2.17

1.80±2.49

2.20±2.05

1.00±1.73

number of

urinations

2.60±1.52

4.20±0.84

4.00±1.73

2.20±1.64

distance 0-3 min [m]

109.44±18.48

106.48±12.37

162.06±40.87*

125.40±39.00

number of rearings

0-3 min

13.60±4.22

12.80±2.77

18.60±8.20

19.20±7.09

Table 9. VL3. Main study. Open field observationsmales (satellite groups)measurement 1

Parameter

Group

0 SAT

n = 5

3 SAT

n = 5

involuntary clonic

movement

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

gait

no changes

no changes

arousal

[34]4

no changes

stereotypies

no changes

no changes

number of defecations

2.20±1.79

2.20±1.92

number of

urinations

1.60±1.52

1.60±0.55

distance 0-3 min [m]

96.42±28.44

96.06±17.15

number of rearings

0-3 min

8.20±1.92

11.60±3.91

Table 10. VL3. Main study. Open field observations - females (satellite groups)measurement 1

Parameter

Group

0 SAT

n = 5

3 SAT

n = 5

involuntary clonic

movement

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

gait

no changes

no changes

arousal

[44]4

no changes

stereotypies

no changes

no changes

number of defecations

0.00±0.00

1.00±1.41

number of

urinations

1.60±0.55

1.20±0.84

distance 0-3 min [m]

106.62±17.52

115.86±4.27

number of rearings

0-3 min

15.00±8.34

19.80±6.69

 

Table 11. VL3. Main study. Open field observations – males (satellite groups) – measurement 2

Parameter

Group

0 SAT

n = 5

3 SAT

n = 5

involuntary clonic

movement

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

gait

no changes

no changes

arousal

[31]2, [32]2, [34]1

[31]2, [32]1, [33]1

[34]2, [35]2

stereotypies

no changes

no changes

number of defecations

0.80±1.10

2.20±2.28

number of

urinations

1.60±0.55

2.80±0.84*

distance 0-3 min [m]

64.82±24.68

44.40±19.49

number of rearings

0-3 min

2.80±3.11

0.60±1.34

 

Table 12. VL3. Main study. Open field observations – females (satellite groups) – measurement 2

Parameter

Group

0 SAT

n = 5

3 SAT

n = 5

involuntary clonic

movement

no changes

no changes

nvoluntary tonic

movement

no changes

no changes

gait

no changes

no changes

arousal

[42]1, [44]2

[42]4, [43]1, [45]1

 

stereotypies

no changes

no changes

number of defecations

1.40±1.95

0.80±1.79

number of

urinations

2.80±2.95

2.60±1.52

distance 0-3 min [m]

96.78±55.48

111.34±68.45

number of rearings

0-3 min

3.80±2.68

9.60±5.73

Table 13. VL3. Main study. Reactions to stimuli

PAIN RESPONSE LATENCY [s]

Group

0

n = 5

1

n = 5

2

n = 5

3

n = 5

Males

3.44±0.27

3.62 ±0.19

3.58±0.45

3.40±0.32

Females

2.94±0.40

3.16±0.30

3.06±0.36

3.24±0.09

  No changes on approach response, touch response, sound response, pinna reflex.

Table 14. VL3. Main study. Reactions to stimuli (satellite groups)

PAIN RESPONSE LATENCY [s]

Group

0 SAT

n = 5

3 SAT

n = 5

Males – measurement 1

3.26±0.11

3.42±0.38

Females - measurement 1

3.24±0.29

2.42±0.23*

Males – measurement 2

2.92±0.39

2.98±0.16

Females – measurement 2

2.54±0.68

2.40±0.72

No changes on approach response, touch response, sound response, pinna reflex.

x± SD

nnumber of test animals

*– statistically significant difference at p ≤ 0.05- Student's t-test

 

Table 15. VL3. Main study. Limb grip strength

 

Group

 Parameter

0

n = 5

1

n = 5

2

n = 5

3

n = 5

Males

forelimb grip

strength [p]

1208.20± 114.02

1234.50± 241.70

1030.50± 46.81

1221.60± 106.38

hindlimb grip

strength [p]

176.60± 33.96

167.60± 42.67

134.70± 45.15

140.30± 28.20

Females

forelimb grip

strength [p]

1194.50± 241.89

1159.20± 138.32

1134.20± 61.33

1263.10± 79.53

hindlimb grip

strength [p]

187.80± 47.64

182.60± 54.94

193.10± 47.25

180.60± 7.67

 

Table 16. VL3. Main study. Limb grip strength – satellite groups

 

Group

Parameter 

0 SAT

n = 5

3 SAT

n = 5

Males – measurement 1

forelimb grip strength [p]

1447.00±143.15

1422.30±116.27

hindlimb grip strength [p]

174.90±54.84

186.00±46.09

Females - measurement 1

forelimb grip strength [p]

1176.70±169.79

1206.30±110.01

hindlimb grip strength [p]

187.80±47.64

160.60±39.50

Males – measurement 2

forelimb grip strength [p]

1322.40±368.82

1423.30±185.26

hindlimb grip strength [p]

175.60±25.91

182.00±11.09

Females – measurement 2

forelimb grip strength [p]

1072.60±136.42

1357.40±195.27*

hindlimb grip strength [p]

133.20±29.48

142.20±29.91

x± SD

nnumber of test animals

*- statistically significant difference at p ≤ 0.05- Student's t-test

Table 17. VL3. Main study. Results of haematological and coagulological examination – males

Parameter

Group

0

n= 10

1

n = 10

2

 n = 10

3

n = 10

HEMOGLOBIN (g/L)

154.30 ± 5.46

153.40 ± 2.72

153.70 ± 4.72

153.30 ± 5.70

HEMATOCRIT (1/1)

0.43 ± 0.02

0.42 ± 0.01

0.42 ± 0.02

0.42 ± 0.02

ERYTHROCYTES (x 1012/L)

8.98 ± 0.26

8.98 ± 0.44

8.96 ± 0.28

8.92 ± 0.31

MCV (fL)

47.70 ± 1.38

47.17 ± 1.46

47.26 ± 1.43

46.99 ± 1.72

MCH (pg)

17.17 ± 0.42

17.12 ± 0.65

17.17 ± 0.40

17.17 ± 0.40

MCHC (g/L)

360.10 ± 8.86

362.20 ± 6.39

363.50 ± 6.92

366.20 ± 5.92

THROMBOCYTES (x 109/L)

667.90 ± 68.70

644.70 ± 55.69

    648.50 ± 54.15

622.60 ± 55.46

LEUKOCYTES (x 109/L)

4.32 ± 1.24

4.07 ± 0.91

5.12 ± 0.94

3.77 ± 0.84

RETICULOCYTES (1/1)

0.019 ± 0.006

0.017 ± 0.004

0.015 ± 0.005

0.020 ± 0.005

HEINZ BODIES (‰)

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

 

0

n=5

1

n=5

2

n=5

3

n=5

PT (s)

12.78 ± 0.69

12.90 ± 0.87

12.36 ± 0.98

11.82 ± 1.01

APTT (s)

52.40 ± 2.41

51.40 ± 4.04

50.20 ± 7.85

47.40 ± 7.16

x± SD;

n - number of test animals

Table 18. VL3. Main study. Results of hematological and coagulological examination – females

Parameter

Group

0

n= 10

1

n = 10

2

 n = 10

3

n = 10

HEMOGLOBIN (g/L)

147.40 ± 4.65

141.08 ± 5.79*

147.08 ± 4.32

150.09 ± 6.67

HEMATOCRIT (1/1)

0.41 ± 0.01

0.39 ± 0.02*

0.40 ± 0.02

0.41 ± 0.02

ERYTHROCYTES (x 1012/L)

7.85 ± 0.33

7.37 ± 0.34*

7.67 ± 0.28

8.06 ± 0.55

MCV (fL)

51.76 ± 1.30

52.24 ± 1.64

52.38 ± 2.24

        50.82 ± 1.08

MCH (pg)

18.81 ± 0.52

19.17 ± 0.45

     19.20 ± 0.71

18.65 ± 0.59

MCHC (g/L)

363.20 ± 5.43

366.83 ± 4.73

366.50 ± 6.36

367.45 ± 5.37

THROMBOCYTES (x 109/L)

724.40 ± 53.19

664.42 ± 87.80

693.83 ± 43.16

756.36 ± 37.99   

LEUKOCYTES (x 109/L)

4.75 ± 1.23

3.94 ± 1.16

      4.03 ± 1.53

4.82 ± 0.97

RETICULOCYTES (1/1)

0.018 ± 0.004

0.017 ± 0.004

0.015 ± 0.004

       0.017 ± 0.005

HEINZ BODIES (‰)

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00

 

0

n=5

1

n=5

2

n=5

3

n=5

PT (s)

9.66 ± 0.13

9.64 ± 0.05

9.94 ± 0.45

       9.64 ± 0.09

APTT (s)

47.80 ± 8.38

53.60 ± 1.52

52.80 ± 1.10

53.80 ± 2.59

x± SD;

n - number of test animals

* -statistically significant difference with p ≤ 0.05; Dunnett's test;

Table 19. VL3. Main study. Results of hematological and coagulological examination – males (satellite groups)

Parameter

GROUP

0 SAT

n=10

3 SAT

n=10

HEMOGLOBIN

g/L

149.00±4.92

152.30±4.76

HEMATOCRIT

1/1

0.42 ± 0.02

0.42 ±0.02

ERYTHROCYTES

x 1012/L

8.800.41

9.02 ± 0.27

MCV

fL

47.23± 1.67

46.62± 1.33

MCH

pg

16.94± 0.53

16.90± 0.33

MCHC

g/L

358.70± 7.07

362.50±6.57

THROMBOCYTES

x 109/L

609.40± 70.84

662.50± 22.11*

LEUKOCYTES

x 109/L

3.450.66

3.47 ±0.96

RETICULOCYTES

1/1

0.020± 0.003

0.020± 0.003

HEINZ BODIES

%〇

0.000.00

0.00 ±0.00

 

 

0 SAT

3 SAT

 

 

n=5

n=5

PT

s

12.12± 0.97

12.24± 0.65

APTT

s

50.80± 5.12

54.00± 3.08

Table 20. VL3. Main study. Results of hematological and coagulological examination – females (satellite groups)

Parameter

GROUP

0 SAT

n=10

3 SAT

n=10

HEMOGLOBIN

g/L

145.40± 5.48

149.00±6.13

HEMATOCRIT

1/1

0.41 ± 0.02

0.41 ± 0.02

ERYTHROCYTES

x 1012/L

8.01 ± 0.37

8.12 ±0.35

MCV

fL

51.28± 1.95

50.53± 1.88

MCH

pg

18.17± 0.47

18.36± 0.50

MCHC

g/L

354.40± 7.85

363.70±6.27*

THROMBOCYTES

x 109/L

614.80± 46.17

672.60± 64.14*

LEUKOCYTES

x 109/L

2.63 ± 0.83

2.31 ± 0.56

RETICULOCYTES

1/1

0.018±0.003

0.019± 0.002

HEINZ BODIES

%〇

0.00 ± 0.00

0.00 ±0.00

 

 

0 SAT

3 SAT

 

 

n=5

n=5

PT

s

9.96 ± 0.21

10.04± 0.48

APTT

s

51.80± 3.90

48.60±2.51

 Table 21. VL3. Main study. Results of hematological examination - males Percentage content of leukocytes (leukocytogram)

Parameter

GROUP

0

n=10

1

n=10

2

n=10

3

n=10

NEUTROCYTES

1/1

0.230.06

0.220.09

0.230.06

0.220.05

EOSINOCYTES

1/1

0.01 ± 0.01

0.000.01

0.000.00

0.000.00

LYMPHOCYTES

1/1

0.760.06

0.770.09

0.770.06

0.780.05

MONOCYTES

1/1

0.000.00

0.000.00

0.000.00

0.000.00

OTHER CELLS

1/1

0.000.01

0.000.00

0.000.01

0.000.00

 Table 22. VL3. Main study. Results of hematological examination - females Percentage content of leukocytes (leukocytogram)

Parameter

GROUP

0

n=10

1

n=12

2

n=12

3

n=11

NEUTROCYTES

1/1

0.460.13

0.480.06

0.380.17

0.480.11

EOSINOCYTES

1/1

0.000.00

0.000.00

0.000.00

0.000.00

LYMPHOCYTES

1/1

0.540.13

0.510.06

0.610.17

0.520.11

MONOCYTES

1/1

0.000.00

0.000.00

0.000.00

0.000.00

OTHER CELLS

1/1

0.000.01

0.000.00

0.010.01

0.000.00

Table 23. VL3. Main study. Results of hematological examination - (satellite groups) Percentage content of leukocytes (leukocytogram)

 

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=10

3 SAT

n=10

0 SAT

n=10

3 SAT

n=10

NEUTROCYTES

1/1

0.190.05

0.210.04

0.140.06

0.160.05

EOSINOCYTES

1/1

0.01 ± 0.01

0.010.01

0.010.01

0.000.00

LYMPHOCYTES

1/1

0.81 ± 0.05

0.790.04

0.850.06

0.830.05

MONOCYTES

1/1

0.000.00

0.000.00

0.000.00

0.000.00

OTHER CELLS

1/1

0.000.00

0.000.00

0.000.00

0.010.01

Table 24. VL3. Main study. Results of hematological studies - bone marrow examination Erythrocyte system - males

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

PROERYTHROBLASTS 1/1

0.013± 0.007

0.011 ±0.002

0.013±0.004

0.013±0.004

BASOPHILIC

ERYTHROBLASTS              1/1

0.015± 0.003

0.016± 0.010

0.018±0.006

0.014±0.004

POLYCHROMATOPHILIC ERYTHROBLASTS              1/1

0.170± 0.022

0.156± 0.006

0.150±0.020

0.154±0.011

ORTHOCHROMATIC ERYTHROBLASTS              1/1

0.206± 0.018

0.223± 0.035

0.219±0.019

0.208 ± 0.021

TOTAL                               1/1

0.405± 0.037

0.406± 0.034

0.401 ±0.030

0.390±0.033

 

Table 25. VL3. Main study. Results of hematological studies - bone marrow examination Erythrocyte system - females

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

PROERYTHROBLASTS 1/1

0.012± 0.003

0.010± 0.002

0.014±0.007

0.011 ±0.003

BASOPHILIC

ERYTHROBLASTS              1/1

0.014± 0.004

0.014± 0.005

0.014±0.009

0.014±0.004

POLYCHROMATOPHILIC ERYTHROBLASTS              1/1

0.165± 0.019

0.170± 0.016

0.156±0.029

0.166±0.011

ORTHOCHROMATIC ERYTHROBLASTS              1/1

0.218± 0.028

0.239± 0.013

0.230±0.033

0.240±0.010

TOTAL                               1/1

0.410± 0.043

0.432± 0.018

0.414±0.063

0.430 ± 0.020

Table 26. VL3. Main study. Results of hematological studies - bone marrow examination Erythrocyte system - (satellite groups)

Parameter

 

GROUP (males)

GROUP (females)

 

0 SAT

n=5

3 SAT

n=5

0 SAT

n=5

3 SAT

n=5

PROERYTHROBLASTS

1/1

0.011 ± 0.003

0.006 ± 0.002*

0.010± 0.002

0.010± 0.004

BASOPHILIC

ERYTHROBLASTS

1/1

0.016± 0.005

0.015± 0.004

0.015± 0.005

0.013± 0.005

POLYCHROMATOPHILIC

ERYTHROBLASTS

1/1

0.159± 0.024

0.158± 0.014

0.152± 0.019

0.175± 0.015

ORTHOCHROMATIC

ERYTHROBLASTS

1/1

0.210± 0.024

0.213± 0.031

0.211 ± 0.027

0.206± 0.015

TOTAL

1/1

0.397± 0.038

0.392± 0.038

0.388± 0.039

0.404± 0.026

Table 27. VL3. Main study. Results of hematological studies - bone marrow examination Leukocyte system – males

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

MYELOBLASTS                  1/1

0.011 ± 0.007

0.016± 0.008

0.012±0.005

0.014±0.004

PROMYELOCYTES            1/1

0.022± 0.006

0.030± 0.008

0.025±0.003

0.022 ± 0.002

ORTHOCHROMATOPHILIC MYELOCYTES                    1/1

0.035± 0.005

0.033± 0.008

0.030±0.005

0.031 ±0.003

ACIDOPHILIC

MYELOCYTES                    1/1

0.003± 0.001

0.004± 0.001

0.003±0.001

0.003±0.001

ORTHOCHROMATOPHILIC METAMYELOCYTES          1/1

0.044± 0.005

0.042± 0.002

0.046 ± 0.005

0.045±0.003

ACIDOPHILIC

METAMYELOCYTES          1/1

0.010± 0.004

0.008± 0.005

0.014±0.005

0.010±0.005

ROD NEUTROPHILS           1/1

0.042± 0.008

0.043±0.012

0.038±0.012

0.042±0.012

ROD EOSINOPHILS           1/1

0.006± 0.002

0.006± 0.002

0.006 ± 0.004

0.003±0.001

FILAMENTED

NEUTROPHILS                   1/1

0.169± 0.027

0.147± 0.019

0.169±0.035

0.170±0.027

FILAMENTED

EOSINOPHILS                    1/1

0.018± 0.009

0.018± 0.009

0.025±0.010

0.011 ±0.003

BASOPHILS                       1/1

0.002± 0.001

0.003± 0.001

0.002±0.001

0.003±0.001

TOTAL                                1/1

0.362± 0.027

0.349± 0.019

0.369 ± 0.040

0.353±0.029

Leukocyte system/erythrocyte system quantitative ratio

0.903± 0.130

0.866± 0.092

0.929±0.160

0.916±0.158

 

Table 28. VL3. Main study. Results of hematological studies - bone marrow examination Leukocyte system - females

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

MYELOBLASTS                  1/1

0.011 ± 0.003

0.014± 0.002

0.013±0.003

0.014±0.004

PROMYELOCYTES            1/1

0.023± 0.007

0.022± 0.004

0.022 ± 0.005

0.026 ± 0.007

ORTHOCHROMATOPHILIC MYELOCYTES                    1/1

0.036± 0.006

0.034± 0.006

0.030±0.005

0.036±0.007

ACIDOPHILIC

MYELOCYTES                    1/1

0.004± 0.002

0.004± 0.003

0.003±0.001

0.004 ± 0.002

ORTHOCHROMATOPHILIC METAMYELOCYTES          1/1

0.046± 0.006

0.048± 0.014

0.042 ± 0.008

0.051 ±0.014

ACIDOPHILIC

METAMYELOCYTES          1/1

0.014± 0.004

0.013± 0.004

0.014±0.010

0.009±0.003

ROD NEUTROPHILS           1/1

0.058± 0.008

0.052± 0.020

0.063±0.013

0.050±0.019

ROD EOSINOPHILS           1/1

0.004± 0.003

0.005± 0.003

0.009±0.009

0.003±0.001

FILAMENTED

NEUTROPHILS                   1/1

0.171 ± 0.011

0.170± 0.021

0.173±0.037

0.180±0.019

FILAMENTED

EOSINOPHILS                    1/1

0.026± 0.008

0.019± 0.010

0.018±0.011

0.022±0.010

BASOPHILS                       1/1

0.002± 0.000

0.002± 0.001

0.002±0.001

0.002±0.001

TOTAL                                1/1

0.394± 0.018

0.384± 0.019

0.388±0.043

0.398±0.017

Leukocyte system/erythrocyte system quantitative ratio

0.973± 0.140

0.891 ± 0.078

0.946 ± 0.086

0.928±0.083

 

Table 29. VL3. Main study. Results of hematological studies - bone marrow examination Leukocyte system - (satellite groups)

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=5

3 SAT

n=5

0 SAT

n=5

3 SAT

n=5

MYELOBLASTS                  1/1

0.015± 0.007

0.012± 0.004

0.012± 0.003

0.013± 0.003

PROMYELOCYTES            1/1

0.024± 0.006

0.023± 0.008

0.024± 0.005

0.023± 0.005

ORTHOCHROMATOPHILIC MYELOCYTES                    1/1

0.027± 0.007

0.028± 0.006

0.033± 0.005

0.032± 0.005

ACIDOPHILIC

MYELOCYTES                    1/1

0.004± 0.002

0.003± 0.001

0.003± 0.001

0.004± 0.002

ORTHOCHROMATOPHILIC METAMYELOCYTES          1/1

0.046± 0.018

0.050± 0.012

0.046± 0.010

0.046± 0.007

ACIDOPHILIC

METAMYELOCYTES          1/1

0.013± 0.008

0.007± 0.003

0.018± 0.013

0.020± 0.010

ROD NEUTROPHILS           1/1

0.043± 0.009

0.040± 0.013

0.049± 0.012

0.048± 0.008

ROD EOSINOPHILS           1/1

0.004± 0.002

0.002± 0.001

0.005± 0.004

0.006± 0.004

FILAMENTED

NEUTROPHILS                   1/1

0.163± 0.037

0.165± 0.019

0.150± 0.017

0.146± 0.012

FILAMENTED

EOSINOPHILS                   1/1

0.029± 0.005

0.018± 0.008*

0.022± 0.010

0.026± 0.005

BASOPHILS                       1/1

0.002± 0.001

0.002± 0.001

0.003± 0.001

0.002± 0.001

TOTAL                                1/1

0.370± 0.023

0.351 ± 0.018

0.364± 0.029

0.366± 0.026

Leukocyte system/erythrocyte system quantitative ratio

0.944± 0.143

0.905± 0.124

0.948± 0.131

0.911 ± 0.102

 

Table 30. VL3. Main study. Results of hematological studies - bone marrow examination Different cells - males

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

LYMPHOCYTES                 1/1

0.187± 0.031

0.204± 0.031

0.180±0.019

0.217±0.023

MONOCYTES                     1/1

0.011 ± 0.003

0.009± 0.007

0.012±0.003

0.009±0.005

PLASMOCYTES                 1/1

0.005± 0.003

0.005± 0.003

0.008±0.005

0.003±0.001

MEGAKARYOCYTES         1/1

0.003± 0.001

0.004± 0.003

0.003±0.001

0.002±0.001

OTHER CELLS                   1/1

0.026± 0.005

0.023± 0.008

0.027 ± 0.006

0.026 ± 0.006

TOTAL                               1/1

0.233± 0.031

0.245± 0.034

0.230 ± 0.021

0.257 ± 0.023

 

Table 31. VL3. Main study. Results of hematological studies - bone marrow examination Different cells - females

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

LYMPHOCYTES                 1/1

0.148± 0.021

0.141 ± 0.008

0.158±0.105

0.130±0.019

MONOCYTES                     1/1

0.013± 0.005

0.012± 0.003

0.013±0.007

0.012±0.005

PLASMOCYTES                 1/1

0.005± 0.004

0.004± 0.002

0.003±0.001

0.003±0.001

MEGAKARYOCYTES         1/1

0.002± 0.001

0.003± 0.001

0.003±0.001

0.002±0.001

OTHER CELLS                   1/1

0.028± 0.006

0.024± 0.004

0.022 ± 0.005

0.025±0.008

TOTAL                               1/1

0.196± 0.026

0.184± 0.010

0.199±0.101

0.172±0.009

 

Table 32. VL3. Main study. Results of hematological studies - bone marrow examination Different cells - (satellite groups)

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=5

3 SAT

n=5

0 SAT

n=5

3 SAT

n=5

LYMPHOCYTES

1/1

0.187± 0.016

0.211 ± 0.022

0.210± 0.036

0.185± 0.024

MONOCYTES

1/1

0.009± 0.004

0.010± 0.004

0.009± 0.002

0.012± 0.009

PLASMOCYTES

1/1

0.004± 0.002

0.004± 0.002

0.003± 0.001

0.005± 0.004

MEGAKARYOCYTES

1/1

0.003± 0.001

0.004± 0.002

0.003± 0.001

0.004± 0.002

OTHER CELLS

1/1

0.030± 0.005

0.026± 0.005

0.023± 0.006

0.024± 0.006

TOTAL

1/1

0.233± 0.024

0.257± 0.022

0.248± 0.042

0.230± 0.029

Table 33. VL3. Main study. Results of blood serum biochemical examination - males

Parameter

GROUP

0

n=10

1

n=10

2

n=10

3

n=10

TOTAL PROTEIN

g/L

60.73±2.03

61.53±2.11

61.63±2.80

62.01 ±3.07

ALBUMIN

g/L

32.56± 0.79

32.61 ± 1.02

32.51 ± 1.13

32.71 ± 1.36

GLOBULIN

g/L

28.17± 1.30

28.92± 1.54

29.12± 1.91

29.30± 1.88

A/G RATIO

1.16± 0.03

1.13± 0.06

1.12± 0.05

1.12± 0.04

GLUCOSE

mmol/L

8.79 ± 1.78

8.83 ± 1.37

8.69 ± 0.69

8.88 ± 1.31

CHOLESTEROL

mmol/L

1.67 ± 0.36

1.58 ± 0.28

1.76± 0.17

1.83 ± 0.25

UREA NITROGEN

mmol/L

4.80 ± 0.44

4.69 ± 0.46

4.41 ± 0.53

5.04 ± 0.67

CREATININE

pmol/L

32.30± 3.86

29.90± 5.07

30.60±3.89

30.50±3.81

BILE ACIDS

pmol/L

18.30± 12.07

12.95± 8.86

15.67±9.13

24.76± 12.48

BILIRUBIN

pmol/L

3.91 ± 0.53

3.40 ± 0.24

3.40 ± 0.60

3.74 ± 0.84

SODIUM

mmol/L

144.50±2.46

145.50± 0.97

145.10± 1.29

146.00± 1.33

POTASSIUM

mmol/L

4.16 ± 0.28

4.24 ± 0.32

4.09 ± 0.20

4.21 ± 0.40

CHLORIDES

mmol/L

105.10±2.02

106.80± 1.40*

107.30± 1.06*

107.00± 1.41*

CALCIUM

mmol/L

2.46 ± 0.08

2.48 ± 0.06

2.46 ± 0.05

2.54 ±0.05*

x± SD;

n - number of test animals

* - statistically significant difference with p < 0.05; Dunnett's test;

Table 34.VL3. Main study. Results of blood serum biochemical examination - females

Parameter

GROUP

0

n=12

1

n=12

2

n=12

3

n=11

TOTAL PROTEIN

g/L

56.32±4.91

55.752.91

57.604.10

55.532.82

ALBUMIN

g/L

30.77± 3.11

30.151.72

31.682.64

30.421.34

GLOBULIN

g/L

25.55± 1.96

25.601.71

25.921.65

25.111.93

A/G RATIO

1.200.06

1.180.08

1.220.06

1.220.09

GLUCOSE

mmol/L

7.461.96

7.181.67

7.241.26

7.401.90

CHOLESTEROL

mmol/L

2.420.52

2.780.51

2.200.63

2.680.54

UREA NITROGEN

mmol/L

7.23 ±2.29

6.671.33

6.251.46

8.771.90

CREATININE

pmol/L

36.33± 3.20

33.332.57

33.083.60

40.825.53*

BILE ACIDS

pmol/L

41.80± 32.74

43.1228.28

39.4925.46

79.07102.36

BILIRUBIN

pmol/L

5.04 ±2.31

5.211.61

4.341.03

5.020.83

SODIUM

mmol/L

139.75± 1.96

139.672.10

141.501.62

140.551.81

POTASSIUM

mmol/L

3.940.71

4.190.40

3.960.22

4.150.40

CHLORIDES

mmol/L

98.92± 3.80

99.173.27

101.753.65

97.552.21

CALCIUM

mmol/L

2.480.06

2.460.08

2.490.10

2.520.04

x± SD;

n - number of test animals

* - statistically significant difference with p < 0.05; Dunnett's test

Table 35. VL3. Main study. Results of blood serum biochemical examination - (satellite groups)

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=10

3 SAT

n=10

0 SAT

n=10

3 SAT

n=10

TOTAL PROTEIN

g/L

61.07± 1.42

63.102.10*

0 SAT

n=10

3 SAT

n=10

ALBUMIN

g/L

31.68± 1.03

32.441.00

60.97± 1.40

61.972.63

GLOBULIN

g/L

29.39± 1.39

30.661.36

34.77± 1.29

35.041.58

A/G RATIO

1.080.07

1.060.04

26.20± 0.65

26.931.52

GLUCOSE

mmol/L

8.291.27

7.150.76*

1.330.06

1.300.07

CHOLESTEROL

mmol/L

2.11 ± 0.37

2.070.36

5.91 ± 0.94

5.880.97

UREA NITROGEN

mmol/L

5.320.49

5.350.46

1.690.46

1.530.39

CREATININE

pmol/L

31.90± 5.82

28.204.44

5.990.75

5.320.60*

BILE ACIDS

pmol/L

21.72± 16.99

19.579.32

34.10±4.41

31.504.28

BILIRUBIN

pmol/L

4.100.54

3.810.46

36.94±20.24

27.9818.13

SODIUM

mmol/L

144.80± 1.69

145.901.37

4.731.12

4.311.09

POTASSIUM

mmol/L

4.450.56

4.250.28

143.80± 0.79

145.100.88*

CHLORIDES

mmol/L

105.101.79

105.201.03

3.920.35

3.950.31

CALCIUM

mmol/L

2.490.07

2.540.05

105.600.84

106.401.26

 

 

 

 

2.480.09

2.440.05

fSD

n - number of test animals

* - statistically significant difference with p < 0.05; Student's t-test;

Table 36. VL3. Main study. Results of blood serum enzymatic examination - males

Parameter

GROUP

0

n=10

1

n=10

2

n=10

3

n=10

AST               IU

152.90±49.99

119.10±20.46

120.10±34.76

137.10±57.92

ALT               IU

38.70±7.86

37.90± 7.20

43.70± 14.44

46.50± 18.34

AP                 IU

159.80±23.54

133.80± 17.78*

133.30±24.23*

149.30± 16.94

fSD

n - number of test animals

* - statistically significant difference with p < 0.05; Dunnett's test;

 

Table 37. VL3. Main study. Results of blood serum enzymatic examination - females

Parameter

GROUP

0

n=12

1

n=12

2

n=12

3

n=11

AST               IU

193.67±62.23

183.67± 81.79

125.75±28.44*

179.64±57.77

ALT               IU

70.75± 39.81

86.25±47.83

52.00± 18.47

73.73±23.95

AP                 IU

135.75±42.63

173.50±61.36

149.08±66.80

158.55±42.32

fSD

n - number of test animals

* - statistically significant difference with p < 0.05; Dunnett's test;

 

Table 38. VL3. Main study. Results of blood serum enzymatic examination - (satellite groups)

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=10

3 SAT

n=10

0 SAT

n=10

3 SAT

n=10

AST

IU

153.50± 37.19

107.50± 18.49*

148.70± 33.72

133.00± 31.61

ALT

IU

42.40± 15.46

34.40± 8.75

29.40± 7.06

28.10± 7.71

AP

IU

103.30± 12.56

93.40± 10.94

51.00± 9.82

56.30± 15.64

fSD;

n - number of test animals

* - statistically significant difference with p < 0.05; Student's t-test;

Table 39. VL3. Main study. Results of general urine examination - males

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

VOLUME                        mL

14.40±6.80

12.004.95

16.009.90

17.4010.33

SPECIFIC GRAVITY

1.020.01

1.020.01

1.020.01

1.020.01

pH

6.900.22

6.800.27

6.900.22

6.800.27

PROTEIN                        g/L

0.300.41

0.260.09

0.180.16

0.100.12

GLUCOSE              mmol/L

0.000.00

0.000.00

0.000.00

0.000.00

KETONE BODIES mmol/L

0.300.67

0.100.22

0.300.27

0.200.27

BILIRUBINc

0.000.00

0.000.00

0.000.00

0.000.00

BLOOD                     Ery/pL

0.000.00

0.000.00

2.004.47

0.000.00

UROBILINOGEN pmol/L

3.200.00

3.200.00

3.200.00

3.200.00

LEUKOCYTES          Leu/pL

0.000.00

0.000.00

0.000.00

0.000.00

xSD

n - number of test animals C - qualitative test

 

Table 40. VL3. Main study. Results of general urine examination - females


Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

VOLUME                        mL

11.20± 3.90

14.803.35

13.604.34

15.203.03

SPECIFIC GRAVITY

1.030.00

1.030.00

1.030.00

1.030.00

pH

6.000.35

6.200.57

6.300.57

6.100.55

PROTEIN                        g/L

0.200.14

0.040.05*

0.060.05*

0.040.05*

GLUCOSE              mmol/L

0.000.00

0.000.00

0.000.00

0.000.00

KETONE BODIES mmol/L

0.200.27

0.300.27

0.200.27

0.000.00

BILIRUBINc

0.000.00

0.000.00

0.000.00

0.000.00

BLOOD                     Ery/pL

0.000.00

0.000.00

0.000.00

16.0035.78

UROBILINOGEN pmol/L

3.200.00

3.200.00

3.200.00

3.200.00

LEUKOCYTES          Leu/pL

0.000.00

0.000.00

0.000.00

0.000.00

 

x ± SD;

n - number of test animals C - qualitative test

* - statistically significant difference with p < 0.05; Dunnett's test;

Table 41. VL3. Main study. Results of general urine examination - males (satellite groups)

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=5

3 SAT

n=5

0 SAT

n=5

3 SAT

n=5

VOLUME

mL

21.60± 5.90

13.40±6.99

16.20±6.87

12.603.13

SPECIFIC GRAVITY

1.02 ± 0.00

1.02 ±0.00

1.020.00

1.020.00

pH

7.10 ± 0.22

7.10 ±0.22

6.80 ± 0.27

6.500.35

PROTEIN

g/L

0.08 ± 0.04

0.54 ± 0.43*

0.000.00

0.000.00

GLUCOSE

mmol/L

0.00 ± 0.00

0.00 ±0.00

0.000.00

0.000.00

KETONE BODIES

mmol/L

1.30 ± 0.45

1.30 ±0.45

0.000.00

0.000.00

BILIRUBINc

0.00 ± 0.00

0.00 ±0.00

0.000.00

0.000.00

BLOOD

Ery/pL

2.00 ±4.47

0.00 ±0.00

0.000.00

0.000.00

UROBILINOGEN

pmol/L

3.20 ± 0.00

3.20 ±0.00

3.200.00

3.200.00

LEUKOCYTES

Leu/pL

0.00 ± 0.00

6.00 ± 8.22

0.000.00

0.000.00

Table 42. VL3. Main study. Results of urine sediment examination - males

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

SQUAMOUS EPITHELIA

1.200.45

1.000.00

1.400.55

1.200.45

COLUMNAR EPITHELIA

0.000.00

0.000.00

0.000.00

0.000.00

LEUKOCYTES

1.000.00

1.000.00

1.000.00

1.000.00

ERYTHROCYTES

1.200.45

1.200.45

1.200.45

1.400.55

BACTERIA

3.600.55

4.000.71

4.200.84

3.800.45

CRYSTALS

1.801.10

1.401.34

1.201.10

2.001.22

 f± SD;

n - number of test animals

Scale of quantitative evaluation of urine sediment elements in single animals in group: 1-single/slide, 2 - single/HPF (HPF-per High Power Field), 3 - few/HPF, 4 - quite numerous/HPF, 5 - numerous/HPF, 6-very numerous/HPF, 7-plantifull/HPF

 

Table 43. VL3. Main study. Results of urine sediment examination - females

Parameter

GROUP

0

n=5

1

n=5

2

n=5

3

n=5

SQUAMOUS EPITHELIA

1.000.00

1.000.00

1.000.00

1.000.00

COLUMNAR EPITHELIA

0.000.00

0.000.00

0.000.00

0.000.00

LEUKOCYTES

1.000.00

1.000.00

1.000.00

1.000.00

ERYTHROCYTES

1.200.45

1.000.00

1.000.00

1.200.45

BACTERIA

2.601.34

2.600.89

3.000.71

2.800.84

CRYSTALS

0.60 ± 1.34

1.601.67

1.801.79

1.601.52

x± SD;

n - number of test animals

Scale of quantitative evaluation of urine sediment elements in single animals in group:

1-single/slide, 2 - single/HPF (HPF-per High Power Field), 3 - few/HPF, 4 - quite numerous/HPF, 5 - numerous/HPF, 6-very numerous/HPF, 7-plantifull/HPF

Table 44. VL3. Main study. Results of urine sediment examination - (satellite groups) 

Parameter

GROUP (males)

GROUP (females)

0 SAT

n=5

3 SAT

n=5

0 SAT

n=5

3 SAT

n=5

SQUAMOUS EPITHELIA

1.200.45

1.200.45

1.00 ± 0.00

1.00 ±0.00

COLUMNAR EPITHELIA

0.000.00

0.000.00

0.00 ± 0.00

0.00 ±0.00

LEUKOCYTES

1.000.00

1.000.00

1.00 ± 0.00

1.00 ±0.00

ERYTHROCYTES

1.200.45

1.200.45

1.20 ± 0.45

1.20 ±0.45

BACTERIA

4.200.84

2.400.89*

2.80 ± 1.30

3.40 ± 1.14

CRYSTALS

0.601.34

2.600.89

1.00 ±2.24

0.00 ±0.00

x± SD;

n - number of test animals

Scale of quantitative evaluation of urine sediment elements in single animals in group:

1-single/slide, 2 - single/HPF (HPF-per High Power Field), 3 - few/HPF, 4 - quite numerous/HPF, 5 - numerous/HPF, 6-very numerous/HPF, 7-plantifull/HPF

Table 45. VL3. Main study. Results of hormonal examination - adult males

Parameter

GROUP

0

n=10

1

n=10

2

n=10

3

n=10

TT4

4.88 ±0.48

5.13 ± 0.63

4.91 ± 0.43

4.94 ± 0.64

 

Table 46. VL3. Main study. Results of hormonal examination - adult males (satellite groups)

 

GROUP

Parameter

0 SAT

3 SAT

 

n=10

n=10

TT4

4.98 ± 0.64

5.56 ±0.77

 

Table 47. VL3. Main study. Gross lesions: euthanized animals

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

organ

Type of change

0

1

2

3

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

Trachea

filled with foamy liquid

 

2

[14,21]

 

2

[17,18]

 

1

[13]

 

 

 

ecchymosis of 1/3 organ area

_

_

_

1

[17]

_

_

_

_

Thymus

petechiae

_

1

[17]

_

1

[18]

_

1

[16]

_

1

[22]

 

decreased

_

1

[17]

-

2

[16,17]

_

1

[13]

_

_

 

increased

2

[6,10]

1

[17]

3

[3,6,9]

5

[11,14,16,18,

22]

-

2

[13,17]

2

[6,10]

1

[11]

 

bright

_

1

[20]

_

1

[18]

_

1

[16]

_

1

[22]

Lungs

bright foci above the surface

_

3

[11,19,20]

_

5

[12,16,17,

18,19]

_

1

[20]

_

1

[22]

 

foci

collapsed under the surface

_

_

_

1

[20]

_

_

_

_

 

red

_

_

_

1

[22]

_

_

_

_

 

Table 47 cont. VL3. Main study.Gross lesions: euthanized animals

Examined

organ

Type of change

GROUP / sex / number of animals / computer numbers of animals

0

1

2

3

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

Lungs

marbled

2

[2,10]

3

[14,18,21]

_

2

[16,21]

_

5

[13,15,19,21,

22]

1

[10]

4

[11,13,14,

18]

dark red

1

[6]

_

3

[3,6,9]

-

-

-

_

1

[17]

dark red foci

1

[2]

2

[17,20]

_

1

[18]

_

1

[16]

_

_

dark red area

1

[8]

6

[12,14,15,16,

21,22]

1

m

2

[15,20]

2

[3,4]

2

[14,17]

_

4

[11,12,14,15]

Heart

slightly

increased

1

[10]

_

_

1

[18]

_

1

[18]

_

_

Cecum

congestion

_

_

-

1

[15]

-

-

_

_

Ovaries

congestion

_

_

-

-

-

1

[16]

_

_

[]-computer numbers of animals with gross changes n - number of tested animals

 

Table 47 cont. VL3. Main study.Gross lesions: euthanized animals

Examined

organ

Type of change

GROUP / sex / number of animals / computer numbers of animals

0

1

2

3

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

Lungs

marbled

2

[2,10]

3

[14,18,21]

_

2

[16,21]

_

5

[13,15,19,21,

22]

1

[10]

4

[11,13,14,

18]

dark red

1

[6]

_

3

[3,6,9]

-

-

-

_

1

[17]

dark red foci

1

[2]

2

[17,20]

_

1

[18]

_

1

[16]

_

_

dark red area

1

[8]

6

[12,14,15,16,

21,22]

1

m

2

[15,20]

2

[3,4]

2

[14,17]

_

4

[11,12,14,15]

Heart

slightly

increased

1

[10]

_

_

1

[18]

_

1

[18]

_

_

Cecum

congestion

_

_

-

1

[15]

-

-

_

_

Ovaries

congestion

_

_

-

-

-

1

[16]

_

_

 

Table 47 cont. VL3. Main study.Gross lesions: euthanized animals

Examined

organ

Type of change

GROUP / sex / number of animals / computer numbers of animals

0

1

2

3

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

males

n=10

females

n=12

Uterus

congestion

_

1

[17]

-

1

[16]

-

2

[13,16]

_

1

[22]

partially

cyanosis

_

_

_

_

_

1

[16]

_

_

expanded

_

-

_

_

_

1

[16]

_

1

[22]

thickened

wall

_

_

_

_

_

_

_

1

[22]

filled with clear liquid

_

_

_

_

_

_

_

1

[22]

cyanosis of the horns

_

_

-

1

[12]

-

3

[19,20,21]

_

_

Small intestine

congestion

_

_

_

1

[15]

_

_

_

_

 

Table 48.VL3. Main study. Gross lesions: euthanized animals (satellite groups)

 

 

GROUP / sex / number of animals / computer numbers of animals

 

 

0 SAT

3 SAT

Examined organ

Type of change

males

n=10

females

n=10

males

n=10

females

n=10

Trachea

filled with foamy liquid

1

[31]

1

[48]

3

[32,34,40]

1

[47]

Thymus

petechiae

3

[31,33,35]

5

[41,45,48,49,50]

5

[32,34,35,36,37]

4

[41,43,48,49]

decreased

1

[37]

1

[48]

1

[37]

_

 

bright red

4

[31,33,34,39]

1

[43]

3

[33,35,38]

_

 

bright red foci

1

[33]

1

[48]

_

_

 

bright red foci above the surface

2

[31,39]

2

[43,50]

3

[33,35,39]

2

[41,48]

Lungs

dark red foci collapsed under the surface

_

_

1

[33]

_

 

increased

2

[32,36]

1

[48]

2

[32,34]

1

[47]

 

marbled

6

[32,35,36,37,39,

40]

1

[46]

7

[31,32,33,34,37,

39,40]

1

[47]

 

Table 48 cont. VL3. Main study.Gross lesions: euthanized animals (satellite groups)

 

 

GROUP / sex / number of animals / computer numbers of animals

 

 

0 SAT

3 SAT

Examined organ

Type of change

males

n=10

females

n=10

males

n=10

females

n=10

 

dark red

_

_

1

[36]

Lungs

dark red foci

3

[31,33,34]

6

[44,45,47,48,49,

50]

1

[38]

8

[41,42,43,44,45,

46,48,49]

 

dark red foci under the surface

_

_

1

[39]

 

doughy

consistency

_

_

1

[50]

Heart

increased

4

[35,36,37,40]

_

2

[34,37]

congestion

_

_

2

[36,38]

Liver

lesion- 7mm long, yellowish, grainy, on the visceral side of the left lobe

_

1

[46]

Spleen

increased

1

[35]

Cecum

congestion

1

[36]

_

 

Table 48 cont. VL3. Main study.Gross lesions: euthanized animals (satellite groups)

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined organ

Type of change

0 SAT

3 SAT

males

females

males

females

 

 

n=10

n=10

n=10

n=10

Colon

petechiae

1

[35]

Mandibular lymph nodes

petechiae

_

1

[34]

2

[41,43]

Kidneys

increased

2

[35,40]

Ovaries

dark red foci

_

1

[45]

1

[48]

 

congestion

_

1

[43]

1

[48]

Uterus

slightly expanded

4

[43,44,48,50]

2

[47,49]

 

filled with

 

4

 

2

 

clear liquid

_

[43,44,48,50]

[47,49]

 

Table 48 cont. VL3. Main study.Gross lesions: euthanized animals (satellite groups) 

Examined organ

Type of change

GROUP / sex / number of animals / computer numbers of animals

0 SAT

3SAT

males

n=10

females

n=10

males

n=10

females

n=10

lesion-

yellowish,elastic, size 5x2mm

in subcutaneous tissue of cervical area above the salivary gland

_

_

1

[31]

_

lesion - yellowish,

5 mm long located in fat tissue in left ovary area

_

_

_

1

[41]

lesion - yellowish,

5 mm long located in fat tissue in pelvis cavity inlet area

_

_

_

1

[46]

Cranial cavity filled with blood

_

_

1

[37]

_

Table 49. VL3. Main study. Absolute weights of internal organs [ mg ] - males. 

 

Group

Examined organ

0

n=10

1

n=10

2

n=10

3

n=10

Brain

2047.400

85.481

1999.700

134.953

2096.300

80.760

2073.200

73.295

Pituitary gland

11.400

1.838

10.600

1.578

10.800

1.874

8.800

1.751*

Thyroid

24.800

5.770

23.700

3.683

25.800

3.084

19.900

4.999

Thymus

327.000

50.859

299.500

45.887

283.800

40.881

268.500

66.304

Heart

1124.200

302.274

1012.500

124.886

1064.500

96.709

980.300

97.803

Liver

11540.600

1760.413

11315.800

970.273

11666.100

2188.889

11380.600

1101.959

Spleen

731.100

94.090

647.100

77.229

655.900

66.534

611.400

84.733*

Kidneys

2606.200

250.519

2605.000

213.370

2591.100

339.513

2633.500

200.172

Adrenals

83.700

10.636

79.700

10.489

78.600

14.416

75.800

12.515

Testicles

3655.600

179.424

3575.400

226.663

3590.500

285.645

3677.700

233.694

Epidydimides

1415.400

115.627

1386.800

117.212

1387.400

154.952

1414.200

187.585

Prostate with vesicular and coagulating glands

2143.800

260.990

2244.000

235.569

2290.300

195.046

2363.500

408.443

Levator ani

310.000

19.299

308.800

41.886

302.000

31.337

280.900

38.240

Bulbocavernosus muscle complex

784.900

80.622

767.500

97.903

792.100

72.301

810.200

93.866

Cowper’s glans

108.300

19.482

101.200

14.038

105.800

17.492

112.600

15.291

Glans penis

90.900

14.708

92.400

8.140

99.700

13.200

95.300

12.996

 Y ± SD

* - statistically significant difference at p < 0.05 - Dunnett's test n - number of tested animals

Table 50. VL3. Main study. Absolute weights of internal organs [ mg ] - females.

 

Group

Examined organ

0

n=12

1

n=12

2

n=12

3

n=12

 

Brain

1898.583

80.4357

1905.250

72.227

1909.667

72.089

1896.417

114.773

Pituitary gland

15.417

1.831955

14.833

1.946

13.250

1.485*

13.417

1.443*

Thyroid

17.917

 

4.521833

18.167

 

2.517

17.417

 

3.397

18.500

 

3.090

Thymus

240.750

101.7771

184.917

53.294

220.333

71.970

211.500

80.636

Heart

795.000

92.63222

813.417

89.118

793.750

65.840

841.417

89.465

Liver

10809.250

1819.769

11455.500

1494.417

10139.333

1436.985

11069.250

1751.459

Spleen

615.250

52.42505

621.417

95.102

631.250

80.462

622.750

108.084

Kidneys

1868.667

203.5826

1885.083

162.555

1867.667

133.189

1847.583

190.692

Adrenals

94.833

10.18763

94.000

13.253

91.667

11.781

97.917

9.605

Ovaries

125.417

30.81457

116.667

16.472

126.417

33.489

123.167

14.721

Uterus with cervix

547.750

215.9192

454.833

100.221

537.167

173.530

531.250

206.144

f ± SD

* - statistically significant difference at p < 0.05 - Dunnett's test n - number of tested animals

Table 51. VL3. Main study. Relative weights of internal organs [%] - males.

 

Group

Examined organ

 

0

 

 

1

 

 

2

 

 

3

 

 

 

n=10

 

 

n=10

 

 

n=10

 

 

n=10

 

Brain

0.496

0.043

0.491

0.054

0.511

0.049

0.514

0.046

Pituitary gland

0.0006

0.0001

0.0005

0.0001

0.0005

0.0001

0.0004

0.0001*

Thyroid

0.006

0.001

0.006

0.001

0.006

0.001

0.005

0.001*

Thymus

0.079

0.009

0.073

0.011

0.069

0.010

0.066

0.016

Heart

0.271

0.073

0.247

0.016

0.258

0.014

0.242

0.013

Liver

2.765

0.216

2.770

0.225

2.811

0.326

2.806

0.166

Spleen

0.176

0.014

0.158

0.015*

0.159

0.016*

0.150

0.014*

Kidneys

0.629

0.062

0.638

0.051

0.627

0.056

0.650

0.030

Adrenals

0.020

0.002

0.020

0.003

0.019

0.003

0.019

0.002

Testicles

0.884

0.070

0.877

0.079

0.873

0.088

0.910

0.074

Epidydimides

0.342

0.026

0.340

0.028

0.336

0.024

0.349

0.037

Prostate with vesicular and

0.518

0.063

0.551

0.069

0.556

0.048

0.582

0.088

coagulating glands

 

 

 

 

 

 

 

 

 

 

 

 

Levator ani

0.075

0.010

0.075

0.009

0.073

0.008

0.069

0.006

Bulbocavernosus muscle complex

0.190

0.020

0.189

0.032

0.193

0.020

0.200

0.014

Cowper’s glans

0.026

0.005

0.025

0.003

0.026

0.004

0.028

0.003

Glans penis

0.022

0.005

0.023

0.003

0.024

0.003

0.023

0.002

f ± SD

* - statistically significant difference at p < 0.05 - Dunnett's test n - number of tested animals

 

Table 52. VL3. Main study. Relative weights of internal organs [%] - females.

Examined organ

Group

0

n=12

1

n=12

2

n=12

3

n=12

Brain

0.672

0.031

0.662

0.029

0.688

0.049

0.668

0.050

Pituitary gland

0.005

0.001

0.005

0.001

0.005

0.001

0.005

0.001

Thyroid

0.006

0.001

0.006

0.001

0.006

0.001

0.007

0.001

Thymus

0.085

0.034

0.064

0.018

0.079

0.025

0.076

0.035

Heart

0.281

0.025

0.282

0.023

0.285

0.012

0.296

0.030

Liver

3.819

0.615

3.968

0.439

3.641

0.461

3.873

0.444

Spleen

0.218

0.017

0.215

0.030

0.227

0.027

0.218

0.029

Kidneys

0.660

0.055

0.654

0.048

0.673

0.061

0.650

0.060

Adrenals

0.034

0.003

0.033

0.004

0.033

0.005

0.034

0.004

Ovaries

0.044

0.012

0.041

0.006

0.046

0.012

0.043

0.005

Uterus with cervix

0.193

0.071

0.158

0.037

0.194

0.065

0.190

0.090

Table 53. VL3. Main study. Absolute weights of internal organs [mg] - males (satellite groups).

 

Group

Examined organ

 

0 SAT

 

 

3SAT

 

 

 

n=10

 

 

n=10

 

Brain

2020.600

108.063

2004.400

137.999

Pituitary gland

9.300

1.494

9.900

1.969

Thyroid

24.300

3.368

23.900

3.784

Thymus

295.400

36.692

340.100

71.191

Heart

1046.300

159.712

1096.100

164.658

Liver

12122.900

2528.654

12430.000

1738.979

Spleen

737.200

157.512

787.500

133.114

Kidneys

2720.000

285.898

2842.400

236.942

Adrenals

77.400

12.158

76.400

9.947

Testicles

3576.700

245.092

3498.200

238.550

Epidydimides

1493.700

116.091

1446.300

131.374

Prostate with vesicular and coagulating glands

2407.000

307.769

2434.000

340.063

Levator ani

303.000

49.169

294.900

32.959

Bulbocavernosus muscle complex

767.200

106.542

787.300

75.918

Cowper’s glans

112.300

19.097

112.500

30.064

Glans penis

89.300

15.492

88.100

13.420

 

Table 54 VL3. Main study. Absolute weights of internal organs [mg] - females (satellite groups).

Examined organ

Group

0 SAT

n=10

3SAT

n=10

Brain

1935.700

110.800

1943.300

88.000

Pituitary gland

13.900

4.458

13.000

2.055

Thyroid

12.700

3.129

12.800

3.615

Thymus

299.100

125.152

334.700

87.987

Heart

658.800

67.952

678.900

56.011

Liver

6631.700

657.827

6540.500

824.436

Spleen

497.800

23.976

512.800

56.233

Kidneys

1648.500

160.506

1745.900

194.600

Adrenals

81.200

7.885

87.900

8.736

Ovaries

162.200

21.175

173.800

24.275

Uterus with cervix

704.300

293.645

733.100

149.730

Table 55.VL3. Main study. Relative weights of internal organs [%] - males (satellite groups).

 

Group

Examined organ

 

0 SAT

 

 

3SAT

 

 

 

n=10

 

 

n=10

 

Brain

0.478

0.064

0.461

0.047

Pituitary gland

0.000

0.0001

0.000

0.000

Thyroid

0.006

0.001

0.005

0.001

Thymus

0.070

0.011

0.078

0.017

Heart

0.244

0.022

0.250

0.026

Liver

2.812

0.342

2.833

0.241

Spleen

0.171

0.021

0.180

0.026

Kidneys

0.636

0.025

0.650

0.032

Adrenals

0.018

0.003

0.017

0.002

Testicles

0.842

0.085

0.801

0.043

Epidydimides

0.351

0.030

0.331

0.027

Prostate with vesicular and coagulating glands

0.567

0.087

0.556

0.069

Levator ani

0.071

0.008

0.068

0.007

Bulbocavernosus muscle complex

0.180

0.022

0.181

0.021

Cowper’s glans

0.026

0.005

0.026

0.007

Glans penis

0.021

0.005

0.020

0.003

 

Table 56.VL3. Main study. Relative weights of internal organs [%] - females (satellite groups).

Examined organ

Group

0 SAT

n=10

3SAT

n=10

Brain

0.745

0.048

0.751

0.051

Pituitary gland

0.005

0.002

0.005

0.001

Thyroid

0.005

0.001

0.005

0.001

Thymus

0.114

0.043

0.128

0.032

Heart

0.253

0.017

0.261

0.011

Liver

2.548

0.188

2.509

0.162

Spleen

0.192

0.016

0.198

0.023

Kidneys

0.633

0.038

0.671

0.042*

Adrenals

0.031

0.003

0.034

0.003

Ovaries

0.062

0.007

0.067

0.010

Uterus with cervix

0.269

0.108

0.285

0.066

* - statistically significant difference at p < 0.05 - student's T-test n - number of tested animals

Table 57. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Pituitary

microcyst

2

[1,4]

-

-

-

-

-

-

Thyroid

congestion

-

-

-

-

-

-

1

[9]

parathyroid

hypertrophy

-

1

P2]

-

-

-

-

-

 

congestion

1

[2]

-

-

1

[18]

-

-

2

[3,9]

Thymus

erythrocytorrhagia

-

-

-

-

-

-

1

[3]

effusion

-

1

[22]

-

1

[17]

 

 

-

 

lymphocytic tissue depletion

-

1

[17]

-

-

-

1

[13]

-

 

erythrocytorrhagia

3

[1,2,10]

-

-

1

[18]

-

-

1

[8]

 

leukocyte infiltration

1

[10]

-

-

 

-

-

1

[10]

 

Heart

foci of fibrosis

1

[10]

-

-

-

-

-

-

 

foci of fatty degeneration

1

[10]

-

-

 

-

-

-

 

left ventricular myocardium hypertrophy

-

-

-

1

[18]

-

1

[18]

-

 

Table 57 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Heart

left and right ventricular myocardium hypertrophy

1

[10]

-

-

-

-

-

-

 

congestion

1

[6]

-

3

[3,6,9]

2

[19,20]

1

[2]

2

[14,22]

2

[1,5]

2

[11,13]

 

erythrocytorrhagia

5

P6,8,9,10]

9

[12,13,14,

15,16,18,

20,21,22]

4

[3,6,7,9]

10

[11,14,15,

16,17,18,

19,20,21,

22]

3

P,3,4]

9

[13,14,15,

16,17,19,

20,21,22]

6

[1,2,4,5,7,

10]

8

[11,12,13,

14,15,17,

18,22]

 

edema

3

P6,10]

4

[13,14,18,

21]

3

[3,6,9]

2

[11,17]

-

2

[17,22]

2

[5,10]

1

[18]

Lungs

emphysema

4

[2,4,8,10]

9

[11,13,14,

15,17,19,

20,21,22]

-

10

[11,12,14,

15,16,18,

19,20,21,

22]

1

[2]

3

[13,14,21]

5

[1,4,5,6,10]

3

[11,18,22]

 

foamy cell infiltration

2

[7,8]

7

[11,15,17,

18,20,21,

22]

-

3

[11,16,17]

-

4

[13,19,20,

21]

1

[6]

1

[18]

 

lymphocyte

infiltration

1

[8]

-

-

-

-

-

1

[10]

 

leukocyte infiltration

1

[10]

-

-

-

-

-

-

 

Table 57 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

 

congestion

1

[10]

2

[15,17]

-

-

-

-

1

[9]

 

effusion

1

[10]

-

-

-

-

-

-

Liver

bile ducts proliferation

1

[10]

-

-

-

-

-

-

 

Browicz-Kupffer cell proliferation

1

[10]

-

-

-

-

-

-

 

hepatocyte fine- droplet fatty degeneration

1

[3]

-

-

-

-

-

-

Spleen

congestion

-

1

[18]

-

-

-

-

-

depletion of the white pulp

-

2

[17,18]

-

-

-

-

-

Pancreas

islets of Langerhans hypertrophy

1

1

P1]

-

-

-

-

3

[4,6,8]

ducts proliferation

-

1

P1]

-

-

-

-

-

Jejunum

lymphatic follicle reactive hyperplasia

1

[5]

-

-

-

-

-

-

Ileum

lymphocyte

infiltration

-

1

[11]

-

-

-

-

-

  

Table 57 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0f

1m

2m

3f

organ / tissue

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Cecum

lymphatic follicle reactive hyperplasia

1

[7]

-

-

-

-

-

-

1

[14]

Colon

lymphatic follicle reactive hyperplasia

1

[4]

-

-

-

-

-

2

P,4]

1

[14]

 

erythrocytorrhagia

1

[8]

1

[15]

-

-

-

-

1

[2]

 

glomeruli congestion

6

[1,2,3,5a

7]

4

[12,17,20,

22]

-

-

-

-

10

[1,2,3,4,5,6,7

8,9,10]

6

[13,14,15,

17,20,22]

 

lymphocyte

infiltration

-

2

[12,21]

-

-

-

-

1

[8]

Kidneys

glomeruli

hypertrophy

9

[1,2,3,5,6,7,8

,9,10]

7

[12,15,17,

19,20,21,

22]

-

-

-

-

9

[1,3,4,5,6,7,8

9,10]

9

[12,13,14,

15,17,19,

20,21,22]

glomeruli

atrophy

4

[1,8,9,10]

5

[15,17,18,

20,21]

-

-

-

-

10

[1,2,3,4,5,6,7

8,9,10]

8

[12,13,14,

15,19,20,

21,22]

 

Bowman's capsule extension

1

[9]

2

[18,21]

-

-

-

-

6

[2,3,5,6,8,9]

 

retention microcysts

-

2

[12,15]

-

-

-

-

1

[8]

2

[14,21]

 

hyaline casts

-

1

[12]

-

-

-

-

-

 

 Table 57 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0f

1m

2m

3f

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Adrenals

left adrenal gland atrophy with medulla microcyst; right adrenal gland hyperplasia

-

-

-

-

-

-

-

1

[11]

Urinary bladder

lymphocyte

infiltration

-

-

-

-

-

-

5

[1,3,5,6,7]

Prostate

congestion

2

[1,9]

 

-

-

-

-

2

[2,8]

 

edema

2

[1,9]

-

-

-

-

-

-

lymphocyte

infiltration

5

[1,3,7,9,10]

-

3

P,4,9]

-

6

[2,4,5,7,8,9]

-

6

[2,3,5,8,9,

10]

Uterus

congestion

-

-

-

1

[16]

-

2

[16,20]

-

effusion

-

-

-

2

[12,16]

-

2

[20,21]

-

fibroid

-

-

-

2

[12,16]

-

1

[20]

-

Mandibular lymph nodes

congestion

1

[6]

-

-

-

-

-

-

hyperplasia

1

[6]

-

-

-

-

-

-

lymphocytic tissue depletion

-

1

[17]

-

-

-

-

-

 

Table 57 cont. VL3. Main study. Histopathological lesions: group 0, group 1,group 2, group 3.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0f

1m

2m

3f

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Males

n=10

Females

n=12

Mesenteric lymph nodes

congestion

-

-

-

-

-

-

1

[6]

hyperplasia

1

[6]

-

-

-

-

-

1

[6]

lymphocytic tissue depletion

-

1

[17]

-

-

-

-

-

[]-computer numbers of animals in which the histopathological lesions were found n - number of tested animals f - full histopathology

m - histopathology of thyroid, testicles, ovaries with oviducts, epididymides, prostate, seminal vesicles, coagulating glands and gross lesions were conducted

Table 58. VL3. Main study. Histopathological lesions: group 0 SAT, group 3 SAT.

Examined organ / tissue

Type of change

GROUP / sex / number of animals / computer numbers of animals

0 SATf

3 SATf

Males

n=10

Females

n=10

Males

n=10

Females

n=10

Pituitary

hypertrophy

-

1

[44]

-

-

microcyst

2

[35,40]

-

-

-

Thymus

congestion

2

[37,38]

-

2

[36,38]

1

[49]

effusion

1

[31]

3

[45,48,49]

2

[34,36]

4

[42,43,48,49]

Heart

congestion

1

[36]

-

3

[36,37,38]

-

erythrocytorrhagia

1

[36]

-

4

[36,37,38,40]

1

[46]

foci of fibrosis

2

[36,38]

-

3

[34,37,40]

-

left ventricular myocardium hypertrophy

3

[36,37,40]

-

2

[34,37]

-

 

Table 58 cont. VL3. Main study. Histopathological lesions: group 0 SAT, group 3 SAT.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0 SATf

3 SATf

organ / tissue

Males

n=10

Females

n=10

Males

n=10

Females

n=10

 

congestion

4

[32,35,39,40]

-

4

[34,36,37,40]

1

[47]

 

erythrocytorrhagia

8

[31,32,33,34,35, 37,39,40]

9

[41,43,44,45,46,

47,48,49,50]

9

[31,32,33,34,36,

37,38,39,40]

9

[41,42,43,44,45,

46,47,48,49]

 

edema

3

[31,32,33]

-

4

[32,34,37,40]

1

[47]

Lungs

emphysema

5

[31,32,36,37,39]

4

[43,44,48,50]

4

[31,33,35,40]

4

[46,48,49,50]

 

foamy cell infiltration

-

1

[50]

1

[36]

1

[41]

 

lymphocyte infiltration

1

[40]

-

-

-

 

inflammatory

infiltration

1

[35]

-

-

-

 

foci of fibrosis

1

[40]

-

-

-

 

congestion

1

[35]

-

1

[37]

-

Liver

effusion

-

-

1

[37]

-

 

Browicz-Kupffer cell proliferation

1

[34]

1

[46]

-

-

 

focus of fibrosis

-

1

[46]

-

-

 

Table 58 cont. VL3. Main study. Histopathological lesions: group 0 SAT, group 3 SAT.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0 SATf

3 SATf

organ / tissue

Males

n=10

Females

n=10

Males

n=10

Females

n=10

Spleen

congestion

1

[37]

-

1

[37]

-

hemosiderin deposits

-

-

1

[35]

2

[47,48]

 

islets of Langerhans congestion

2

[35,37]

-

2

[35,37]

2

[45,48]

Pancreas

islets of Langerhans hypertrophy

7

[33,34,35,36,37,

38,401

2

[41,50]

3

[31,35,37]

3

[42,45,48]

lymphocyte infiltration

2

[35,36]

1

[41]

-

-

 

foci of atrophy of lobules with fibrosis

-

-

1

[35]

-

 

ducts proliferation

-

-

1

[37]

-

Jejunum

lymphatic follicle reactive hyperplasia

-

1

[43]

-

-

Cecum

lymphatic follicle reactive hyperplasia

1

[34]

-

2

[34,36]

3

[41,42,45]

Colon

lymphatic follicle reactive hyperplasia

-

1

[50]

-

-

lymphocyte infiltration

-

1

[48]

-

1

[50]

 

Table 58 cont. VL3. Main study. Histopathological lesions: group 0 SAT, group 3 SAT.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0 SATf

3 SATf

organ / tissue

Males

n=10

Females

n=10

Males

n=10

Females

n=10

 

glomeruli congestion

5

[35,36,37,38,40]

4

[41,45,47,50]

5

[32,33,34,35,37]

6

[43,44,45,47,48,

50]

Kidneys

glomeruli hypertrophy

6

[32,35,36,37,38,

40]

4

[41,45,47,50]

6

[32,33,34,35,37,

39]

5

[44,45,47,48,50]

glomeruli atrophy

3

[32,37,38]

2

[45,47]

3

[34,35,39]

3

[44,48,50]

 

Bowman's capsule extension

1

[32]

-

1

[39]

-

 

retention microcysts

1

[37]

2

[45,47]

-

-

Urinary bladder

lymphocyte infiltration

-

-

1

[34]

-

Prostate

congestion

1

[38]

-

-

-

lymphocyte infiltration

3

[32,34,38]

-

2

[35,40]

-

Ovaries

congestion

-

1

[43]

-

-

 

Table 58 cont. VL3. Main study. Histopathological lesions: group 0 SAT, group 3 SAT.

 

 

GROUP / sex / number of animals / computer numbers of animals

Examined

Type of change

0 SATf

3 SATf

organ / tissue

Males

n=10

Females

n=10

Males

n=10

Females

n=10

 

congestion

-

-

1

[34]

1

[41]

Mandibular

erythrocytorrhagia

-

-

-

1

[41]

lymph nodes

effusion

-

-

1

[34]

1

[43]

 

lymphocytic tissue reactive hyperplasia

-

1

[42]

-

-

Nodule

(gross lesion isolated from the subcutaneous tissue of cervical area above the salivary gland)

lipoma

-

-

1

[31]

-

Nodule

(gross lesion isolated from the fat tissue of the left ovary area)

lipoma

-

-

-

1

[41]

Nodule

(gross lesion isolated from the fat tissue of the pelvis cavity inlet area)

lipoma

-

-

-

1

[46]


Applicant's summary and conclusion

Conclusions:
The oral 28d-NOAEL in rats was determined to be 300 mg/kg bw/day since no adverse effects were observed at the highest dose tested.
Executive summary:

A Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in rats according to the OECD Guideline No. 422 (2016) with GLPs. The study involved the determination of the effects of the test item on body weights, food intake, behaviour, clinical, haematological, coagulation, biochemical, enzymatic, urine, and hormonal parameters and the evaluation of gross and histopathological lesions in tissues and internal organs. Two dose range finding tests and the main study were conducted. The first Dose range finding stated that 7-day exposure of rats to VL3 at the doses of 250, 500 and 1000 mg/kg b.w./day cause one treatment-related change, i.e. decrease in number of leukocytes (males from group 1 and females from group 1 and 3).The second Dose-range finding test revealed that 7-day exposure of rats to VL3 at the doses of 31.3, 62.5 and 125.0 mg/kg b.w./day did not cause treatment-related changes. The main study was performed on 60 males and 68 females of Wistar rats treated with vehicle control or with 33.3, 100, 300 mg/kg b.w./day of test item. The animals received the test item/vehicle control by gavage once a day (7 days/week). After two weeks of initial treatment, the animals were mated. The test item/vehicle control was administered to males for 28 days (2 weeks prior to mating, during the mating and post-mating period). Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (about 51-59 days, depending on the duration of the mating and pregnancy period). Furthermore, two satellite groups were used;: a control group and one treated group which was given VL3 at the dose of 300 mg/kg b.w/day. The satellite groups were not be mated. Males from groups 0 SAT and 3 SAT were treated for 28 days. Females from groups 0 SAT and 3 SAT were treated for 53 days (on average as long as females from groups 0, 1, 2 and 3). After that, males and females from satellite groups were observed for 14 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects. During the experiment, no mortality of the adults was observed. There were no differences in physical appearance and behaviour of adult animals between the treated and the control groups. No treatment-related clinical signs, ophthalmic alterations or significant differences in body weights and food intake were observed. Statistical analysis of the clinical-chemical examinations results obtained in males and females of group 1, 2 and 3 and satellite groups revealed some statistically significant changes however all of them were considered as not related with the administration of the test item. There were gross changes observed in control group 0, treated groups 1, 2 and 3 and satellite groups 0 SAT and 3 SAT. However, these changes occurred regardless of the degree of exposure to the test item (this changes were observed both in control and treated groups). Statistical analysis of absolute and relative weights of internal organs revealed statistically significant changes but these organ weight disorders seem to be accidental. The macroscopic and the histological examinations of the rats treated with the test item at the doses of 33.3, 100, and 300 mg/kg b.w./day did not reveal any toxic effects. Additionally, based on results, it was concluded that the test item is not immunotoxic for rats.

In conclusion, the treatment of adult (male and female) rats with the test item at the dose levels of 33.3, 100 and 300 mg/kg bw/day by the repeated oral administration, revealed no adverse effects. Based on the results of clinical studies, clinical-chemical, pathomorphological and histopathological examinations, the no observed adverse effect level (NOAEL) of the test item for systemic toxicity was determined to be 300 mg/kg b.w./day.