tri{[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy}(vinyl)silane

Endpoint summary

Description of key information

Key study: Test method OECD 422. GLP study. 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.

Key study: Test method OECD 408 and EU Method B.26. GLP study. The NOAEL for the subchronic repeated dose toxicity of the test item in rats by oral route was determined to be 250 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26.08.2020-24.03-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

During the experiment the humidity dropped below 30% or exceed 70% (22.1 – 92.9%) a few times as well as the temperature dropped below 20°C (15.9 – 22.5°C). This changes were temporary and did not influence on the study course.

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(Cmdb: Wi; outbred)

Details on species / strain selection:

The animal species (Rattus norvegicus) is recognized by OECD guideline No. 408 as a recommended test system for repeated dose oral toxicity study in rodents

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok kept behind a breeding barrier (unit number in the register of units entitled to husbandry of laboratory animals: 043)
- Age at study initiation: 6-7 weeks
- Weight at study initiation:
Group 0: males 210.1g (193g – 236g); females 155.8g (143g – 177g)
Group 1: males 210.2g (190g – 229g); females 155.8g (146g – 175g)
Group 2: males 210.1g (194g – 233g); females 155.8g (144g – 176g)
Group 3: males 210.1g (192g – 227g); females 155.8g (142g – 168g)
Group 0 SAT: males 197.3g (180g – 218g); females 160.9g (151g – 175g)
Group 3 SAT: males 197.4g (188g – 218g); females 160.9g (151g – 172g)
- Fasting period before study: no
- Housing: The animals were kept in air-conditioned cages (58x37x21) with a plastic bottom and covered with wire-bar lids and UV-sterilized wood chips were used as bedding.
- Diet: Altromin 1324 P TPF” (Phytoestrogen-poor, Total Pathogen Free) standard granulated fodder produced by Altromin Spezialfutter GmbH & Co. KG, Lage, Germany (batch numbers: 5/20, 6/20)
- Water: Drinking tap water ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Ad libitum access to the “Altromin 1324 P TPF” (Phytoestrogen-poor, Total Pathogen Free) standard granulated fodder produced by Altromin Spezialfutter GmbH & Co. KG, Lage, Germany (batch numbers: 5/20, 6/20) (Appendix No. 8) and drinking tap water.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.9 – 22.5°C
- Humidity (%): 22.1 – 92.9%
- Air changes (per hr): about 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Batch number / expiry date: 20259 / 05.2021, 20342 / 06.2021; Storage / stability: stored at room temperature (15-25 °C); Manufacturer: Oleofarm Sp. z o.o., ul. Mokronoska 8, 52-407 Wrocław; Packaging: brown glass bottles, volume 0.5 L

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose of 62.5 mg/kg b.w./day: There was 12.5 mg VL3 in 1 mL (13.6 mg in 1g) of corn oil solution.
Dose of 250 mg/kg b.w./day: There was 50 mg VL3 in 1 mL (54.4 mg in 1g) of corn oil solution.
Dose of 1000 mg/kg b.w./day: There was 200 mg VL3 in 1 mL (217.7 mg in 1g) of corn oil solution.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is soluble in inert organic solvents like corn oil
- Concentration in vehicle: 12.5, 50 and 200 mg/mL
- Amount of vehicle (if gavage): The volume of the dose formulation (X) administered to each animal was calculated according to the formula: X= 0.5 mL x animal body weight [g]/100 g.
- Lot/batch no. (if required): 20259
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Equipment:
Gas Chromatograph (6890N network GC System, Agilent)
Usual laboratory material (flasks, pipettes, balances)

Analytical method:
The concentration of VL3 was chemically determined with gas chromatographic method with FID. The validation was involved the assessment of the range of linearity of the analytical graph, specificity, precision (repeatability), recovery, and limits of quantification and detection. The test item was used as the standard.

Method validation:
The method was validated at two fortification levels (5.0 mg/g and 50 mg/g) for corn oil. For each fortification level and matrix, five replicates were prepared and analysed. Additionally, two replicates of unfortified samples were analysed (control samples).
The method validation conducted to prove the validity of the applied method showed that the method was suitable to determine VL3 in corn oil. The mean recoveries for matrix ranged from 96.6% to 100.6%. Therefore, the obtained recovery values fulfill the legal requirement for recovery values (SANCO/3029/99 rev. 4).
For analyte, the relative standard deviations (RSD) were below 10% and no significant peak occurred at the retention time, so absence of interference was confirmed.

Storage Stability of Samples:
The experiments demonstrate that VL3 was not stable in corn oil over a time period of 4 days, when stored at ambient temperature (day 0 result of recovery is 98.6%, day 4 result of recovery is 10.7% (result below LOD i.e. 1.5 mg/g).

Chemical analysis during the study:
Test item formulations were transferred thrice for chemical analysis on: 28.08.2020, 08.10.2020 and 23.11.2020. The mean recovery values for test samples ranged from 92.4 to 103.6%. All results were in the range from 80 to 120%, therefore the acceptance criteria for test samples for toxicological study on animals was met. The results of the determination of VL3 in test samples are presented in Table 3.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Animals from all groups were treated with the test item/control item once daily, seven days a week (the first dosing day = day 0).

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

GROUP 0 AND SAT0

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

GROUP 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

GROUP 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

GROUP 3 AND SAT 3

No. of animals per sex per dose:

10 males and 10 females in each group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels as well as vehicle were established based on existing data according to OECD guideline No. 422. In this guideline, the doses were: 33.3 mg/kg b.w., 100 mg/kg b.w. and 300 mg/kg b.w. where no test item- related lesions in parental animals were stated. Dose of 1000 mg/kg b.w./day is the accepted dose by OECD to be the limit dose up to which adverse effects should be checked. Due to longer time of exposition, higher level of doses with 4-fold intervals in current study results of the study should allow for estimating the NOAEL or LOAEL.
- Rationale for animal assignment (if not random): The animals were randomly divided according to stratification by body weight into four experimental groups and two satellite groups.
- Fasting period before blood sampling for clinical biochemistry: 15 hours
- Post-exposure recovery period in satellite groups: 21 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day usually at the beginning and end of each day
- Cage side observations checked: mortality and morbidity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the test item / vehicle administration on day 0 and then once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the beginning of the experiment (day 0) and then twice a week during the experiment and on the last day of experiment

FOOD CONSUMPTION: Food consumption was measured, although it is not a feeding study.
Food in cages was weighted to determine food intake once a week during the experiment.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the study begins and at the end of the study (on the 89th day for groups 0, 1, 2 and 3 or on the 110th day for groups 0 SAT and 3 SAT). In case of the satellite groups, it also took place after the end of the test item / vehicle administration (on the 89th day)
- Dose groups that were examined: 0, 1, 2, 3, 0 SAT and 3 SAT

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at main study termination (90th day) and on satellite animals on the day of recovery sacrifice (111th day)
- Anaesthetic used for blood collection: Yes, xylazine-ketamine mixture at a dose of: 10 mg/kg b.w. of xylazine and 100 mg/kg b.w. of ketamine
- Animals fasted: Yes
- How many animals: All of them
- Parameters examined: leukocyte count, erythrocyte count, thrombocyte count, level of hemoglobin, hematocrit value. The morphological results contain the calculated erythrocyte indices such as: mean corpuscular volume (MCV), mean corpuscular hemoglobin weight (MCH), mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at main study termination (90th day) and on satellite animals on the day of recovery sacrifice (111th day)
- Animals fasted: Yes
- How many animals: All of them
- Parameters examined: total protein, albumin, level of globulin, total cholesterol, urea nitrogen, creatinine, glucose, bilirubin, calcium, sodium, potassium, chlorides, inorganic phosphorus, high - density lipoproteins (HDL) and low - density lipoproteins (LDL).
- Enzymatic examinations: The activity of the following enzymes were determined in serum: − aspartate aminotransferase (AST).
− alanine aminotransferase (ALT).
− alkaline phosphatase (AP).
− gamma - glutamyl transferase (GGT).

SERUM HORMONES: Yes
- Time of blood sample collection: at a comparable time of the day
- Animals fasted: Not specified
- How many animals: All of them
- The level of thyroxine (T4), triiodothyronine (T3) in serum was determined ELISA method using Calbiotech kits: Mouse/Rat Triiodothyronine (T3) ELISA , Mouse/Rat Thyroxine (T4) ELISA. The level of thyroid stimulating hormone (TSH) in serum was determined ELISA. All hormones determinations were made using FLUOstar Omega reader.

URINALYSIS: Yes
- Time schedule for collection of urine: at main study termination (90th day) and on satellite animals on the day of recovery sacrifice (111th day)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume of urine (mL), urine colour, specific gravity, pH, protein (g/L), glucose (mmol/L), ketone bodies (mmol/L), bilirubin (qualitative test), blood (Ery/μL), urobilinogen (μmol/L), leukocyte count (leu/μL) and nitrites (qualitative test).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Groups 0, 1, 2 and 3: during the thirteenth week of the experiment (between the 85th and 89th day). Satellite groups: during the thirteenth week of the experiment - measurement 1 (between the 85th and 89th day) and at the end of the sixteenth week of the experiment - measurement 2 (between 104th and 110th day)
- Dose groups that were examined: 0, 1, 2, 3, 0 SAT and 3 SAT
- Battery of functions tested: open field observations, evaluation of responses to simuli, measurement of the fore- and hindlimb grip strength, evaluation of locomotor activity.

IMMUNOLOGY: No

OTHER:
Bone marrow examinations: Bone marrow was obtained from a dissected femur after animal euthanasia. The collected bone marrow pellet was transferred to an eppendorf tube containing 50μl FBS. After a few minutes and gently mixing, a smear was made. The slides were stained May-Grunwald-Giemsa method and were qualitatively and quantitatively evaluated through the determination of the number of individual nuclear cells per 1000 test cells.
The number of following cells were determined:
- within the erythrocyte system,
- within the leukocyte system,
- within the range of different cells: lymphocytes, monocytes, plasmocytes, megakaryocytes and other cells
Bone marrow was assessed in males and females from the control groups i.e. group 0 and recovery group 0 SAT and highest dosage group i.e. group 3 and recovery group 3 SAT. Microscopic analysis was not extended to groups 1 and 2, as no treatment-related changes were found in groups 3 and 3 SAT.

Coagulation examinations: Prothrombin time (PT) and activated partial thromboplastin time (APTT) was determined in plasma collected in tubes containing anticoagulant sodium citrate 3.2% at a 1:9 ratio (sodium citrate:blood). PT and APTT evaluation was based on the changes of the optical density measured chronometrically with the use of the Coag Chrom 3003 coagulometer and BioKsel reagents.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals which survived the experiment after the collection of blood from the heart for the clinical-chemical examinations, and were euthanized with a xylazine-ketamine mixture at a dose of 20 mg/kg b.w. of xylazine and 200 mg/kg b.w. of ketamine, as well as one female euthanized for humanitarian reasons during the experiment, were subjected to post-mortem examinations. Gross examination which was carried out on all animals participating in the experiment, consisted of thorough observations of the external body surface and all orifices, all natural apertures as well as the cranial, thoracic and abdominal cavities with their content. From each animal from which the blood was taken for clinical-chemical tests, one femur was collected for bone marrow examination.
From all females involved in the experiment during gross examination vaginal smears were collected and estrus cycle of all females was determinated.
After the end of experiment, absolute weights of particular internal organs of all surviving animals were determined. The organs were as follows: brain, thymus, heart, liver, spleen, kidneys, adrenal glands, testes, epididymides, uterus, ovaries, thyroid with parathyroid, prostate with the seminal vesicles and coagulating glands, pituitary gland.
The relative weight of internal organs were calculated on the basis of the absolute organ weight with reference to the live animals’ body weight after fasting.

HISTOPATHOLOGY: Yes

The following organs and tissues of all animals were preserved for histopathological examinations: brain (representative regions including cerebrum, cerebellum and medulla/pons), pituitary, spinal cord (cervical, mid-thoracic and lumbar), eyes with the optic nerve (if changes were observed during ophthalmological examinations), mandibular salivary glands, stomach, small and large intestines (including Peyer's patches if macroscopical visible) i.e.: duodenum, jejunum, ileum, cecum, colon, and rectum; liver, pancreas, kidneys, adrenal glands, urinary bladder, spleen, heart, aorta, thymus, lymph nodes (mandibular and mesenteric), thyroid with parathyroid, oesophagus, trachea, lungs, ovaries, uterus with cervix, vagina, vaginal smears, testes, epididymides, prostate with seminal vesicles and coagulating glands, skeletal muscle including peripheral nerve (sciatic or tibial), femur, skin, mammary gland and all tissues showing abnormality. Collected organs and tissues were fixed in a 10% solution of formalin, except the testes and epididymides which were fixed in modified Davidson’s fluid. Bones were subjected to a decalcification.
Preserved samples of all collected organs and tissues were embedded in paraffin, stained with hematoxylin and eosin, and evaluated under a light microscope.
Due to no observed changes in ophthalmological examinations in all surviving animals, histopathological examination of eyes with optic nerves was not conducted. It was conducted only in the female euthanized for humanitarian reasons during the experiment, due to lack of ophthalmological examination.
Full histopathological examination was performed on all preserved organs and tissues of all animals from the control and high dose groups (group 0 and group 3). All gross lesions were examined.
Due to occurrence of the treatment-related lesions in urinary bladder and kidneys of animals from high dose group (group 3), histopathological examination of these organs from other dosage groups, as well as from satellite groups, was conducted.

Statistics:

The results obtained in the study were subjected to statistical analysis, using following programs: Excel 2007 and STATISTICA 10.0 PL.
The results are presented in tables in the form of average values and standard deviation.
The treated groups i.e. groups 1, 2, and 3 were compared to the control group (group 0).
The course of the statistical analysis (group 0, 1, 2, 3) was as follows:
- first of all, the normality of the distribution was examined using the Shapiro-Wilk test and the homogeneity of variance by the Brown-Forsythe test,
- results which were characterized by normal distribution and homogeneous variances were compiled using a one-way analysis of variance followed by Dunnett's test,
- in the absence of normality of distribution or non-homogeneous variances, the nonparametric Kruskal-Wallis test was used, followed by Dunnett's test.
The treated satellite groups i.e. 3 SAT were compared to the satellite control groups (group 0 SAT). The following statistical tests were used to assess intergroup relationships for groups 0 SAT and 3 SAT:
- first of all, the normality of the distribution was examined using the Shapiro-Wilk test and the homogeneity of variance by the Brown-Forsythe test,
- results which were characterized by normal distribution and homogeneous variances were compiled using the Student’s,
- parameters characterized by non-normal distribution were compiled using Mann-Whitney U test,
- parameters characterized by normal distribution and non-homogenous variance were compiled by Cochrane-Cox test.
Ordinal variables (part of urine parameters) were analyzed using Kruskal-Wallis test followed by Dunnett's test (main groups) or Mann-Whitney U-test (satellite groups).
The weights of internal organs are presented in tables as absolute values as well as relative values (with reference to the body weight of live animals and to weight of brain), in the form of average values and standard deviation.
The obtained results were analyzed statistically at p ≤ 0.05

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Group 0:
− 2 males and 1 female showed thinning of coat on one or both cheeks at some point during the experiment and the female posteriorly showed alopecia on the left cheek.

Group 1:
− 1 male showed thinning of coat on the right cheek and 1 female showed thinning of coat on both sides of neck and then alopecia on both sides of neck.

Group 3:
− 1 male showed thinning of coat on both forearms.
− 2 males: showed thinning of coat on both cheeks and one of them alopecia on the left cheek.
− 1 female showed thinning of coat on both sides of neck and then alopecia on both sides of neck
− 3 females showed thinning of coat on one or both cheeks.
− 1 female showed thinning of coat on both forearms.

Group 0 SAT:
− 1 female showed thinning of coat on the right thoracic limb, then alopecia on the right thoracic limb and on the right side of abdomen and finally then alopecia on the right thoracic limb, on the right side of abdomen and the left forearm.

Group 3 SAT:
− 1 male showed alopecia on both forearms.
− 1 male showed thinning of coat on both sides of loins.
− 1 female showed thinning of coat on the right cheek.
− 1 female showed dejection, distinct decrease of locomotor activity, no intake of food and water. Later, this animal lied on its abdomen motionless, with no reaction to external stimuli and catching and no intake of food and water, pallor of the skin and irises, bristled coat and hypothermia. It was euthanized.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

MALES:
- Significant increase of average food intake was found in the males of groups 1 and 3 (12th week), in the group 2 (10th week), and group 3 SAT (6th, 8th, 12th, 13th, 14th and 15th week).
- Significant decrease of average food intake was found in the males of group 3 SAT(2nd week).

FEMALES:
- Significant increase of average food intake was found in the females of group 1 (4th week), and group 3 SAT (15th week).
- Significant decrease of average food intake was found in the females of group 3 SAT in (1st week).

These effects are considered to be accidental because lack of confirmation in body weight measurements.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES:
- Decrease in the MCV, MCH and reticulocytes values in group 3.

FEMALES:
- Decrease in the erythrocytes number and MCHC value and increase in the MCV value and number of leukocytesin group 3 SAT.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

-Increase in the potassium concentration in group 3 SAT.

Endocrine findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

MALES:
-Significant decrease in pH value and increase in the number of transitional epithelia and leukocytes in group 3.

FEMALES:
- Increase in the number of transitional epithelia and leukocytes in group 3 and decrease in the amount of ammonium- magnesium phosphates in group 3 SAT.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- Significant decrease of the horizontal locomotor activity and vertical locomotor activity in males from group 3 SAT, probable related to the increase of potassium levels.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Significant increase of weight in males from group 3 in kidneys.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Significant amount of males with thickened wall in urinary bladder from group 3 (4) compared to the rest of the main groups (0)

-Significant amount of females with thickened wall in urinary bladder from group 3 (6) compared to the rest of the main groups (0)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES:
- Group 3: Significant amount with moderate or severe papillary hyperplasia of urothelium in urinary bladder and left kidney, and hyperplasia of papilla epithelium effects in both kidneys.
- Group 3: Significant amount with minimal, slight or moderate renal pelvis and papillary hyperplasia of urothelium effects in both kidneys.
- Group 3 and 3SAT: Significant amount of basophilic (renal) tubules was observed in both kidneys.

FEMALES:
- Group 3: Significant amount with severe papillary hyperplasia of urothelium in urinary bladder
- Group 3: Significant amount with slight, moderate or severe papillary hyperplasia of urothelium and hyperplasia of epithelium effects in both kidneys.
- Group 3: Significant amount with minimal, slight or moderate renal pelvis and papillary hyperplasia of urothelium effects in both kidneys.

Histopathological findings: neoplastic:

not examined

Details on results:

ENZYMATIC EXAMINATIONS:
-Increase in the AST and ALT activity in males in group 3 SAT compared to the control group 0 SAT.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 4. Mortality and clinical observations – main groups

Parameter	GROUP / DOSE [mg/kg b.w./day]
	0 / 0		1 / 62.5		2 / 250		3 / 1000
	males	females	males	females	males	females	males	females
Animals in group	10	10	10	10	10	10	10	10
Death of animals	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
Euthanized animals	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10	0 / 10
Animals with clinical signs	2 / 10	1 / 10	1 / 10	1 / 10	0 / 10	0 / 10	3 / 10	5 / 10
CLINICAL SIGNS
Survived animals
Thinning coat or alopecia of particular part of body	2 / 10	1 / 10	1 / 10	1 / 10	0 / 10	0 / 10	3 / 10	5 / 10

 

Table 5. Mortality and clinical observations – satellite groups

Parameter		GROUP / DOSE [mg/kg b.w./day]
		0 SAT / 0		3 SAT / 1000
	males		females	males	females
Animals in group	10		10	10	10
Death of animals	0 / 10		0 / 10	0 / 10	0 / 10
Euthanized animals	0 / 10		0 / 10	0 / 10	1 / 10
Animals with clinical signs	0 / 10		1 / 10	2 / 10	2 / 10
CLINICAL SIGNS
Survived animals
Thinning coat or alopecia of particular part of body	0 / 10		1 / 10	2 / 10	1 / 10
Euthanized animals
dejection and distinct decrease of locomotor activity, animal lied on its abdomen motionless, no reaction to external stimuli and catching, no intake of food and water, pallor of the skin and irises, bristled coat and hypothermia	0 / 10		0 / 10	0 / 10	1 / 10

 

Table 6. Average food intake [g/100 g b.w./day] – main groups 

Week	GROUP / DOSE [mg/kg b.w./day]
	0 / 0		1 / 62.5		2 / 250		3 / 1000
	♂ n=10	♀ n=10	♂ n=10	♀ n=10	♂ n=10	♀ n=10	♂ n=10	♀ n=10
1	9.25 ± 0.21	8.60 ± 0.15	9.14 ± 0.27	8.69 ± 0.23	9.21 ± 0.28	8.60 ± 0.43	8.98 ± 0.23	8.75 ± 0.53
2	8.11 ± 0.09	8.11 ± 0.20	8.02 ± 0.20	8.37 ± 0.18	8.34 ± 0.53	7.92 ± 0.29	7.97 ± 0.34	8.04 ± 0.39
3	7.18 ± 0.37	7.61 ± 0.49	7.00 ± 0.33	7.84 ± 0.30	7.34 ± 0.31	7.32 ± 0.26	7.18 ± 0.25	7.49 ± 0.19
4	6.26 ± 0.36	6.90 ± 0.24	6.31 ± 0.28	7.34 ± 0.38*	6.55 ± 0.20	6.76 ± 0.33	6.44 ± 0.25	6.95 ± 0.40
5	5.75 ± 0.31	6.38 ± 0.33	5.83 ± 0.30	6.81 ± 0.59	5.85 ± 0.09	6.19 ± 0.46	5.91 ± 0.26	6.48 ± 0.28
6	5.50 ± 0.09	6.28 ± 0.30	5.55 ± 0.22	6.50 ± 0.24	5.41 ± 0.08	6.08 ± 0.37	5.44 ± 0.25	6.23 ± 0.26
7	5.30 ± 0.22	5.81 ± 0.20	5.29 ± 0.22	6.18 ± 0.37	5.27 ± 0.09	5.78 ± 0.37	5.29 ± 0.26	5.79 ± 0.16
8	4.90 ± 0.32	5.46 ± 0.20	4.99 ± 0.22	5.75 ± 0.45	4.96 ± 0.14	5.39 ± 0.34	4.98 ± 0.32	5.41 ± 0.13
9	4.76 ± 0.22	5.24 ± 0.11	4.80 ± 0.27	5.46 ± 0.23	4.58 ± 0.14	5.31 ± 0.39	4.66 ± 0.28	5.33 ± 0.09
10	4.63 ± 0.22	5.26 ± 0.15	4.59 ± 0.17	5.49 ± 0.17	4.38 ± 0.14*	5.21 ± 0.45	4.52 ± 0.14	5.31 ± 0.14
11	4.43 ± 0.17	5.08 ± 0.31	4.47 ± 0.07	5.32 ± 0.37	4.30 ± 0.12	4.99 ± 0.36	4.46 ± 0.18	5.09 ± 0.07
12	4.16 ± 0.27	5.17 ± 0.13	4.39 ± 0.11*	5.20 ± 0.25	4.17 ± 0.18	4.96 ± 0.41	4.44 ± 0.24*	5.10 ± 0.40
13	4.48 ± 0.18	5.38 ± 0.50	4.56 ± 0.03	5.41 ± 0.56	4.29 ± 0.24	5.32 ± 0.46	4.63 ± 0.30	5.07 ± 0.13

^x ± SD

n - number of animals per group

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

 

Table 7. Average food intake [g/100 g b.w./day] – satellite groups

Week	GROUP / DOSE [mg/kg b.w./day]
	0 SAT / 0		3 SAT / 1000
	♂ n=10	♀ n=10	♂ n=10	♀ n=10
1	9.60 ± 0.05	9.01 ± 0.22	9.41 ± 0.28	8.66 ± 0.32**
2	8.64 ± 0.18	8.30 ± 0.11	8.31 ± 0.26*	8.27 ± 0.36
3	7.47 ± 0.13	7.79 ± 0.18	7.62 ± 0.24	7.95 ± 0.15
4	6.88 ± 0.20	7.16 ± 0.25	6.75 ± 0.07	7.17 ± 0.19
5	6.21 ± 0.25	6.55 ± 0.29	6.15 ± 0.11	6.53 ± 0.28
6	5.54 ± 0.13	6.28 ± 0.43	5.72 ± 0.06**	6.44 ± 0.22
7	5.33 ± 0.03	5.90 ± 0.46	5.34 ± 0.18	6.19 ± 0.13
8	5.06 ± 0.05	5.56 ± 0.37	5.13 ± 0.08**	5.68 ± 0.19
9	4.77 ± 0.15	5.23 ± 0.25	4.84 ± 0.05	5.46 ± 0.29
10	4.54 ± 0.13	5.35 ± 0.35	4.51 ± 0.11	5.45 ± 0.06
11	4.45 ± 0.20	5.33 ± 0.32	4.58 ± 0.08	5.39 ± 0.32
12	4.31 ± 0.04	5.00 ± 0.27	4.49 ± 0.11**	5.01 ± 0.19
13	4.34 ± 0.18	4.92 ± 0.10	4.56 ± 0.11**	5.01 ± 0.25
14	4.90 ± 0.05	5.60 ± 0.24	5.32 ± 0.22**	5.81 ± 0.13
15	4.84 ± 0.26	5.66 ± 0.22	5.22 ± 0.18**	5.84 ± 0.10**
16	5.22 ± 0.17	5.85 ± 0.21	5.36 ± 0.21	6.02 ± 0.20#

^x ± SD

n - number of animals per group

* - statistically significant difference with p ≤ 0.05; Student’s t-test;

** - statistically significant difference with p ≤ 0.05; Mann-Whitney U test;   # - n=7 

 

Table 8. Urine sediment examinations - main groups 

Parameter		GROUP / DOSE [mg/kg b.w.]
	0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
		males			females
	n=9^#	n=10	n=10	n=10	n=8^#	n=9^#	n=9^#	n=9^#
SQUAMOUS EPITHELIA	1.33 ± 0.50	1.40 ± 0.52	1.30 ± 0.48	1.40 ± 0.52	1.00 ± 0.00	1.22 ± 0.44	1.22 ± 0.44	1.44 ± 0.53
TRANSITIONAL EPITHELIA	0.00 ± 0.00	0.10 ± 0.32	0.00 ± 0.00	1.00 ± 0.82*	0.00 ± 0.00	0.22 ± 0.44	0.11 ± 0.33	0.78 ± 0.44*
LEUKOCYTES	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00	2.50 ± 0.71*	1.00 ± 0.00	1.00 ± 0.00	1.00 ± 0.00	2.00 ± 0.50*
ERYTHROCYTES	1.33 ± 0.50	1.30 ±    0.48	1.40 ± 0.52	1.40 ± 0.52	1.13 ± 0.35	1.22 ± 0.44	1.33 ± 0.50	1.56 ± 0.53
BACTERIA	3.11 ± 0.78	2.90 ± 0.74	3.00 ± 0.82	3.50 ± 0.71	3.13 ± 1.13	2.78 ± 1.09	2.78 ± 0.44	2.67 ± 0.71
AMORPHOUS URATES	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
AMORPHOUS PHOSPHATES	0.00 ± 0.00	0.00 ± 0.00	1.20 ± 1.99	1.10 ± 1.79	0.38 ± 1.06	1.22 ± 1.86	0.33 ± 1.00	0.00 ± 0.00
AM. MAG. PHOSPHATES	2.11 ± 1.45	2.70 ± 0.82	2.50 ± 1.35	0.90 ± 1.20	1.13 ± 1.13	1.67 ± 0.87	1.89 ± 1.27	0.56 ± 1.13

 

Table 9. Urine sediment examinations - satellite groups

Parameter		GROUP / DOSE [mg/kg b.w.]
	0SAT/0		3SAT/1000	0SAT/0	3SAT/1000
		males		females
	n=10		n=10	n=10	n=9
SQUAMOUS EPITHELIA	1.10 ± 0.32		1.20 ± 0.42	1.10 ± 0.32	1.33 ± 0.50
TRANSITIONAL EPITHELIA	0.00 ± 0.00		0.00 ± 0.00	0.00 ± 0.00	0.22 ± 0.44
LEUKOCYTES	1.00 ± 0.00		1.10 ± 0.32	1.00 ± 0.00	1.00 ± 0.00
ERYTHROCYTES	1.20 ± 0.42		1.00 ± 0.00	1.10 ± 0.32	1.11 ± 0.33
BACTERIA	3.90 ± 0.74		3.20 ± 0.92	3.30 ± 1.06	3.00 ± 1.22
AMORPHOUS URATES	0.00 ± 0.00		0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
AMORPHOUS PHOSPHATES	1.30 ± 1.70		1.00 ± 1.63	1.30 ± 2.11	0.67 ± 1.32
AM. MAG. PHOSPHATES	2.30 ± 0.82		1.50 ± 1.35	2.10 ± 0.57	0.78 ± 1.20**

** - statistically significant difference with p ≤ 0.05; Mann-Whitney U test

 

Table 10. Locomotor activity – satellite groups

Parameter			GROUP / DOSE [mg/kg b.w./day]
	0 SAT / 0 (measurement 1)		3 SAT / 1000  (measurement 1)		0 SAT / 0  (measurement 2)		3 SAT / 1000 (measurement 2)
	♂ n=10	♀ n=10	♂ n=10	♀ n=10	♂ n=10	♀ n=10	♂ n=10	♀ n=10
DISTANCE 0- 30MIN [m]	555.48 ± 120.95	630.76 ± 128.61	580.48 ± 130.47	670.88 ± 151.01	610.24 ± 93.32	626.40 ± 115.83	480.58 ± 55.26 *	602.03 ± 142.87
DISTANCE 0- 10MIN [m]	304.65 ± 35.52	329.55 ± 59.65	314.60 ± 71.50	328.80 ± 40.00	289.03 ± 59.91	304.18 ± 57.29	236.53 ± 33.50 *	291.60 ± 48.12
DISTANCE 10-20MIN [m]	169.04 ± 62.26	192.42 ± 54.80	165.10 ± 55.19	187.68 ± 56.46	182.28 ± 46.59	170.03 ± 47.26	164.18 ± 42.17	178.57 ± 72.34
DISTANCE 20-30MIN [m]	81.77 ± 51.37	108.80 ± 45.60	100.84 ± 68.51	154.53 ± 74.05	138.93 ± 39.09	152.22 ± 55.01	79.87 ± 48.19*	131.86 ± 42.35
NUMBER OF REARINGS 0-30MIN	63.60 ± 18.88	86.50 ± 31.81	71.90 ± 14.72	105.20 ± 30.11	71.40 ± 19.67	88.30 ± 17.53	42.80 ± 12.86*	102.10 ± 47.51
NUMBER OF REARINGS 0-10MIN	40.40 ± 6.96	49.40 ± 17.28	49.00 ± 13.09	56.60 ± 13.95	43.80 ± 15.28	46.80 ± 13.85	26.60 ± 7.44 *	51.70 ± 22.03
NUMBER OF REARINGS 10-20MIN	17.10 ± 11.79	22.40 ± 8.33	14.10 ± 8.28	26.80 ± 13.11	17.00 ± 7.72	19.70 ± 7.09	10.50 ± 9.06	32.50 ± 21.88
NUMBER OF REARINGS 20-30MIN	6.30 ± 6.88	14.90 ± 11.61	8.90 ± 9.62	21.80 ± 16.19	10.90 ± 4.56	21.80 ± 11.97	5.70 ± 7.90	17.90 ± 11.23

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

 

Table 11. Hematological and coagulological examinations - main groups

Parameter	GROUP/DOSE [mg/kg b.w.]
	0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
	males				females
	n=10	n=10	n=10	n=10	n=10	n=10	n=8^#	n=10
HEMOGLOBIN [g/L]	153.10 ± 4.48	153.80 ± 3.65	154.90  ± 3.73	149.80  ± 3.91	146.60 ± 4.12	148.10   ± 5.53	147.00 ± 4.81	143.80 ± 8.57
HEMATOCRIT [1/1]	0.429  ± 0.017	0.428  ± 0.010	0.431    ± 0.013	0.418    ± 0.012	0.418   ± 0.012	0.414     ± 0.020	0.412   ± 0.016	0.411   ± 0.027
ERYTHROCYTES  [x 10^12/L]	8.91    ± 0.42	8.96    ± 0.18	9.09      ± 0.30	9.20      ± 0.33	8.04    ± 0.25	8.17      ± 0.32	8.11    ± 0.16	8.06     ± 0.55
MCV [fL]	48.12   ± 0.99	47.75   ± 0.56	47.41    ± 1.10	45.50    ± 1.27*	51.95   ± 0.74	50.70     ± 1.36	50.83   ± 1.79	51.04   ± 1.26
MCH [pg]	17.20  ± 0.38	17.15  ± 0.24	17.04    ± 0.40	16.30    ± 0.39*	18.26   ± 0.27	18.16     ± 0.38	18.11   ± 0.51	17.84   ± 0.46
MCHC [g/L]	357.50 ± 4.74	359.40 ± 3.47	359.90  ± 3.57	358.40  ± 3.17	351.70 ± 2.75	358.20   ± 5.73	356.75 ± 8.66	349.90 ± 8.50
LEUKOCYTES     [x 10^9/L]	4.10    ± 0.51	3.59    ± 0.96	4.38      ± 0.59	4.05      ± 0.93	2.92    ± 0.66	2.65      ± 0.45	3.11    ± 0.76	2.88     ± 0.58
THROMBOCYTES   [x 10^9/L]	507.10 ± 55.42	488.90 ± 42.66	513.70  ± 38.15	508.00  ± 30.01	485.80 ± 37.28	446.80   ± 110.86	482.75 ± 33.96	453.30 ± 90.89
RETICULOCYTES   [1/1]	0.010  ± 0.001	0.009  ± 0.002	0.008    ± 0.002	0.008    ± 0.001*	0.011   ± 0.003	0.011     ± 0.002	0.011   ± 0.003	0.009   ± 0.005
PT [s]	15.99  ± 0.86	14.48  ± 3.13	15.06    ± 1.14	14.91    ± 1.38	n=9^#	13.11     ± 1.00	13.36   ± 1.37	n=8^#
					12.99   ± 1.07			13.85   ± 1.69
APTT [s]	24.09  ± 8.94	26.02  ± 5.58	29.35    ± 17.12	25.10    ± 4.25	n=9^#	19.77     ± 3.70	22.58   ± 5.61	n=8^#
					20.70   ± 5.26			21.19   ± 5.62

x ± SD; n - number of test animals; 

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

# - n<10 because of clot in the sample

 

Table 12. Haematological and coagulological examinations - satellite groups 

Parameter	GROUP/DOSE [mg/kg b.w.]
	0SAT/0	3SAT/1000	0SAT/0	3SAT/1000
	males		females
	n=10	n=9^#	n=10	n=9
HEMOGLOBIN [g/L]	154.80 ± 2.90	154.33 ± 5.74	151.40 ± 3.47	149.33 ± 2.06
HEMATOCRIT [1/1]	0.438 ± 0.014	0.441± 0.016	0.431 ± 0.014	0.433 ± 0.005
ERYTHROCYTES [x 10^12/L]	8.98 ± 0.26	9.07 ± 0.36	8.41 ± 0.23	8.16 ± 0.13*
MCV [fL]	48.71 ± 1.20	48.58 ± 0.67	51.27 ± 0.95	53.11 ± 0.86*
MCH [pg]	17.25 ± 0.32	17.03 ± 0.24	18.01 ± 0.29	18.30 ± 0.35
MCHC [g/L]	354.40 ± 6.45	350.56 ± 5.03	351.80 ± 5.73	345.00 ± 5.15*
LEUKOCYTES     [x 10^9/L]	3.38 ± 0.42	3.74 ± 0.57	2.33 ± 0.43	2.96 ± 0.51*
THROMBOCYTES   [x 10^9/L]	553.00 ± 38.24	515.56 ± 56.96	531.70 ± 33.13	507.33 ± 50.20
RETICULOCYTES   [1/1]	0.007 ± 0.001	0.007 ± 0.002	0.009 ± 0.002	0.009 ± 0.002
PT [s]	16.13 ± 1.35	16.01 ± 0.91	15.05 ± 0.75	14.67 ± 0.88
APTT [s]	30.43 ± 6.92	28.94 ± 6.15	26.82 ± 13.18	24.12 ± 7.12

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

 

Table 13. Blood serum biochemical examinations - main groups 

Parameter		GROUP / DOSE [mg/kg b.w.]
	0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
		males			females
	n=10	n=10	n=10	n=10	n=10	n=10	n=10	n=10
TOTAL PROTEIN  [g/L]	58.40 ±1.75	59.16 ±1.54	59.25 ±2.28	58.14 ±1.34	64. 59 ±2.70	61.98 ±3.05	63.84 ±2.53	60.57 ±3.91*
ALBUMIN [g/L]	31.90 ± 1.71	31.68 ±1.78	32.18 ± 1.94	30.93 ±1.46	36.02 ±1.69	34.83 ±1.57	35.49 ±1.81	33.58 ±3.36
GLOBULIN [g/L]	26.50 ±2.18	27.48 ±2.20	27.07 ±2.07	27.21 ±2.43	28.57 ±1.35	27.15 ±1.70	28.35 ±2.05	26.99 ±1.25
A/G RATIO	1.22 ±0.15	1.16 ±0.14	1.20 ±0.13	1.15 ±0.14	1.26 ±0.06	1.28 ±0.06	1.26 ±0.11	1.24 ±0.12
GLUCOSE [mmol/L]	9.51 ±2.08	9.25 ±1.68	10.39 ± 1.75	9.93 ±2.40	8.56 ±2.36	8.10 ±1.38	7.48 ±0.91	7.41 ±0.74
CHOLESTEROL  [mmol/L]	1.76 ±0.24	1.70 ±0.14	1.84 ±0.30	1.86 ±0.31	1.53 ±0.36	1.32 ±0.28	1.36 ±0.39	1.27 ±0.28
HDL [mmol/L]	1.15 ±0.15	1.14 ±0.11	1.24 ±0.21	1.22 ±0.19	1.08 ±0.25	0.93 ±0.18	0.94 ±0.29	0.85 ±0.21
LDL [mmol/L]	0.38 ±0.14	0.56 ±0.30	0.39 ±0.13	0.36 ±0.09	0.42 ±0.27	0.45 ±0.27	0.42 ±0.19	0.46 ±0.21
UREA NITROGEN [mmol/L]	4.95 ±0.75	4.91 ±0.52	4.75 ±0.58	5.66 ±0.33*	5.25 ±1.19	5.97 ±0.86	5.52 ±0.60	6.03 ±1.17
CREATININE  [μmol/L]	28.20 ± 4.37	28.00 ± 2.11	28.90 ± 5.65	30.40 ±4.45	33.40 ± 3.31	31.20 ± 2.35	26.80 ±3.19*	33.20 ±8.26
BILIRUBIN  [μmol/L]	3.78 ±0.46	3.78 ±0.64	3.82 ±0.63	3.98 ±0.48	4.77 ±2.03	4.64 ±1.58	4.71 ±1.27	5.82 ±3.08
SODIUM [mmol/L]	144.30 ± 2.11	144.90 ± 1.60	146.40 ±1.78*	146.00 ±1.63	145.10 ±1.52	144.30 ±1.34	145.70 ±1.16	144.70 ±1.25
POTASSIUM  [mmol/L]	4.21 ±0.33	4.16 ±0.21	4.21 ±0.30	4.38 ±0.32	3.91 ±0.48	4.06 ±0.42	4.03 ±0.36	4.26 ±0.36
CHLORIDES [mmol/L]	104.30 ± 1.25	104.50 ±0.71	104.50 ±2.07	103.70 ±2.06	105.20 ±1.32	106.70 ±1.95	106.60 ±1.58	105.90 ±1.97
CALCIUM [mmol/L]	2.39 ±0.08	2.43 ±0.07	2.48 ±0.08	2.42 ±0.07	2.55 ±0.07	2.48 ±0.08	2.55 ±0.06	2.44 ±0.08*
INORGANIC PHOSPHOROUS [mmol/L]	1.71 ±0.19	1.67 ±0.12	1.80 ±0.21	1.99 ±0.19*	1.52 ±0.28	1.59 ±0.26	1.88 ±0.18*	1.76 ±0.22

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

 

Table 14. Blood serum biochemical examinations - satellite groups 

Parameter	GROUP / DOSE [mg/kg b.w.]
	0SAT/0		3SAT/1000	0SAT/0		3SAT/1000
	males			females
	n=10		n=10	n=10		n=9
TOTAL PROTEIN  [g/L]	63.18 ± 1.50		61.56 ± 2.72	63.59 ± 3.15		64.34 ± 2.08
ALBUMIN [g/L]	33.67 ± 1.10		32.67 ± 1.50	35.78 ± 1.35		36.72 ± 1.23
GLOBULIN [g/L]	29.51 ± 0.94		28.89 ± 1.48	27.81 ± 2.03		27.62 ± 1.30
A/G RATIO	1.14 ± 0.05		1.13 ± 0.05	1.29 ± 0.07		1.33 ± 0.06
GLUCOSE  [mmol/L]	9.00 ± 1.00		8.84 ± 1.11	7.13 ± 0.93		7.30 ± 0.77
CHOLESTEROL   [mmol/L]	1.98 ± 0.24		1.99 ± 0.18	1.67 ± 0.36		1.90 ± 0.28
HDL [mmol/L]	1.33 ± 0.15		1.38 ± 0.15	1.11 ± 0.20		1.22 ± 0.16
LDL [mmol/L]	0.40 ± 0.06		0.36 ± 0.05	0.40 ± 0.15		0.41 ± 0.08
UREA NITROGEN [mmol/L]	6.55 ± 0.93		6.31 ± 0.89	7.08 ± 1.24		7.33 ± 1.06
CREATININE   [μmol/L]	30.50 ± 6.04		29.00 ± 5.16	35.20 ± 5.49		38.11 ± 4.81
BILIRUBIN  [μmol/L]	4.18 ± 0.42		4.45 ± 0.53	4.46 ± 1.05		5.00 ± 0.92
SODIUM  [mmol/L]	136.80 ± 2.30		137.60 ± 0.97	143.90 ± 1.10		143.44 ± 0.88
POTASSIUM [mmol/L]	3.82 ± 0.14		4.14 ± 0.24*	3.83 ± 0.25		3.73 ± 0.39
CHLORIDES  [mmol/L]	97.40 ± 1.35		98.50 ± 1.27	104.80 ± 1.75		103.22 ± 0.97**
CALCIUM [mmol/L]	2.42 ± 0.05		2.40 ± 0.06	2.52 ± 0.06		2.47 ± 0.08
INORGANIC PHOSPHOROUS [mmol/L]	1.87 ± 0.18		1.91 ± 0.20	1.68 ± 0.15		1.54 ± 0.27

x ± SD; 

n - number of test animals

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

** - statistically significant difference with p ≤ 0.05; Mann-Whitney U test;  

 

Table 15. Relative organ-to-body weight of internal organs [%] – main groups

Examined organ	GROUP / DOSE [mg/kg b.w./day]
	0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
	males				females
	n=10	n=10	n=10	n=10	n=10	n=10	n=10	n=10
Brain with cerebellum	0.493 ±0.029	0.491 ±0.038	0.471 ±0.038	0.515 ±0.045	0.795 ±0.044	0.757 ±0.061	0.782 ±0.068	0.774 ±0.049
Pituitary gland	0.002 ±0.0004	0.002 ±0.001	0.002 ±0.001	0.002 ±0.0004	0.007 ±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	0.006 ±0.001	0.008 ±0.002	0.007 ±0.002	0.008 ±0.001	0.008 ±0.001
Thymus	0.084 ±0.022	0.097 ±0.018	0.082 ±0.015	0.073 ±0.015	0.124 ±0.020	0.130 ±0.015	0.126 ±0.022	0.117 ±0.025
Heart	0.239 ±0.015	0.256 ±0.016	0.248 ±0.013	0.264* ±0.018	0.289 ±0.040	0.281 ±0.023	0.278 ±0.029	0.281 ±0.025
Liver	2.700 ±0.279	2.978 ±0.291	2.922 ±0.212	2.977 ±0.223	2.980 ±0.227	2.863 ±0.295	2.709 ±0.345	2.862 ±0.178
Spleen	0.131 ±0.015	0.151 ±0.011	0.143 ±0.019	0.156* ±0.025	0.206 ±0.014	0.202 ±0.022	0.206 ±0.038	0.208 ±0.026
Kidneys	0.578 ±0.032	0.598 ±0.035	0.580 ±0.038	0.672* ±0.061	0.678 ±0.057	0.648 ±0.061	0.653 ±0.056	0.641 ±0.032
Adrenal glands	0.017 ±0.002	0.017 ±0.003	0.016 ±0.001	0.016 ±0.002	0.038 ±0.004	0.035 ±0.004	0.035 ±0.005	0.035 ±0.005
Testicles	0.865 ±0.054	0.873 ±0.064	0.869 ±0.053	0.936 ±0.106	-	-	-	-
Epididymides	0.340 ±0.035	0.341 ±0.027	0.327 ±0.024	0.355 ±0.028	-	-	-	-
Prostate with seminal vesicles and coagulating glands	0.461 ±0.083	0.487 ±0.056	0.443 ±0.083	0.464 ±0.047	-	-	-	-
Ovaries	-	-	-	-	0.050 ±0.007	0.047 ±0.006	0.047 ±0.007	0.048 ±0.007
Uterus with cervix	-	-	-	-	0.249 ±0.056	0.280 ±0.132	0.273 ±0.108	0.283 ±0.132

n- number of tested animals;          x ± SD;

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

 

Table 16.Histopathological examination - main groups

Examined organ	Type of changes	Degree of severity (numerical score)	GROUP / DOSE [mg/kg b.w./day] / number of animals
			0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
			males				females
			n=10	n=10	n=10	n=10	n=10	n=10	n=10	n=10
Urinary bladder	hyperplasia urothelium, papillary, diffuse	minimal [1]	-	-	2	-	-	-	4	-
		slight [2]	-	-	-	-	-	-	2	-
		severe [4]	-	-	-	10	-	-	-	10
	proteinaceous plug	present [+]	1	-	-	-	-	-	-	-
	infiltration/s, mononuclear cells	minimal [1]	-	-	2	3	-	-	-	2
		slight [2]	-	-	-	-	-	-	-	2
		moderate [3]	-	-	-	-	-	-	-	1
	oedema	present [+]	-	-	-	-	1	-	-	1

ns – examination not scheduled, n – number of animals in group

Examined organ	Type of changes	Degree of severity (numerical score)	GROUP / DOSE [mg/kg b.w./day] / number of animals
			0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
			males				females
			n=10	n=10	n=10	n=10	n=10	n=10	n=10	n=10
Left kidney	hemorrhage/s	minimal [1]	1	2	3	1	2	5	4	3
		slight [2]	3	7	6	7	4	4	2	3
		moderate [3]	6	1	-	2	4	1	2	1
	renal tubules, basophilia	present [+]	2	1	1	6	2	-	-	3
	renal tubules, degeneration, focal	present [+]	-	-	-	-	1	-	-	-
	renal tubules, degeneration, diffuse	present [+]	1	1	1	-	-	-	2	-
	infiltration/s, mononuclear cells, focal	minimal [1]	2	2	4	2	-	1	-	-
		slight [2]	-	-	-	-	-	1	1	-

ns – examination not scheduled, n – number of animals in group

Examined organ	Type of changes	Degree of severity (numerical score)	GROUP / DOSE [mg/kg b.w./day] / number of animals
			0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
			males				females
			n=10	n=10	n=10	n=10	n=10	n=10	n=10	n=10
Left kidney	cyst/s	present [+]	4	2	1	1	1	-	2	2
	mineralization, focal	present [+]	-	-	-	-	7	5	7	5
	renal pelvis, urothelium, hyperplasia, papillary, diffuse	minimal [1]	-	-	-	1	-	-	-	3
		slight [2]	-	-	-	5	-	-	-	2
		moderate [3]	-	-	-	4	-	-	-	-
	papilla, hyperplasia, epithelium, papillary diffuse	minimal [1]	-	-	-	1	-	-	-	2
		slight [2]	-	-	-	3	-	-	-	2
		moderate [3]	-	-	-	2	-	-	-	2
		severe [4]	-	-	-	4	-	-	-	1
	fibrosis	present [+]	1	-	-	-	-	1	1	-
Right kidney	hemorrhage/s	minimal [1]	1	1	1	-	-	1	1	3
		slight [2]	4	2	4	1	1	6	5	4
		moderate [3]	5	6	5	7	8	3	4	2
	renal tubules, basophilia	present [+]	3	2	2	8	2	1	1	1
	renal tubules, degeneration, diffuse	present [+]	-	1	-	1	-	-	1	-
	infiltration/s, mononuclear cells, focal	minimal [1]	4	3	2	2	2	-	-	3
		slight [2]	-	-	-	2	-	-	1	-

n – number of animals in group

Examined organ	Type of changes	Degree of severity (numerical score)	GROUP / DOSE [mg/kg b.w./day] / number of animals
			0/0	1/62.5	2/250	3/1000	0/0	1/62.5	2/250	3/1000
			males				females
			n=10	n=10	n=10	n=10	n=10	n=10	n=10	n=10
Right kidney	cyst/s	present [+]	3	7	3	3	2	5	4	3
	papilla, atrophy	present [+]	-	-	1	-	-	-	-	-
	fibrosis	present [+]	-	-	-	2	-	-	-	-
	renal pelvis, urothelium, hyperplasia, papillary, diffuse	minimal [1]	-	-	-	1	-	-	-	3
		slight [2]	-	-	-	3	-	-	-	3
		moderate [3]	-	-	-	5	-	-	-	1
	papilla, hyperplasia, epithelium, papillary diffuse	minimal [1]	-	-	-	5	-	-	-	3
		slight [2]	-	-	-	1	-	-	-	1
		moderate [3]	-	-	-	5	-	-	-	1
		severe [4]	-	-	-	2	-	-	-	-
	mineralization, focal	present [+]	-	1	-	-	-	5	6	8
	subscapular, necrosis,  focal	present [+]	-	-	1	-	-	-	-	-
	fibrosis	present [+]	-	-	-	-	-	-	-	1

n – number of animals in group

 

Table 17. Histopathological examination - satellite groups

Examined organ	Type of changes	Degree of severity (numerical score)	GROUP / DOSE [mg/kg b.w./day] / number of animals
			0SAT/0	3SAT/1000	0SAT/0	3SAT/1000
			males		females
			n=10	n=10	n=10	n=10
Urinary bladder	hyperplasia urothelium, papillary, diffuse	minimal [1]	-	-	-	3
		slight [2]	-	7	-	5
		moderate [3]	-	3	-	-
	infiltration/s, mononuclear cells	minimal [1]	-	1	-	-
	oedema	present [+]	-	-	-	1
Left kidney	hemorrhage/s	minimal [1]	1	3	6	2
		slight [2]	4	4	1	5
		moderate [3]	3	1	1	2
	renal tubules, basophilia	present [+]	-	3	-	1
	renal tubules, degeneration, focal	present [+]	-	1	3	-
	renal tubules, degeneration, diffuse	minimal [1]	3	-	-	-
	infiltration/s, mononuclear cells, focal	minimal [1]	5	2	1	-
	cyst/s	present [+]	1	3	-	3
	papilla, atrophy	present [+]	-	-	-	1

n – number of animals in group

Examined organ	Type of changes	Degree of severity (numerical score)	GROUP / DOSE [mg/kg b.w./day] / number of animals
			0SAT/0	3SAT/1000	0SAT/0	3SAT/1000
			males		females
			n=10	n=10	n=10	n=10
Left kidney	renal tubule, dilation	present [+]	1	-	2	1
	mineralization, focal	present [+]	-	-	7	6
	hyperplasia, papillary, diffuse, epithelium papilla	minimal [1]	-	6	-	3
		slight [2]	-	1	-	4
		moderate [3]	-	-	-	1
	renal pelvis, urothelium, hyperplasia, papillary, diffuse	minimal [1]	-	3	-	4
		slight [2]	-	1	-	2
	glomerulus, epithelium hyperplasia	present [+]	1	-	-	-
Right kidney	hemorrhage/s	minimal [1]	2	2	1	1
		slight [2]	3	4	8	4
		moderate [3]	5	3	1	4
	renal tubules, basophilia	present [+]	1	4	1	1
	renal tubules, degeneration, focal	present [+]	-	-	1	-
	renal tubules, degeneration, diffuse	present [+]	2	-	-	-
	infiltration/s, mononuclear cells, focal	minimal [1]	3	5	2	-
		slight [2]	1	-	-	-
		moderate [3]	1	-	-	-
	cyst/s	present [+]	5	6	2	2
	pelvis dilation	present [+]	-	-	-	-
	renal tubule, dilation	present [+]	1	-	1	1
	mineralization, focal	present [+]	-	-	7	9
	hyperplasia, papillary, diffuse, epithelium papilla	minimal [1]	-	4	-	1
		slight [2]	-	3	-	2
		moderate [3]	-	-	-	2
	renal pelvis, urothelium, hyperplasia, papillary, diffuse	minimal [1]	-	3	-	6
		slight [2]	-	4	-	1
	glomerulus, epithelium hyperplasia	present [+]	1	-	-	-

n – number of animals in group

Conclusions:

The NOAEL for the sub-chronic repeated dose toxicity of the test item in rats by oral route was determined to be 250 mg/kg bw/day.

Executive summary:

A 90-day repeated dose toxicity study was conducted with the test item according to OECD Guideline 408 and EU Method B.26 (GLP study). The test item was exposed to 120 Wistar rats (60 males, 60 females), which were divided into four groups main groups, one control and three treated groups, and two satellite groups. There were 10 males and 10 females in each group. The doses of the test item were 62.5, 250 and 1000 mg/kg b.w./day. During the study, clinical signs, mortality, body weights, and food intake were recorded. Moreover, ophthalmic examinations and behavioural studies were conducted. Behavioural studies involved observations of animal behaviour in an open field, responses to stimuli, locomotor activity, and fore- and hindlimb grip strength. At the end of the experiment, all animals were subjected to hematological examinations of peripheral blood and bone marrow, coagulation examinations, biochemical and enzymatic examinations of serum, general urinalyses, examinations of urine sediment and hormone examinations. After the end of experiment, absolute weights of particular internal organs of all surviving animals were determined. An extensive amount of organs and tissues were preserved for histopathological examinations.

On the basis of the results of the clinical examinations, the test item induced a decrease in the vertical locomotor activity (males group 3 SAT), which may be related to an increase in the amount of potassium, as found in the clinical-chemical examinations. In the post-mortem examination, it was observed papillary hyperplasia of urothelium in urinary bladder and kidneys as well as hyperplasia of papilla epithelium in kidneys - in males and females of group 3, a significant increase of relative weight of kidneys - in males of group 3 and the presence of basophilic tubules - in males of group group 3 and 3SAT.

It may be concluded that the test item related changes concerned especially kidneys and urinary bladder at high dose 1000 mg/kg b.w/day, which were observed in males and females. Despite the reversibility of some of them, changes resulted in an adverse effect on animal well-being, morphology and physiology during the study and also after recovery period. Based on this, the No Observed Adverse Effect Level (NOAEL) of the test item in rats of this study was determined to be 250 mg/kg b.w./day for males and females.

Reference 2

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 or test system 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

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

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

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.

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.

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.

Key result

Critical effects observed:

no

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

n–number 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 observations–males (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

n–number 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

n–number 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 10^12/L	8.80土0.41	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 10^9/L	609.40± 70.84	662.50± 22.11*
LEUKOCYTES	x 10^9/L	3.45土0.66	3.47 ±0.96
RETICULOCYTES	1/1	0.020± 0.003	0.020± 0.003
HEINZ BODIES	%〇	0.00土0.00	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 10^12/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 10^9/L	614.80± 46.17	672.60± 64.14*
LEUKOCYTES	x 10^9/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	0.23土0.06	0.22土0.09	0.23土0.06	0.22土0.05
EOSINOCYTES	1/1	0.01 ± 0.01	0.00土0.01	0.00土0.00	0.00土0.00
LYMPHOCYTES	1/1	0.76土0.06	0.77土0.09	0.77土0.06	0.78土0.05
MONOCYTES	1/1	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
OTHER CELLS	1/1	0.00土0.01	0.00土0.00	0.00土0.01	0.00土0.00

 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	0.46土0.13	0.48土0.06	0.38土0.17	0.48土0.11
EOSINOCYTES	1/1	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
LYMPHOCYTES	1/1	0.54土0.13	0.51土0.06	0.61土0.17	0.52土0.11
MONOCYTES	1/1	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
OTHER CELLS	1/1	0.00土0.01	0.00土0.00	0.01土0.01	0.00土0.00

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	0.19土0.05	0.21土0.04	0.14土0.06	0.16土0.05
EOSINOCYTES	1/1	0.01 ± 0.01	0.01土0.01	0.01土0.01	0.00土0.00
LYMPHOCYTES	1/1	0.81 ± 0.05	0.79土0.04	0.85土0.06	0.83土0.05
MONOCYTES	1/1	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
OTHER CELLS	1/1	0.00土0.00	0.00土0.00	0.00土0.00	0.01土0.01

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	55.75土2.91	57.60土4.10	55.53土2.82
ALBUMIN	g/L	30.77± 3.11	30.15土1.72	31.68土2.64	30.42土1.34
GLOBULIN	g/L	25.55± 1.96	25.60土1.71	25.92土1.65	25.11土1.93
A/G RATIO		1.20土0.06	1.18土0.08	1.22土0.06	1.22土0.09
GLUCOSE	mmol/L	7.46土1.96	7.18土1.67	7.24土1.26	7.40土1.90
CHOLESTEROL	mmol/L	2.42土0.52	2.78土0.51	2.20土0.63	2.68土0.54
UREA NITROGEN	mmol/L	7.23 ±2.29	6.67土1.33	6.25土1.46	8.77土1.90
CREATININE	pmol/L	36.33± 3.20	33.33土2.57	33.08土3.60	40.82土5.53*
BILE ACIDS	pmol/L	41.80± 32.74	43.12土28.28	39.49土25.46	79.07土102.36
BILIRUBIN	pmol/L	5.04 ±2.31	5.21土1.61	4.34土1.03	5.02土0.83
SODIUM	mmol/L	139.75± 1.96	139.67土2.10	141.50土1.62	140.55土1.81
POTASSIUM	mmol/L	3.94土0.71	4.19土0.40	3.96土0.22	4.15土0.40
CHLORIDES	mmol/L	98.92± 3.80	99.17土3.27	101.75土3.65	97.55土2.21
CALCIUM	mmol/L	2.48土0.06	2.46土0.08	2.49土0.10	2.52土0.04

^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	63.10土2.10*	0 SAT n=10	3 SAT n=10
ALBUMIN	g/L	31.68± 1.03	32.44土1.00	60.97± 1.40	61.97土2.63
GLOBULIN	g/L	29.39± 1.39	30.66土1.36	34.77± 1.29	35.04土1.58
A/G RATIO		1.08土0.07	1.06土0.04	26.20± 0.65	26.93土1.52
GLUCOSE	mmol/L	8.29土1.27	7.15土0.76*	1.33土0.06	1.30土0.07
CHOLESTEROL	mmol/L	2.11 ± 0.37	2.07土0.36	5.91 ± 0.94	5.88土0.97
UREA NITROGEN	mmol/L	5.32土0.49	5.35土0.46	1.69土0.46	1.53土0.39
CREATININE	pmol/L	31.90± 5.82	28.20土4.44	5.99土0.75	5.32土0.60*
BILE ACIDS	pmol/L	21.72± 16.99	19.57土9.32	34.10±4.41	31.50土4.28
BILIRUBIN	pmol/L	4.10土0.54	3.81土0.46	36.94±20.24	27.98土18.13
SODIUM	mmol/L	144.80± 1.69	145.90土1.37	4.73土1.12	4.31土1.09
POTASSIUM	mmol/L	4.45土0.56	4.25土0.28	143.80± 0.79	145.10土0.88*
CHLORIDES	mmol/L	105.10土1.79	105.20土1.03	3.92土0.35	3.95土0.31
CALCIUM	mmol/L	2.49土0.07	2.54土0.05	105.60土0.84	106.40土1.26
				2.48土0.09	2.44土0.05

f土SD

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

^f土SD

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

^f土SD

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

^f土SD;

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	12.00土4.95	16.00土9.90	17.40土10.33
SPECIFIC GRAVITY	1.02土0.01	1.02土0.01	1.02土0.01	1.02土0.01
pH	6.90土0.22	6.80土0.27	6.90土0.22	6.80土0.27
PROTEIN                        g/L	0.30土0.41	0.26土0.09	0.18土0.16	0.10土0.12
GLUCOSE              mmol/L	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
KETONE BODIES mmol/L	0.30土0.67	0.10土0.22	0.30土0.27	0.20土0.27
BILIRUBIN^c	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
BLOOD                     Ery/pL	0.00土0.00	0.00土0.00	2.00土4.47	0.00土0.00
UROBILINOGEN pmol/L	3.20土0.00	3.20土0.00	3.20土0.00	3.20土0.00
LEUKOCYTES          Leu/pL	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00

^x土SD

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	14.80土3.35	13.60土4.34	15.20土3.03
SPECIFIC GRAVITY	1.03土0.00	1.03土0.00	1.03土0.00	1.03土0.00
pH	6.00土0.35	6.20土0.57	6.30土0.57	6.10土0.55
PROTEIN                        g/L	0.20土0.14	0.04土0.05*	0.06土0.05*	0.04土0.05*
GLUCOSE              mmol/L	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
KETONE BODIES mmol/L	0.20土0.27	0.30土0.27	0.20土0.27	0.00土0.00
BILIRUBIN^c	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
BLOOD                     Ery/pL	0.00土0.00	0.00土0.00	0.00土0.00	16.00土35.78
UROBILINOGEN pmol/L	3.20土0.00	3.20土0.00	3.20土0.00	3.20土0.00
LEUKOCYTES          Leu/pL	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00

 

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	12.60土3.13
SPECIFIC GRAVITY		1.02 ± 0.00	1.02 ±0.00	1.02土0.00	1.02土0.00
pH		7.10 ± 0.22	7.10 ±0.22	6.80 ± 0.27	6.50土0.35
PROTEIN	g/L	0.08 ± 0.04	0.54 ± 0.43*	0.00土0.00	0.00土0.00
GLUCOSE	mmol/L	0.00 ± 0.00	0.00 ±0.00	0.00土0.00	0.00土0.00
KETONE BODIES	mmol/L	1.30 ± 0.45	1.30 ±0.45	0.00土0.00	0.00土0.00
BILIRUBIN^c		0.00 ± 0.00	0.00 ±0.00	0.00土0.00	0.00土0.00
BLOOD	Ery/pL	2.00 ±4.47	0.00 ±0.00	0.00土0.00	0.00土0.00
UROBILINOGEN	pmol/L	3.20 ± 0.00	3.20 ±0.00	3.20土0.00	3.20土0.00
LEUKOCYTES	Leu/pL	0.00 ± 0.00	6.00 ± 8.22	0.00土0.00	0.00土0.00

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	1.20土0.45	1.00土0.00	1.40土0.55	1.20土0.45
COLUMNAR EPITHELIA	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
LEUKOCYTES	1.00土0.00	1.00土0.00	1.00土0.00	1.00土0.00
ERYTHROCYTES	1.20土0.45	1.20土0.45	1.20土0.45	1.40土0.55
BACTERIA	3.60土0.55	4.00土0.71	4.20土0.84	3.80土0.45
CRYSTALS	1.80土1.10	1.40土1.34	1.20土1.10	2.00土1.22

 ^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	1.00土0.00	1.00土0.00	1.00土0.00	1.00土0.00
COLUMNAR EPITHELIA	0.00土0.00	0.00土0.00	0.00土0.00	0.00土0.00
LEUKOCYTES	1.00土0.00	1.00土0.00	1.00土0.00	1.00土0.00
ERYTHROCYTES	1.20土0.45	1.00土0.00	1.00土0.00	1.20土0.45
BACTERIA	2.60土1.34	2.60土0.89	3.00土0.71	2.80土0.84
CRYSTALS	0.60 ± 1.34	1.60土1.67	1.80土1.79	1.60土1.52

^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	1.20土0.45	1.20土0.45	1.00 ± 0.00	1.00 ±0.00
COLUMNAR EPITHELIA	0.00土0.00	0.00土0.00	0.00 ± 0.00	0.00 ±0.00
LEUKOCYTES	1.00土0.00	1.00土0.00	1.00 ± 0.00	1.00 ±0.00
ERYTHROCYTES	1.20土0.45	1.20土0.45	1.20 ± 0.45	1.20 ±0.45
BACTERIA	4.20土0.84	2.40土0.89*	2.80 ± 1.30	3.40 ± 1.14
CRYSTALS	0.60土1.34	2.60土0.89	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]	一
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]	一
	doughy consistency	_	_	一	1 [50]
Heart	increased	4 [35,36,37,40]	_	2 [34,37]	一
	congestion	_	_	2 [36,38]	一
Liver	lesion- 7mm long, yellowish, grainy, on the visceral side of the left lobe	_	1 [46]	一	一
Spleen	increased	1 [35]	一	一	一
Cecum	congestion	1 [36]	_	一	一

 

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]	一	一	一
Mandibular lymph nodes	petechiae	_	一	1 [34]	2 [41,43]
Kidneys	increased	2 [35,40]	一	一	一
Ovaries	dark red foci	_	1 [45]	一	1 [48]
	congestion	_	1 [43]	一	1 [48]
Uterus	slightly expanded	一	4 [43,44,48,50]	一	2 [47,49]
	filled with		4		2
	clear liquid	^_	[43,44,48,50]	^一	[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	土	85.481	1999.700	土	134.953	2096.300	土	80.760	2073.200	土	73.295
Pituitary gland	11.400	土	1.838	10.600	土	1.578	10.800	土	1.874	8.800	土	1.751*
Thyroid	24.800	土	5.770	23.700	土	3.683	25.800	土	3.084	19.900	土	4.999
Thymus	327.000	土	50.859	299.500	土	45.887	283.800	土	40.881	268.500	土	66.304
Heart	1124.200	土	302.274	1012.500	土	124.886	1064.500	土	96.709	980.300	土	97.803
Liver	11540.600	土	1760.413	11315.800	土	970.273	11666.100	土	2188.889	11380.600	土	1101.959
Spleen	731.100	土	94.090	647.100	土	77.229	655.900	土	66.534	611.400	土	84.733*
Kidneys	2606.200	土	250.519	2605.000	土	213.370	2591.100	土	339.513	2633.500	土	200.172
Adrenals	83.700	土	10.636	79.700	土	10.489	78.600	土	14.416	75.800	土	12.515
Testicles	3655.600	土	179.424	3575.400	土	226.663	3590.500	土	285.645	3677.700	土	233.694
Epidydimides	1415.400	土	115.627	1386.800	土	117.212	1387.400	土	154.952	1414.200	土	187.585
Prostate with vesicular and coagulating glands	2143.800	土	260.990	2244.000	土	235.569	2290.300	土	195.046	2363.500	土	408.443
Levator ani	310.000	土	19.299	308.800	土	41.886	302.000	土	31.337	280.900	土	38.240
Bulbocavernosus muscle complex	784.900	土	80.622	767.500	土	97.903	792.100	土	72.301	810.200	土	93.866
Cowper’s glans	108.300	土	19.482	101.200	土	14.038	105.800	土	17.492	112.600	土	15.291
Glans penis	90.900	土	14.708	92.400	土	8.140	99.700	土	13.200	95.300	土	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	土	80.4357	1905.250	土	72.227	1909.667	土	72.089	1896.417	土	114.773
Pituitary gland	15.417	土	1.831955	14.833	土	1.946	13.250	土	1.485*	13.417	土	1.443*
Thyroid	17.917		4.521833	18.167		2.517	17.417		3.397	18.500		3.090
Thymus	240.750	土	101.7771	184.917	土	53.294	220.333	土	71.970	211.500	土	80.636
Heart	795.000	土	92.63222	813.417	土	89.118	793.750	土	65.840	841.417	土	89.465
Liver	10809.250	土	1819.769	11455.500	土	1494.417	10139.333	土	1436.985	11069.250	土	1751.459
Spleen	615.250	土	52.42505	621.417	土	95.102	631.250	土	80.462	622.750	土	108.084
Kidneys	1868.667	土	203.5826	1885.083	土	162.555	1867.667	土	133.189	1847.583	土	190.692
Adrenals	94.833	土	10.18763	94.000	土	13.253	91.667	土	11.781	97.917	土	9.605
Ovaries	125.417	土	30.81457	116.667	土	16.472	126.417	土	33.489	123.167	土	14.721
Uterus with cervix	547.750	土	215.9192	454.833	土	100.221	537.167	土	173.530	531.250	土	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	土	0.043	0.491	土	0.054	0.511	土	0.049	0.514	土	0.046
Pituitary gland	0.0006	土	0.0001	0.0005	土	0.0001	0.0005	土	0.0001	0.0004	土	0.0001*
Thyroid	0.006	土	0.001	0.006	土	0.001	0.006	土	0.001	0.005	土	0.001*
Thymus	0.079	土	0.009	0.073	土	0.011	0.069	土	0.010	0.066	土	0.016
Heart	0.271	土	0.073	0.247	土	0.016	0.258	土	0.014	0.242	土	0.013
Liver	2.765	土	0.216	2.770	土	0.225	2.811	土	0.326	2.806	土	0.166
Spleen	0.176	土	0.014	0.158	土	0.015*	0.159	土	0.016*	0.150	土	0.014*
Kidneys	0.629	土	0.062	0.638	土	0.051	0.627	土	0.056	0.650	土	0.030
Adrenals	0.020	土	0.002	0.020	土	0.003	0.019	土	0.003	0.019	土	0.002
Testicles	0.884	土	0.070	0.877	土	0.079	0.873	土	0.088	0.910	土	0.074
Epidydimides	0.342	土	0.026	0.340	土	0.028	0.336	土	0.024	0.349	土	0.037
Prostate with vesicular and	0.518	土	0.063	0.551	土	0.069	0.556	土	0.048	0.582	土	0.088
coagulating glands
Levator ani	0.075	土	0.010	0.075	土	0.009	0.073	土	0.008	0.069	土	0.006
Bulbocavernosus muscle complex	0.190	土	0.020	0.189	土	0.032	0.193	土	0.020	0.200	土	0.014
Cowper’s glans	0.026	土	0.005	0.025	土	0.003	0.026	土	0.004	0.028	土	0.003
Glans penis	0.022	土	0.005	0.023	土	0.003	0.024	土	0.003	0.023	土	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	土	0.031	0.662	土	0.029	0.688	土	0.049	0.668	土	0.050
Pituitary gland	0.005	土	0.001	0.005	土	0.001	0.005	土	0.001	0.005	土	0.001
Thyroid	0.006	土	0.001	0.006	土	0.001	0.006	土	0.001	0.007	土	0.001
Thymus	0.085	土	0.034	0.064	土	0.018	0.079	土	0.025	0.076	土	0.035
Heart	0.281	土	0.025	0.282	土	0.023	0.285	土	0.012	0.296	土	0.030
Liver	3.819	土	0.615	3.968	土	0.439	3.641	土	0.461	3.873	土	0.444
Spleen	0.218	土	0.017	0.215	土	0.030	0.227	土	0.027	0.218	土	0.029
Kidneys	0.660	土	0.055	0.654	土	0.048	0.673	土	0.061	0.650	土	0.060
Adrenals	0.034	土	0.003	0.033	土	0.004	0.033	土	0.005	0.034	土	0.004
Ovaries	0.044	土	0.012	0.041	土	0.006	0.046	土	0.012	0.043	土	0.005
Uterus with cervix	0.193	土	0.071	0.158	土	0.037	0.194	土	0.065	0.190	土	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	土	108.063	2004.400	土	137.999
Pituitary gland	9.300	土	1.494	9.900	土	1.969
Thyroid	24.300	土	3.368	23.900	土	3.784
Thymus	295.400	土	36.692	340.100	土	71.191
Heart	1046.300	土	159.712	1096.100	土	164.658
Liver	12122.900	土	2528.654	12430.000	土	1738.979
Spleen	737.200	土	157.512	787.500	土	133.114
Kidneys	2720.000	土	285.898	2842.400	土	236.942
Adrenals	77.400	土	12.158	76.400	土	9.947
Testicles	3576.700	土	245.092	3498.200	土	238.550
Epidydimides	1493.700	土	116.091	1446.300	土	131.374
Prostate with vesicular and coagulating glands	2407.000	土	307.769	2434.000	土	340.063
Levator ani	303.000	土	49.169	294.900	土	32.959
Bulbocavernosus muscle complex	767.200	土	106.542	787.300	土	75.918
Cowper’s glans	112.300	土	19.097	112.500	土	30.064
Glans penis	89.300	土	15.492	88.100	土	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	土	110.800	1943.300	土	88.000
Pituitary gland	13.900	土	4.458	13.000	土	2.055
Thyroid	12.700	土	3.129	12.800	土	3.615
Thymus	299.100	土	125.152	334.700	土	87.987
Heart	658.800	土	67.952	678.900	土	56.011
Liver	6631.700	土	657.827	6540.500	土	824.436
Spleen	497.800	土	23.976	512.800	土	56.233
Kidneys	1648.500	土	160.506	1745.900	土	194.600
Adrenals	81.200	土	7.885	87.900	土	8.736
Ovaries	162.200	土	21.175	173.800	土	24.275
Uterus with cervix	704.300	土	293.645	733.100	土	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	土	0.064	0.461	土	0.047
Pituitary gland	0.000	土	0.0001	0.000	土	0.000
Thyroid	0.006	土	0.001	0.005	土	0.001
Thymus	0.070	土	0.011	0.078	土	0.017
Heart	0.244	土	0.022	0.250	土	0.026
Liver	2.812	土	0.342	2.833	土	0.241
Spleen	0.171	土	0.021	0.180	土	0.026
Kidneys	0.636	土	0.025	0.650	土	0.032
Adrenals	0.018	土	0.003	0.017	土	0.002
Testicles	0.842	土	0.085	0.801	土	0.043
Epidydimides	0.351	土	0.030	0.331	土	0.027
Prostate with vesicular and coagulating glands	0.567	土	0.087	0.556	土	0.069
Levator ani	0.071	土	0.008	0.068	土	0.007
Bulbocavernosus muscle complex	0.180	土	0.022	0.181	土	0.021
Cowper’s glans	0.026	土	0.005	0.026	土	0.007
Glans penis	0.021	土	0.005	0.020	土	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	土	0.048	0.751	土	0.051
Pituitary gland	0.005	土	0.002	0.005	土	0.001
Thyroid	0.005	土	0.001	0.005	土	0.001
Thymus	0.114	土	0.043	0.128	土	0.032
Heart	0.253	土	0.017	0.261	土	0.011
Liver	2.548	土	0.188	2.509	土	0.162
Spleen	0.192	土	0.016	0.198	土	0.023
Kidneys	0.633	土	0.038	0.671	土	0.042*
Adrenals	0.031	土	0.003	0.034	土	0.003
Ovaries	0.062	土	0.007	0.067	土	0.010
Uterus with cervix	0.269	土	0.108	0.285	土	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	0^f		1m		2m		3^f
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]	-	-	-	-	-	-	一
Thyroid	congestion	-	-	-	-	-	-	1 [9]	一
	parathyroid hypertrophy	-	1 P2]	-	-	-	-	-	一
	congestion	1 [2]	-	-	1 [18]	-	-	2 [3,9]	一
Thymus	erythrocytorrhagia	-	-	-	-	-	-	1 [3]	一
	effusion	-	1 [22]	-	1 [17]			-	一
	lymphocytic tissue depletion	-	1 [17]	-	-	-	1 [13]	-	一
	erythrocytorrhagia	3 [1,2,10]	-	-	1 [18]	-	-	1 [8]	一
	leukocyte infiltration	1 [10]	-	-		-	-	1 [10]
Heart	foci of fibrosis	1 [10]	-	-	-	-	-	-	一
	foci of fatty degeneration	1 [10]	-	-		-	-	-	一
	left ventricular myocardium hypertrophy	-	-	-	1 [18]	-	1 [18]	-	一

 

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	0^f		1m		2m		3^f
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]	-	-	-	-	-	-	一
	congestion	1 [6]	-	3 [3,6,9]	2 [19,20]	1 [2]	2 [14,22]	2 [1,5]	2 [11,13]
	erythrocytorrhagia	5 P，6,8,9,10]	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 P，6,10]	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]	一
	leukocyte infiltration	1 [10]	-	-	-	-	-	-	一

 

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	0^f		1m		2m		3^f
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]	一
	effusion	1 [10]	-	-	-	-	-	-	一
Liver	bile ducts proliferation	1 [10]	-	-	-	-	-	-	一
	Browicz-Kupffer cell proliferation	1 [10]	-	-	-	-	-	-	一
	hepatocyte fine- droplet fatty degeneration	1 [3]	-	-	-	-	-	-	一
Spleen	congestion	-	1 [18]	-	-	-	-	-	一
	depletion of the white pulp	-	2 [17,18]	-	-	-	-	-	一
Pancreas	islets of Langerhans hypertrophy	1	1 P1]	-	-	-	-	3 [4,6,8]	一
	ducts proliferation	-	1 P1]	-	-	-	-	-	一
Jejunum	lymphatic follicle reactive hyperplasia	1 [5]	-	-	-	-	-	-	一
Ileum	lymphocyte infiltration	-	1 [11]	-	-	-	-	-	一

  

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	0^f		1m		2m		3^f
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]	一
	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]	一
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]	一
	retention microcysts	-	2 [12,15]	-	-	-	-	1 [8]	2 [14,21]
	hyaline casts	-	1 [12]	-	-	-	-	-	一

 

 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
		0^f		1m		2m		3^f
		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]	一
Prostate	congestion	2 [1,9]		-	-	-	-	2 [2,8]
	edema	2 [1,9]	-	-	-	-	-	-	一
	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]	一
Uterus	congestion	-	-	-	1 [16]	-	2 [16,20]	-	一
	effusion	-	-	-	2 [12,16]	-	2 [20,21]	-	一
	fibroid	-	-	-	2 [12,16]	-	1 [20]	-	一
Mandibular lymph nodes	congestion	1 [6]	-	-	-	-	-	-	一
	hyperplasia	1 [6]	-	-	-	-	-	-	一
	lymphocytic tissue depletion	-	1 [17]	-	-	-	-	-	一

 

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
		0^f		1m		2m		3^f
		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]	一
	hyperplasia	1 [6]	-	-	-	-	-	1 [6]	一
	lymphocytic tissue depletion	-	1 [17]	-	-	-	-	-	一

[]-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 SAT^f		3 SAT^f
		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 SAT^f		3 SAT^f
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 SAT^f		3 SAT^f
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 SAT^f		3 SAT^f
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 SAT^f		3 SAT^f
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]

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study has a Klimisch score of 1.

Organ:

bladder

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, the substance is not classified for repeated dose toxicity (STOT RE) in accordance with CLP Regulation (EU) No. 1272/2008.