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

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (2S)-2-{[ethenylbis({[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy})silyl]oxy}propanoate

EC Number:

701-301-6

Molecular formula:

C17H30O9Si

IUPAC Name:

ethyl (2S)-2-{[ethenylbis({[(2S)-1-ethoxy-1-oxopropan-2-yl]oxy})silyl]oxy}propanoate

Test material form:

liquid

Details on test material:

- Stabilisation: in water undergoes hydrolysis

Test animals

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

Administration / exposure

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).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

GROUP 0 AND SAT0

Doses / concentrations 2

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

GROUP 1

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

GROUP 2

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.