Reaction product of p-nonylphenol, formaldehyde and diethanolamine, propoxylated

Endpoint summary

Description of key information

Under the conditions of the repeated-dose oral toxicity study in rats (OECD 408, GLP), the NOAEL for general toxicity was determined to be 37.5 mg/kg bw.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Mar 2018 to 04 Oct 2018

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)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Toxicological Studies, Doświadczalna 27, 43-200 Pszczyna, Poland

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Cmdb: Wi; outbred

Details on species / strain selection:

The animal species (Rattus norvegicus) is recognised 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.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 - 8 weeks at the beginning of the experiment
- Weight at study initiation: On the day of the introduction to the study (day 0), the body weight of individual animals was ± 20% of the average value for each sex:
Group 0 males 242.4 g (220 g – 268 g); females 183.5 g (170 g – 199 g)
Group 4 males 241.4 g (223 g – 288 g); females 183.4 g (175 g – 203 g)
Group 1 males 242.6 g (216 g – 268 g); females 183.5 g (172 g – 199 g)
Group 2 males 242.4 g (223 g – 268 g); females 183.5 g (172 g – 202 g)
Group 0 SAT males 280.0 g (250 g – 304 g); females 174.6 g (171 g – 185 g)
Group 2 SAT males 280.2 g (272 g – 290 g); females 174.6 g (171 g – 182 g).
- Fasting period before study: no
- Housing: The animals were kept 2-3 animals in one cage, each sex was kept separately. The cages had a plastic bottom and covered with wire-bar lids. The dimensions of cages were 58 × 37 × 21 cm (length × width × height). Autoclaved and additionally UV- irradiated dust-free wood chips were used as bedding. In each cage wooden blocks were placed, nesting material and tunnels for laboratory animals were provided.
- Diet: Labofeed H Standard, Zofia Połczyńska Wytwórnia Pasz “Morawski”, Kcynia, ad libitum.
- Water: Tap water, ad libitum
- Acclimation period: minimum of 5 days.
- A general medical-veterinary examination was performed on the day of the introduction of the animals to the quarantine and at the end of it. Only healthy animals were used in the experiment.

DETAILS OF FOOD AND WATER QUALITY:
Each batch of feed and the water was analysed for content and contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 28
- Humidity (%): 45 - 85
- Air changes (per hr): about 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
14 Mar 2018 to 04 Oct 2018

Route of administration:

oral: gavage

Vehicle:

other: Sunflower oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The stability of the solutions of test item in sunflower oil was evaluated. Solutions of the test item for each group of animals were prepared every second day and stored in the fridge at a temperature of 2 – 8°C. The solutions were mixed before the administration to each animal. Solutions of the test item were prepared in the following way:
For 37.5 mg/kg b.w., there were 7.5 mg of Rokopol RF-151 in 1 g of sunflower oil formulation.
For 75 mg/kg b.w., there were 15 mg of Rokopol RF-151 in 1 g of sunflower oil formulation.
For 150 mg/kg b.w., there were 30 mg of Rokopol RF-151 in 1 g of sunflower oil formulation.

- VEHICLE
- Amount of vehicle: The test item / vehicle was given to animals in a constant volume of 0.5 mL/ 100 g b.w. calculated on the basis of body weight determined on weighing days of animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In order to confirm correct preparation of the test item formulation, the solutions of the test item in sunflower oil were analysed for concentration. During treatment one sample was analysed for one dose level from batches prepared on 14-03-2018, 21-03-2018, 07-05-2018, 20-06-2018. The concentration of the test item in a vehicle was chemically determined using High Performance Liquid Chromatography (HPLC), Shimadzu, Prominence LC-2030C with Diode Array Detector (DAD).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, seven days a week (first day of dosing = day 0)

Dose / conc.:

37.5 mg/kg bw/day (actual dose received)

Remarks:

Group 4 (new group after termination of treatment group 3)

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

Group 1

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3 (group terminated for humanitarian reasons)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

Group 2 SAT (used for recovery assessment)

No. of animals per sex per dose:

Group 1, 2, 4: 10 animals/dose/sex
Group 3: 5 animals/dose/sex (terminated)
Group 0 SAT/ Group 2 SAT: 5 animals/dose/sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 75, 150 and 300 mg/kg bw and also the vehicle were selected on the basis of Acute Oral Toxicity Study (Acute Toxic Class Method) (OECD 423) and a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening test (OECD 422). Then the doses were revised on the basis of clinical signs of animals introduced to the group 3 (the dose of 300 mg/kg bw). Clinical signs such as: respiratory murmurs, difficult breathing, diarrhoea, decreased locomotor activity, bristled coat, vocalisation and body weight loss above 20%. The aim was to decrease the dose levels so that the highest dose of 150 mg/kg bw would induce toxicity but not death or severe suffering. The medium dose 75 mg/kg bw and the low dose 37.5 mg/kg bw were fixed with two fold intervals for setting the descending dose levels.
- Rationale for animal assignment: Random
- Rationale for selecting satellite groups: The satellite groups were introduced to assess the potential reversibility of any effect.
- Post-exposure recovery period in satellite groups: 21 days.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, including mortality
- Time schedule: Performed twice a day usually at the beginning and end of each day, or otherwise once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The detailed clinical observations were conducted before the test item / medium administration and then once a week. They involved evaluation of changes in skin, fur, eyes, and mucosa membranes changes, the respiratory, the circulatory, and nervous systems, somatic activity and behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the animals were determined prior to the beginning of the experiment (day 0) and then twice a week during the experiment.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Food in cages was weighted to determine food intake once a week during the experiment.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were conducted before the introduction of the animals to the experiment and before euthanasia. An indirect ophthalmoscope was used. In case of the satellite groups, these examinations were also conducted after the end of the test item/medium administration.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 90th day for groups 0, 4, 1, 2 or on 111th day for groups 0 SAT and 2 SAT.
- Anaesthetic used for blood collection: Yes, xylazine-ketamine mixture at a dose of: 10 mg/kg bw of xylazine and 100 mg/kg bw of ketamine.
- Animals fasted: Yes, 16 hours without access to fodder, but ad libitum access to water.
- How many animals: All survived animals.
- Parameters examined using the Exigo haematological apparatus designed for veterinary studies taking account of the species specificity: leukocyte count, erythrocyte count, thrombocyte count, the level of haemoglobin, the hematocrit value
- Calculated erythrocyte indices included: MCV – Mean Corpuscular Volume, MCH - Mean Corpuscular Haemoglobin weight, MCHC – Mean Corpuscular Haemoglobin Concentration.
- Microscopic parameters examined using the May-Grunwald-Giemsa method: number of reticulocytes.
- Prothrombin time (PT) was determined with the use of test strips for the CoaguChek XS apparatus made by Roche.
- Activated partial thromboplastin time (APTT) was performed on the Hemochron apparatus (ITC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 90th day for groups 0, 4, 1, 2 or on 111th day for groups 0 SAT and 2 SAT.
- Animals fasted: Yes, 16 hours without access to fodder, but ad libitum access to water.
- How many animals: All survived animals.
- Parameters examined performed on the Olympus AU400 biochemistry analyser with the use of the Beckman Coulter reagents, bile acids were determined with the use of Randox reagents are listed in Table 1 in ‘Any other information on materials and methods incl. tables’.

URINALYSIS: Yes
- Time schedule for collection of urine: A day before the euthanasia of animals (on 89th day for groups 0, 4, 1, 2 or on 110th day for groups 0 SAT and 2 SAT).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No, but no access to fodder for 16 hours during housing in the metabolism cages.
- Parameters examined included: 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), nitrites. The general urinalysis was performed with the use of the test strips evaluated and the Clinitek Status reader with automatic reading. The Multistix-10 SG test strips (Bayer) were used, change of colour of test fields was used for a semiquantitative determination of chemical content in urine.
- The urine samples were checked microscopically for: squamous epithelium, cuboidal epithelium, leukocytes , erythrocytes, bacteria, crystals.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: for all groups between day 85 and 89 and for the satellite.
- Dose groups that were examined: All animals from group 0, 4, 1, 2 and groups 0 SAT and 2 SAT.
- Battery of functions tested: behaviour in an open field, responses to stimuli, fore- and hindlimb grip strength, and sensorimotor activity (vertical and horizontal).

IMMUNOLOGY: Yes
- The test item effects on the immune system were evaluated based on results of blood morphology with a picture of peripheral blood and bone marrow, concentration of albumin as an acute phase protein, urea, cholesterol, creatinine, total bilirubin, AST, ALT, AP, total protein, albumin/globulin ratio, histopathological examination of thymus, spleen and lymph nodes as well as absolute and relative weights of thymus and spleen from all surviving animals.

OTHER:
Microscopical examinations of bone-marrow obtained from femur after animal euthanasia through the determination of the number of individual nuclear cells per 1000 test cells included the number of cells was determined: within the erythrocyte system, within the leukocyte system, within the range of different cells (lymphocytes, monocytes, plasmocytes, megakaryocytes and other cells).
Evaluation of bone-marrow was performed in all animals from the control group (group 0, 0 SAT) and high dose group (group 2, 2 SAT). The examination was not extended to animals of all other dosage (group 1 and 2) because all statistically significant changes that were observed were considered to be unrelated to the test item.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Detailed gross examination which included thorough observations of the external body surface and all orifices as well as the cranial, thoracic and abdominal cavities with their content.
After the end of experiment, absolute weights of the following internal organs of all animals were determined: brain with cerebellum, pituitary gland, thymus, heart, liver, spleen, kidneys, adrenal glands, testicles, epididymides, prostate with the seminal vesicles and coagulating glands, ovaries.
The relative weight of internal organs was calculated on the basis of the absolute organ weight with reference to the live animals’ body weight.

HISTOPATHOLOGY: Yes
The following organs and tissues were preserved for histopathological examinations: brain (representative regions including cerebrum, cerebellum and medulla/pons), pituitary, spinal cord (cervical, mid-thoracic and lumbar), eye with the optic nerve if changes were observed during ophthalmological examinations, Harderian gland, tongue, mandibular salivary gland, stomach, duodenum, jejunum, ileum, cecum, colon, liver, pancreas, kidneys, ureters, adrenal glands, urinary bladder, spleen, heart, aorta, thymus, lymph nodes (mandibular and mesenteric), thyroid with parathyroid, oesophagus, larynx, trachea, lungs, ovaries with oviducts, uterus with cervix, vagina, testicles, epididymides, accessory sex glands (prostate with seminal vesicles and coagulating glands), skeletal muscle including peripheral nerve, femur with joint, skin, mammary gland and all detected gross lesions.
Bones after fixation were subjected to a decalcification.
The testicles and epididymides were fixed in modified Davidson’s fluid.
Other collected organs and tissues were fixed in a 10% solution of formalin.
Preserved samples of all collected organs and tissues were embedded in paraffin, stained with haematoxylin and eosin, and evaluated under a light microscope.
Full histopathological examination was performed on preserved organs and tissues of all animals from the control (0 and 0 SAT) and high dose (2 and 2 SAT) groups. The examination was extended to animals of other dosage groups (group 4 and 1) to organs showing treatment-related histopathological changes in the high dose group, i.e. stomach, mesenteric lymph nodes and spleen. All gross lesions were also examined.
The severity of observed pathological lesions were evaluated according to the scale listed in Table 2 in ´Any other information on materials and methods incl. tables’.

Statistics:

The results obtained in the study were subjected to statistical analysis, using following programs: Excel 2007, Excel 2013 and STATISTICA 10.0 PL.
Statistical analysis for males and females was performed separately. The results are presented in tables in the form of average values and standard deviation.
Food intake is summarised in tables, however statistical analysis of the results was not conducted, because the amount of data was insufficient (4 cages in non-satellite groups and 2 cages in 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.
The treated groups i.e. groups 4, 1, 2 were compared to the control group (group 0).
The treated satellite group i.e. 2 SAT was compared to the satellite control group (group 0 SAT), however statistical analysis of the results obtained in the satellite groups was not performed due to insufficient data (number of animals n=4 in the group 2 SAT, both in males and females).
The course of the statistical analysis (group 0, 4, 1, 2) was as follows:
- 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 analysed using a one-way analysis of variance and if necessary followed by Dunnett's test,
- in the absence of normality of distribution or non-homogeneous variances, the nonparametric Kruskal-Wallis test was used, if necessary followed by Dunnett's test.
The obtained results were analysed statistically at p ≤ 0.05.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In 5 males and 5 females at the (originally intended) dose of 300 mg/kg b.w. following clinical changes were observed: respiratory murmurs, difficult breathing, diarrhea, decreased locomotor activity, bristled coat, vocalization, body weight loss above 20%, therefore other animals were not introduced into group 3. Due to these changes all of 10 animals from group 3 (dose 300 mg/kg b.w.) were euthanised for humanitarian reasons.
Clinical signs were observed in 16 animals during the entire experiment. The few clinical signs which occurred in animals during the entire experiment were connected with: skin, eyes and eyelids, respiratory system. The symptoms connected with hair and skin were observed from the 4th week and remained till the end of the experiment. Porphyrin (deposition around the eye) was observed on the 1st and the 8th week of the experiment, therefore can be considered as transient. These changes occurred spontaneously in animals and should not be connected with the test item action.
Respiratory murmurs were observed on the 1st and 13th week of the experiment and may be caused by administering the test item by gavage.
The clinical examinations did not reveal any test item-related effects in animals that died during the course of experiment, with the exception of clinical signs connected with locomotor system, behaviour and reactions to stimuli (dejection, seizures, distinct decrease of locomotor activity) in one male of group 2, that occurred after administration of the test item, before the death of the animal and probably were test item-related.
There were no significant differences in physical appearance and behaviour between the treated groups and the control groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

The administration of the test item to the animals did not cause mortality in group 0 (dose 0 mg/kg bw), 0 SAT (0 mg/kg bw), 4 (dose 37.5 mg/kg bw) and 1 (dose 75 mg/kg bw). Six animals died during the experiment (four animals from group 2 (dose 150 mg/kg bw) and two animals from group 2 SAT (150 mg/kg bw).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The administration of the test item at the doses of 37.5 mg/kg bw and 75 mg/kg bw did not influence body weights of animals in the treated groups.
Statistically significantly lower average body weight in males of group 2 (the dose 150 mg/kg bw), which occurred in the 13th week of the experiment was correlated with body weight loss in two males stated during this time. Moreover, average body weight in the males from group 2 SAT was, at the same time of experiment, 11% lower compared to the group 0 SAT. So the tendency to mild body weight loss in males is clear in both groups at the end of the dosing period and it is probably connected with the test item action (Table 1 and 2 in 'Any other information on results incl. tables').

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Body weight loss was not accompanied by changes in food intake, as food intake was similar in treated groups compared to control groups both in males and females.

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 show any changes in the eyes of the control animals and the treated animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological findings can be found in Tables 3 to 12 in 'Any other information on results incl. tables'.
In case of haematological examinations, the most significant, treatment related change that was found both in males and females, was an increase in the number of leukocytes in group 2. Compared to the control group 0, the number of leukocytes in males increased by 85% and in females by 66%. Despite the lack of statistical analysis of the results of satellite groups, it was visible that this change was persistent. In females from group 2 SAT the number of leukocyte increased by 61% compared to the control group 0 SAT, while in males from group 2 SAT the increase by 21% was observed compared to the control group 0 SAT.
Changes dependent on the test item were also found in the percentage content of leukocytes. In males, dose-related increase in the number of neutrocytes and decrease in the number of lymphocytes in group 1 and 2 was observed. Similar changes were stated in females from group 2. Despite the lack of statistical analysis of the results of satellite groups, it was visible that these changes were persistent. In case of neutrocytes, in females from group 2 SAT this change was more severe, the number of neutrocytes increased by 193% compared to the control group 0 SAT, while in males from group 2 SAT the increase by 92% was observed compared to the control group 0 SAT. In case of lymphocytes, the decrease by about 31%, both in males and females from group 2 SAT was observed compared to the control group 0 SAT.
The changes observed in the percentage of leukocytes were confirmed in the results of the bone marrow examination in the form of the following treatment related changes: increase in the total number of cells in the leukocyte system in males from group 1 and 2, decrease in the total number of different cells, both in males and females from group 2, and decrease in the number of lymphocytes in females from group 1, and both in males and females from group 2. A similar tendency was visible in the results of satellite group despite the lack of statistical analysis.
All the changes discussed above in haematological and bone marrow examinations may indicate the presence of inflammation in animals.
Statistically significant increase in the number of thrombocytes could also be connected with inflammation, however, changes observed in females from group 1 and 2 were constant and did not change with increasing dose, both in group 1 and in group 2, the number of thrombocytes increased by 11%. The increase in the number of thrombocytes in males from group 2 was similar to that of females, about 12%. In the satellite groups 2 SAT, changes in the number of thrombocytes were less intense and opposite, i.e. in males the number of thrombocytes increased by 7%, while in females the number of thrombocytes decreased by 7% compared to the control group 0S AT. Therefore, changes in the number of thrombocytes were not considered to be related to the treatment.
The decrease in the prothrombin time in males from group 2 was considered as not related with the treatment due to isolated nature.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry findings can be found in Tables 13 to 16 in 'Any other information on results incl. tables'.
There were several statistically significant changes in the results of biochemical examinations in group 2, both in males and females. Changes in the concentration of albumin, globulin as well as changes in the A/G ratio were connected with the inflammation and were considered to be related to the test item. A similar tendency in the results of biochemical examinations was visible in the results of satellite groups despite the lack of statistical analysis.
Statistically significant changes in the concentration of electrolytes, i.e. sodium and chlorides, in females from groups 4 and 1 were not related to the test item due to the accidental nature and lack of confirmation in groups with a higher dosage.
No statistically significant changes were found in the results of enzymatic examinations, both in males and females from groups 4, 1, and 2. In case of satellite groups, results of enzymatic examinations in groups 2 SAT were comparable to the results of the control group 0 SAT with the exception of AST activity in males from group 2 SAT. An elevated mean AST activity in males from group 2 SAT was caused by a high reading in a single animal, and it should be considered as accidental.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant changes observed in the results of general urine examinations were not connected with the treatment. Increase in the amount of nitrites in females from group 4 did not occur in the higher dosage groups i.e. 1 and 2. Decrease in the pH value in males from group 2 was not toxicologically relevant. There were no statistically significant changes in the results of urine sediment examinations.
In the results of general and sediment urine examinations in the satellite group 2 SAT, both in males and females, there were no results which can be perceived as treatment related compared to the control group 0 SAT.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The statistically significant decrease of locomotor activity (vertical) was observed in females from group 2 during time intervals 0-30 minutes compared to control group. Lower average of locomotor activity (vertical) was observed in the same time intervals in females from group 2 SAT (measurement 1). Because the measurement of group 2 and 2 SAT was conducted at the same stage of the experiment (before the end of administration of the test item), it can be concluded that this change probably could be connected with the administration of the test item.
The behavioural studies showed that the nervous system functioned properly. The detailed clinical observations made during and after the treatment, and the open field observations did not reveal any muscarinic symptoms, nicotinic symptoms, or central symptoms caused by the test item. Delayed neurotoxicity was not observed.
During the open field observations, single cases of increased and decreased arousal were found. Changes in arousal was observed both in treated and control groups so it should not be connected with the administered material. There were no statistically significant changes in males and females of: number of faecal boluses, number of urine pools, horizontal and vertical locomotor activity (3-minute measurement).
Results of evaluation of reactions to stimuli did not show any harmful influence of the test item on animals. Changes in responses to objects, being touched, sound, and the pinna reflex were not noticed both in males and females from groups 0, 4, 1, 2.
The administration of the test item did not affect latency time of pain reaction in the no satellite groups.
The administration of the test item did not affect fore- and hindlimb grip strength in the treated groups. There were no statistically significant differences in fore- and hindlimb grip strength of males and females between the treated and the control groups.
There were no differences in horizontal locomotor activities of males and females between the treated groups (4, 1, 2) and the control group (0).

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

Findings related to immunological effects can be found in Tables 13 to 16 in 'Any other information on results incl. tables'.
The test item effects on the immune system were evaluated based on results of blood morphology with a picture of peripheral blood and bone marrow, concentration of albumin as an acute phase protein, urea, cholesterol, creatinine, total bilirubin, AST, ALT, AP, total protein, albumin/globulin ratio, histopathological examination of thymus, spleen and lymph nodes as well as absolute and relative weights of thymus and spleen from all animals.
Statistical analyses of the clinical-chemical examinations results showed several statistically significant changes which were perceived as caused by the test item at the doses of 75 mg/kg bw (group 1) and 150 mg/kg bw (group 2) and may indicate inflammation in treated animals, i.e. increase in the number of leukocytes in males and females from group 2; increase in the number of neutrocytes in males from group 1 and 2 and females from group 2; decrease in the number of lymphocytes in males from group 1and 2 and females from group 2; increase in the total number of cells in leukocyte system of bone marrow in males from group 1 and 2; decrease in the number of bone marrow lymphocytes in males from group 2 and females from group 1 and 2; decrease in the total number of different cells in males and females from group 2; decrease in the albumin concentration and A/G ratio in males from group 2; increase in the globulin concentration and decrease in the A/G ratio in females from group 2.
There were no statistically significant changes in absolute and relative weights of thymus and spleen as well as no test item-related pathological changes in thymus, however mesenteric lymph nodes were enlarged in males of group 1 and males and females of group 2 and 2 SAT.
Histopathological evaluation revealed in mesenteric lymph nodes from group 1, 2 and 2 SAT atrophic lymphoid tissue, increased histiocytes, necrosis, enlargement, fibrosis with accompanying in some cases angiogenesis and/or shrinking and/or plasma cell hyperplasia and/or cell degeneration and vacuolation, that most likely are test item related.
White pulp hyperplasia observed in spleen, despite the occurrence in the control group, due to increase in the number of animals as well as severity degree in females in group 2, should be considered as related to the test item. The decreased number and/or severity of white pulp hyperplasia in animals from group 2 SAT indicates possible regression of changes after stopping the administration of the test item.
Therefore it can be concluded that the test item at the dose levels of 75 and 150 mg/kg bw (males and females) affected the immune system of the treated animals.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The only statistically significant increases were stated in relative weights of thyroid, testicles and epididymides in males of group 2 (the dose of 150 mg/kg bw), however no statistically significant changes were stated in absolute weights of internal organs. Organs may change in relative weight in a manner dependent upon body weight rather than as a result of a test item primary toxic effect and at the end of the dosing period the decrease of average body weight in the males of group 2 occurred. Moreover, histopathological evaluation did not reveal corresponding pathological changes in above mentioned organs that could state for toxicity of the test item, hence the decreases of relative weight of thyroid, testicles and epididymides should not be related to the test item (Table 17 to 20 in 'Any other information on results incl. tables').

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The test item also was not responsible for the increase of the amount of visceral fat, that was noticed during gross examinations as excessively obese animals in controls (group 0 and 0 SAT) and low dose group 4, which was rather connected with the type of adminstered vehicle (sunflower oil), as there were only two cases of females in the mid dose and two in the high dose group.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological evaluation revealed in mesenteric lymph nodes from group 1, 2 and 2 SAT atrophic lymphoid tissue, increased histiocytes, necrosis, enlargement, fibrosis with accompanying in some cases angiogenesis and/or shrinking and/or plasma cell hyperplasia and/or cell degeneration and vacuolation, that most likely are test item related.
Minimal increases of histiocytes were observed in mesenteric lymph nodes in group 4 in a small number, in this case it should not be connected with test item due to spontaneously occurring in rats.
White pulp hyperplasia observed in spleen, despite the occurrence in the control group, due to increase in the number of animals, as well as the severity degree in females in group 2, should be considered as related to the test item. The decreased number and/or severity of white pulp hyperplasia in animals from group 2 SAT indicates possible regression of changes after stopping the administration of the test item. In males, because of similarity of occurrence, these lesions may be treated as normal incidental finding and not related to the test item.
Although erosions in stomach are spontaneously occurring in rats, in the fallen animals, a slightly higher intensity of occurrence was observed. In connection with necrosis of mucosa (singular cases in groups with the highest dose) there is possibility of influence of test item on stomach mucosa in the highest dose.
Other findings were incidental and not related to the test material. These findings include histological changes in the thymus, lymph nodes, germ cells, epididymis, salivary gland, prostate, infiltration of leukocytes (in Harderian glands, trachea, larynx, lungs, liver, kidneys, prostate and various parts of digestive tract), pancreas, pituitary pars distalis, muscle fibres degeneration liver, kidney, adrenal glands.
Furthermore, so-called background lesions are often observed in rats. They are related to species, gender or age, and should not be associated with the test item administered to animals. Such changes in the rat include, among others: foci of mineralisation, foci of osteometaplasia, foci of chondrometaplasia, foci of neurometaplasia, aggregation/s of alveolar macrophages (macroscopically seen as light foci bulged above surface of lungs), cysts, bronchoalveolar hyperplasia, atrophy of the renal papillae, hyaline casts, fibroplasia, basophilic tubules, hepatocytes vacuolation, diverticulum in jejunum, accessory adrenocortical nodules, germ cell degeneration, hyperkeratosis and focal glandules dilation in stomach, glandular hyperplasia in duodenum, dilation of the tracheal glands, foci of hepatocyte degeneration, angiogenesis, bile duct hyperplasia, periportal lipid vacuolation, macrovesicular fatty changes, acinar dilation in seminal vesicles and coagulating gland, foci of fatty changes in pancreas or ovaries or adrenal glands or urinary bladder, hyperplasia or hypoplasia of islet cells, hyperplasia or hypoplasia of mammary gland, oedema in prostate. The lack of association with the influence of the test item is evidenced by the presence of the lesions in the control groups and historical data obtained from other 90-day toxicity studies.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

37.5 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

75 mg/kg bw/day (actual dose received)

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Analytical verification of dose concentrations

The calculated ROKOPOL RF-151 average determination were in the range of 84.9% ÷ 98.1% of labelled dosages, so they fulfilled acceptance criteria for biological samples, which are 80.0% ÷ 120.0%.

Table 1 Body weight - treatment groups (mean ± SD)

Week	GROUP / DOSE [mg/kg bw]
	0 / 0		4 / 37.5			1 / 75			2 / 150
	Male n=10	Female n=10		Male n=10	Female  n=10		Male n=10	Female n=10		Male n=10	Female n=10
0	242.40 ± 13.91	183.50 ± 9.25		241.40 ± 20.23	183.40 ± 8.49		242.60 ± 15.12	183.50 ± 8.07		242.40 ± 12.55	183.50 ± 9.44
0.5	267.10 ± 16.06	193.80 ± 11.92		264.40 ± 25.66	194.10 ± 9.49		258.70 ± 14.80	189.80 ± 7.18		251.40 ± 16.36	186.10 ± 11.83
1	289.00 ± 16.18	200.80 ± 11.54		283.00 ± 29.14	200.60 ± 9.59		275.70 ± 15.17	195.80 ± 9.02		270.80 ± 22.75	196.11 ± 10.23 a
1.5	301.90 ± 16.95	205.20 ± 11.32		296.30 ± 32.47	204.50 ± 13.27		289.70 ± 18.83	200.10 ± 10.16		279.00 ± 19.16	203.56 ± 12.38 a
2	316.90 ± 19.63	212.70 ± 11.95		311.80 ± 34.20	211.90 ± 13.90		304.70 ± 22.45	205.50 ± 8.48		294.30 ± 21.03	209.11 ± 11.23 a
2.5	326.10 ± 20.97	214.90 ± 13.58		319.60 ± 37.76	215.90 ± 12.33		313.10 ± 23.35	212.70 ± 8.47		302.10 ± 21.06	213.00 ± 12.51 a
3	337.20 ± 23.20	220.40 ± 15.13		330.30 ± 42.47	220.00 ± 13.56		325.80 ± 23.03	218.80 ± 8.93		316.60 ± 24.80	219.67 ± 14.11 a
3.5	345.70 ± 23.90	225.30 ± 13.57		339.90 ± 44.08	223.70 ± 13.00		332.70 ± 26.06	221.50 ± 9.56		322.20 ± 25.73	223.89 ± 13.13 a
4	354.40 ± 24.84	230.00 ± 14.46		348.00 ± 48.27	229.00 ± 14.15		342.40 ± 27.75	223.00 ± 10.79		333.50 ± 25.43	229.00 ± 11.37 a
4.5	363.20 ± 26.43	234.10 ± 15.58		355.70 ± 50.95	234.70 ± 14.13		351.60 ± 29.02	228.60 ± 9.92		340.30 ± 25.88	233.44 ± 12.45 a
5	370.80 ± 28.90	237.30 ± 16.10		359.90 ± 53.46	238.80 ± 15.19		358.90 ± 29.04	233.50 ± 10.35		344.80 ± 26.44	235.11 ± 13.42 a
5.5	374.10 ± 30.72	238.50 ± 15.87		366.30 ± 53.10	240.60 ± 16.06		366.50 ± 28.59	234.10 ± 12.39		351.50 ± 25.73	238.33 ± 14.89 a
6	380.80 ± 31.29	242.30 ± 16.15		372.60 ± 54.04	243.10 ± 17.14		369.80 ± 28.23	236.60 ± 13.68		353.00 ± 25.98	239.44 ± 15.32 a
6.5	386.00 ± 31.41	244.90 ± 18.02		376.30 ± 54.63	244.80 ± 15.41		373.40 ± 28.41	239.60 ± 13.30		358.60 ± 28.73	241.44 ± 15.96 a
7	393.00 ± 33.22	247.70 ± 18.19		383.50 ± 57.67	248.00 ± 15.75		379.40 ± 28.58	242.90 ± 12.27		358.90 ± 31.15	242.89 ± 16.58 a
7.5	396.20 ± 32.98	250.30 ± 16.85		386.20 ± 56.49	250.80 ± 15.60		383.20 ± 29.69	244.10 ± 12.62		366.90 ± 30.06	245.11 ± 14.09 a
8	399.10 ± 34.99	253.70 ± 16.53		392.00 ± 59.03	257.10 ± 15.74		389.00 ± 31.99	246.60 ± 12.43		371.60 ± 30.04	246.67 ± 13.30 a
8.5	405.40 ± 34.94	256.60 ± 17.75		395.60 ± 58.79	256.90 ± 15.90		393.20 ± 31.67	248.20 ± 10.80		377.30 ± 28.68	250.11 ± 16.21 a
9	409.90 ± 33.39	259.00 ± 17.80		398.90 ± 59.08	257.60 ± 17.35		396.00 ± 31.70	250.10 ± 11.63		378.70 ± 28.79	251.13 ± 14.59 b
9.5	414.70 ± 33.96	258.80 ± 18.42		402.50 ± 57.97	260.40 ± 19.02		401.70 ± 32.54	251.40 ± 12.69		382.40 ± 27.56	250.88 ± 18.03 b
10	418.30 ± 36.44	260.50 ± 17.63		405.90 ± 57.95	261.90 ± 18.79		406.30 ± 33.65	252.80 ± 12.06		384.10 ± 29.26	253.13 ± 18.28 b
10.5	422.90 ± 35.12	259.80 ± 20.09		409.90 ± 58.36	262.60 ± 18.80		409.80 ± 33.46	255.20 ± 12.45		387.20 ± 25.21	256.00 ± 16.97 c
11	427.30 ± 33.44	263.60 ± 19.35		413.10 ± 57.55	265.30 ± 19.42		413.40 ± 34.07	254.90 ± 12.86		390.20 ± 27.97	258.57 ± 17.24 c
11.5	433.10 ± 35.43	263.90 ± 19.42		416.40 ± 58.61	265.90 ± 18.99		415.30 ± 35.35	255.40 ± 10.86		392.00 ± 26.22	259.14 ± 15.52 c
12	437.70 ± 34.26	266.70 ± 18.52		420.60 ± 58.89	268.20 ± 18.68		416.80 ± 36.44	257.40 ± 11.55		388.89 ± 22.71 a	260.57 ± 15.11 c
12.5	441.90 ± 34.75	268.30 ± 19.29		423.10 ± 57.50	270.90 ± 17.79		420.00 ± 38.78	257.10 ± 10.86		391.89 ± 21.50 a	262.71 ± 17.18 c
13	442.90 ± 34.67	269.70 ± 20.76		424.40 ± 57.33	272.10 ± 19.89		422.20 ± 40.52	262.00 ± 12.06		387.78 ± 20.50* a	261.57 ± 16.85 c

n – number of animals in group

* – statistically significant difference at p ≤ 0.05

a - n=9

b - n=8

c - n=7

 

Table 2. Body weight - satellite groups (mean ± SD)

Week	GROUP / DOSE [mg/kg bw]
	0 SAT / 0		2 SAT / 150
	Male n=5	Female n=5	Male n=5	Female n=5
0	280.00 ± 20.09	174.60 ± 5.94	280.20 ± 7.95	174.60 ± 4.34
0.5	294.00 ± 22.97	182.20 ± 3.11	285.40 ± 4.51	174.40 ± 5.13
1	305.80 ± 26.53	192.40 ± 2.88	296.00 ± 4.69	185.60 ± 4.22
1.5	314.80 ± 25.90	194.40 ± 3.51	307.20 ± 4.71	192.80 ± 6.87
2	326.60 ± 27.90	199.60 ± 5.03	316.60 ± 2.51	198.80 ± 5.45
2.5	335.00 ± 27.84	203.80 ± 6.46	322.80 ± 4.38	202.80 ± 5.72
3	339.20 ± 29.49	208.60 ± 8.38	324.00 ± 6.52	208.00 ± 5.00
3.5	349.40 ± 31.17	212.60 ± 3.78	330.20 ± 5.36	213.80 ± 4.32
4	356.20 ± 33.89	217.80 ± 4.87	335.80 ± 6.98	221.20 ± 4.55
4.5	359.00 ± 33.93	223.60 ± 6.58	338.00 ± 4.74	222.40 ± 3.58
5	363.80 ± 36.36	225.20 ± 6.72	340.40 ± 5.68	223.25 ± 3.20 a
5.5	371.00 ± 37.91	228.80 ± 8.70	347.40 ± 7.47	226.25 ± 3.86 a
6	379.80 ± 39.96	230.60 ± 4.77	349.00 ± 7.48	228.50 ± 5.07 a
6.5	384.00 ± 38.60	231.60 ± 6.47	351.60 ± 5.64	231.25 ± 6.40 a
7	392.00 ± 37.97	232.20 ± 8.14	358.40 ± 6.11	235.75 ± 6.85 a
7.5	395.80 ± 38.31	237.20 ± 6.94	362.75 ± 5.68 a	235.50 ± 6.03 a
8	400.20 ± 39.07	239.00 ± 7.35	367.50 ± 7.23 a	238.50 ± 6.03 a
8.5	405.80 ± 39.60	243.40 ± 8.02	372.75 ± 7.72 a	235.25 ± 6.18 a
9	409.80 ± 40.00	244.60 ± 8.85	377.00 ± 6.38 a	239.50 ± 4.65 a
9.5	413.40 ± 39.20	244.40 ± 9.45	380.00 ± 7.16 a	241.75 ± 7.72 a
10	416.60 ± 39.85	246.80 ± 9.76	385.25 ± 6.70 a	243.25 ± 8.26 a
10.5	422.20 ± 39.93	247.00 ± 11.36	388.00 ± 8.12 a	246.50 ± 10.79 a
11	426.20 ± 38.34	247.60 ± 12.78	389.75 ± 5.38 a	253.75 ± 15.86 a
11.5	430.80 ± 37.92	249.60 ± 11.01	389.50 ± 8.27 a	249.25 ± 8.96 a
12	434.00 ± 36.16	253.00 ± 11.20	392.25 ± 11.59 a	251.50 ± 7.33 a
12.5	438.80 ± 35.35	254.60 ± 11.19	393.75 ± 10.31 a	249.75 ± 2.99 a
13	444.00 ± 32.69	255.40 ± 10.78	395.00 ± 10.10 a	251.25 ± 3.86 a
13.5	440.80 ± 36.52	253.80 ± 9.60	399.75 ± 9.11 a	253.00 ± 7.62 a
14	443.40 ± 33.80	256.00 ± 11.31	405.00 ± 10.89 a	257.25 ± 11.18 a
14.5	447.40 ± 34.63	254.40 ± 13.33	407.50 ± 9.68 a	258.50 ± 10.54 a
15	449.40 ± 32.42	255.60 ±12.66	412.00 ± 5.60 a	259.50 ± 11.39 a
15.5	454.00 ± 34.46	259.00 ± 12.39	417.50 ± 7.05 a	260.75 ± 9.84 a
16	448.20 ± 32.56	255.20 ± 11.65	415.00 ± 4.76 a	256.00 ± 9.42 a

n – number of animals in group

a - n=4

No statistical analyses were conducted, because the amount of data was insufficient (4 animals/ group 2 SAT/ sex).

Table 3. Haematological and coagulological examination - treatment groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0/0		4/37.5		1/75		2/150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=9	Male n=9	Female  n=7
HAEMOGLOBIN [g/L]	157.40 ± 6.35	146.10 ± 4.79	157.80 ± 4.13	150.50 ± 4.38	152.20 ± 9.91	145.89 ± 5.44	152.22 ± 6.10	147.86 ± 9.26
HAEMATOCRIT [1/1]	0.45 ± 0.04	0.43 ± 0.03	0.46 ± 0.01	0.46 ± 0.01	0.43 ± 0.04	0.42 ± 0.03	0.43 ± 0.03	0.44 ± 0.04
ERYTHROCYTES [1012/L x]	9.47 ± 0.49	8.43 ± 0.31	9.63 ± 0.22	8.67 ± 0.27	9.32 ± 0.58	8.39 ± 0.40	9.53 ± 0.53	8.67 ± 0.63
MCV [fL]	47.34 ± 1.90	50.95 ± 1.95	48.08 ± 1.00	52.82 ± 1.66	46.31 ± 2.27	50.38 ± 2.15	45.18 ± 2.25	50.19 ± 2.20
MCH [pg]	16.60 ± 0.45	17.34 ± 0.33	16.41 ± 0.34	17.39 ± 0.52	16.31 ± 0.55	17.39 ± 0.62	15.98 ± 0.57	17.06 ± 0.44
MCHC [g/L]	351.60 ± 14.56	341.00 ± 12.52	341.10 ± 4.09	329.60 ± 2.50	353.00 ± 11.13	345.78 ± 11.58	354.22 ± 10.44	340.29 ± 15.43
RETICULOCYTES [1/1]	0.013 ± 0.004	0.013 ± 0.003	0.011 ± 0.003	0.011 ± 0.003	0.014 ± 0.005	0.016 ± 0.004	0.015 ± 0.006	0.016 ± 0.003
THROMBOCYTES [x 109/L]	628.30 ± 26.39	608.80 ± 48.07	653.90 ± 45.92	635.40 ± 52.39	665.20 ± 34.12	677.67 ± 67.49*	701.67 ± 71.67*	675.43 ± 35.86*
LEUKOCYTES [x 109/L]	4.40 ± 0.82	2.79 ± 0.77	4.40 ± 0.83	3.06 ± 0.66	5.64 ± 1.30	3.43 ± 0.51	8.16 ± 2.62*	4.63 ± 0.93*
PT [s]	13.28 ± 0.70	11.12 ± 0.50	13.28 ± 0.99	11.36 ± 0.25	12.70 ± 0.81	10.86 ± 0.66^a	12.36 ± 0.52*	11.24 ± 0.28
APTT [s]	56.80 ± 4.54	58.30 ± 5.77	57.00 ± 10.76	51.80 ± 6.60	55.50 ± 9.47	50.78 ± 7.05	57.11 ± 7.83	56.86 ± 5.34

n - number of test animals

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

A - n=10

 

Table 4. Haematological and coagulological examination - satellite groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0SAT / 0	0SAT / 0	2SAT / 150	2SAT / 150
	Male n=5	Female n=5	Male n=3	Female n=4
HAEMOGLOBIN [g/L]	150.60 ± 6.07	148.40 ± 1.82	155.67 ± 8.62	151.25 ± 1.26
HAEMATOCRIT [1/1]	0.43 ± 0.02	0.42 ± 0.01	0.45 ± 0.02	0.43 ± 0.01
ERYTHROCYTES [1012/L x]	8.98 ± 0.20	8.20 ± 0.31	9.36 ± 0.33	8.65 ± 0.23
MCV [fL]	48.26 ± 1.76	51.66 ± 1.29	48.20 ± 0.95	50.00 ± 1.94
MCH [pg]	16.74 ± 0.46	18.12 ± 0.63	16.63 ± 0.32	17.50 ± 0.58
MCHC [g/L]	347.20 ± 4.76	350.80 ± 5.36	345.33 ± 1.15	350.00 ± 2.16
RETICULOCYTES [1/1]	0.025 ± 0.010	0.018 ± 0.004	0.018 ± 0.005	0.017 ± 0.002
THROMBOCYTES [x 109/L]	658.80 ± 56.44	617.00 ± 71.23	705.67 ± 81.13	590.25 ± 67.29
LEUKOCYTES [x 109/L]	3.06 ± 0.74	2.02 ± 0.43	3.70 ± 0.69	3.25 ± 1.01
PT [s]	13.32 ± 0.77	11.30 ± 0.75	12.70 ± 0.96^a	11.93 ± 0.95
APTT [s]	53.20 ± 7.01	48.60 ± 8.73	46.75 ± 8.81^a	64.25 ± 19.17

n - number of test animals

a - n=4

No statistical analyses were conducted due to insufficient data

 

 

Table 5. Haematological examination - Percentage content of leukocytes (leukocytogram) - treatment groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0/0		4/37.5		1/75		2/150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=9	Male n=9	Female n=7
NEUTROCYTES [1/1]	0.18 ± 0.06	0.17 ± 0.05	0.23 ± 0.07	0.17 ± 0.03	0.36 ± 0.16*	0.25 ± 0.10	0.49 ± 0.13*	0.37 ± 0.17*
EOSINOCYTES [1/1]	0.00 ± 0.01	0.00 ± 0.01	0.01 ± 0.01	0.00 ± 0.00	0.00 ± 0.01	0.00 ± 0.00	0.00 ± 0.01	0.00 ± 0.00
LYMPHOCYTES [1/1]	0.81 ± 0.06	0.82 ± 0.05	0.76 ± 0.08	0.82 ± 0.04	0.63 ± 0.16*	0.74 ± 0.10	0.50 ± 0.12*	0.63 ± 0.17*
MONOCYTES [1/1]	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
OTHER CELLS [1/1]	0.01 ± 0.01	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.01	0.00 ± 0.00	0.01 ± 0.01	0.00 ± 0.01	0.00 ± 0.01

n - number of test animals

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

 

Table 6. Haematological examination - Percentage content of leukocytes (leukocytogram) - satellite groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0SAT / 0	0SAT / 0	2SAT / 150	2SAT / 150
	Male n=5	Female n=5	Male n=3	Female n=4
NEUTROCYTES [1/1]	0.25 ± 0.08	0.14 ± 0.03	0.48 ± 0.17	0.41 ± 0.16
EOSINOCYTES [1/1]	0.01 ± 0.01	0.00 ± 0.00	0.00 ± 0.01	0.00 ± 0.00
LYMPHOCYTES [1/1]	0.74 ± 0.07	0.85 ± 0.03	0.52 ± 0.16	0.59 ± 0.16
MONOCYTES [1/1]	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
OTHER CELLS [1/1]	0.01 ± 0.01	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00

n - number of test animals

No statistical analyses were conducted due to insufficient data

 

Table 7. Results of haematological studies – bone marrow examination Erythrocyte system - treatment groups (mean ± SD)

Parameter		GROUP / DOSE [mg/kg bw]
		0/0		4/37.5		1/75		2/150
		Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=10	Male n=9	Female n=7
PROERYTHROBLAST	[1/1]	0.017 ± 0.004	0.016 ± 0.004	0.017 ± 0.004	0.016 ± 0.003	0.016 ± 0.004	0.016 ± 0.003	0.017 ± 0.003	0.015 ± 0.003
BASOPHILIC ERYTHROBLASTS	[1/1]	0.034 ± 0.003	0.032 ± 0.004	0.031 ± 0.004	0.031 ± 0.004	0.032 ± 0.004	0.034 ± 0.003	0.032 ± 0.003	0.034 ± 0.005
POLYCHROMATOPHILIC ERYTHROBLASTS	[1/1]	0.162 ± 0.008	0.163 ± 0.009	0.163 ± 0.008	0.162 ± 0.007	0.161 ± 0.006	0.162 ± 0.007	0.162 ± 0.008	0.162 ± 0.008
ORTHOCHROMATIC ERYTHROBLASTS	[1/1]	0.182 ± 0.007	0.182 ± 0.005	0.182 ± 0.006	0.181 ± 0.009	0.178 ± 0.004	0.178 ± 0.005	0.182 ± 0.006	0.181 ± 0.005
TOTAL	[1/1]	0.394 ± 0.012	0.393 ± 0.010	0.393 ± 0.006	0.391 ± 0.007	0.387 ± 0.012	0.390 ± 0.010	0.392 ± 0.013	0.393 ± 0.010

n - number of test animals

 

Table 8. Results of haematological studies – bone marrow examination Erythrocyte system - satellite groups (mean ± SD)

Parameter		GROUP / DOSE [mg/kg bw]
		0SAT / 0		2SAT / 150
		Male n=5	Female n=5	Male n=4	Female n=4
PROERYTHROBLAST	[1/1]	0.016 ± 0.003	0.015 ± 0.003	0.016 ± 0.003	0.014 ± 0.002
BASOPHILIC ERYTHROBLASTS	[1/1]	0.034 ± 0.005	0.034 ± 0.002	0.034 ± 0.005	0.033 ± 0.004
POLYCHROMATOPHILIC ERYTHROBLASTS	[1/1]	0.162 ± 0.008	0.161 ± 0.007	0.162 ± 0.005	0.161 ± 0.009
ORTHOCHROMATIC ERYTHROBLASTS	[1/1]	0.181 ± 0.006	0.181 ± 0.007	0.177 ± 0.008	0.177 ± 0.004
TOTAL	[1/1]	0.394 ± 0.015	0.391 ± 0.016	0.389 ± 0.002	0.385 ± 0.012

n - number of test animals

No statistical analyses were conducted due to insufficient data

 

Table 9. Results of haematological studies – bone marrow examination Leukocyte system - treatment group (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0/0		4/37.5		1/75		2/150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=10	Male n=9	Female n=7
MYELOBLASTS	0.016 ±	0.016 ±	0.016 ±	0.016 ±	0.018 ±	0.016 ±	0.017 ±	0.015 ±
[1/1]	0.003	0.003	0.003	0.003	0.003	0.003	0.003	0.003
PROMYELOCYTES	0.028 ±	0.029 ±	0.030 ±	0.029 ±	0.030 ±	0.029 ±	0.031 ±	0.029 ±
[1/1]	0.004	0.004	0.005	0.003	0.003	0.004	0.003	0.004
ORTHOCHROMATOPHILIC MYELOCYTES [1/1]	0.038 ± 0.004	0.035 ± 0.005	0.037 ± 0.005	0.037 ± 0.004	0.039 ± 0.004	0.039 ± 0.003	0.040 ± 0.006	0.038 ± 0.005
ACIDOPHILIC MYELOCYTES [1/1]	0.014 ± 0.002	0.013 ± 0.003	0.014 ± 0.003	0.014 ± 0.002	0.015 ± 0.003	0.014 ± 0.003	0.014 ± 0.002	0.017 ± 0.003
ORTHOCHROMATOPHILIC METAMYELOCYTES [1/1]	0.046 ± 0.004	0.043 ± 0.006	0.043 ± 0.005	0.044 ± 0.004	0.048 ± 0.004	0.046 ± 0.003	0.046 ± 0.005	0.046 ± 0.005
ACIDOPHILIC METAMYELOCYTES [1/1]	0.016 ± 0.003	0.017 ± 0.003	0.017 ± 0.004	0.017 ± 0.003	0.017 ± 0.002	0.018 ± 0.002	0.017 ± 0.003	0.018 ± 0.002
ROD NEUTROPHILS	0.026 ±	0.027 ±	0.028 ±	0.028 ±	0.027 ±	0.028 ±	0.028 ±	0.025 ±
[1/1]	0.004	0.005	0.006	0.003	0.005	0.006	0.003	0.003
ROD EOSINOPHILS	0.014 ±	0.014 ±	0.014 ±	0.014 ±	0.014 ±	0.014 ±	0.014 ±	0.016 ±
[1/1]	0.002	0.002	0.002	0.003	0.002	0.002	0.002	0.003
FILAMENTED NEUTROPHILS [1/1]	0.180 ± 0.008	0.179 ± 0.007	0.183 ± 0.008	0.182 ± 0.006	0.184 ± 0.006	0.182 ± 0.006	0.187 ± 0.006	0.186 ± 0.007
FILAMENTED EOSINOPHILS [1/1]	0.014 ± 0.003	0.015 ± 0.003	0.016 ± 0.003	0.015 ± 0.003	0.016 ± 0.002	0.016 ± 0.002	0.015 ± 0.002	0.015 ± 0.002
BASOPHILS	0.005 ±	0.005 ±	0.005 ±	0.005 ±	0.006 ±	0.006 ±	0.005 ±	0.006 ±
[1/1]	0.002	0.002	0.002	0.002	0.003	0.003	0.002	0.002
TOTAL	0.398 ±	0.392 ±	0.401 ±	0.402 ±	0.412 ±	0.408 ±	0.414 ±	0.412 ±
[1/1]	0.013	0.018	0.010	0.010	0.010*	0.011	0.019*	0.020
Leukocyte system/erythrocyte system quantitative ratio	1.010 ± 0.054	1.000 ± 0.066	1.021 ± 0.036	1.030 ± 0.036	1.067 ± 0.051	1.045 ± 0.047	1.058 ± 0.080	1.051 ± 0.072

n - number of test animals

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

 

Table 10. Results of haematological studies – bone marrow examination. Leukocyte system - satellite groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0SAT / 0		2SAT / 150
	Male n=5	Female n=5	Male n=4	Female  n=4
MYELOBLASTS [1/1]	0.016 ± 0.003	0.016 ± 0.003	0.016 ± 0.003	0.017 ± 0.002
PROMYELOCYTES [1/1]	0.029 ± 0.003	0.030 ± 0.002	0.031 ± 0.004	0.031 ± 0.005
ORTHOCHROMATOPHILIC MYELOCYTES [1/1]	0.037 ± 0.003	0.035 ± 0.005	0.040 ± 0.003	0.039 ± 0.004
ACIDOPHILIC MYELOCYTES [1/1]	0.014 ± 0.002	0.014 ± 0.002	0.014 ± 0.002	0.015 ± 0.002
ORTHOCHROMATOPHILIC METAMYELOCYTES [1/1]	0.046 ± 0.005	0.044 ± 0.005	0.049 ± 0.004	0.047 ± 0.004
ACIDOPHILIC METAMYELOCYTES [1/1]	0.017 ± 0.002	0.017 ± 0.003	0.017 ± 0.002	0.016 ± 0.003
ROD NEUTROPHILS [1/1]	0.026 ± 0.004	0.028 ± 0.003	0.028 ± 0.003	0.026 ± 0.005
ROD EOSINOPHILS [1/1]	0.014 ± 0.002	0.013 ± 0.002	0.014 ± 0.002	0.015 ± 0.004
FILAMENTED NEUTROPHILS [1/1]	0.181 ± 0.008	0.182 ± 0.006	0.183 ± 0.004	0.183 ± 0.007
FILAMENTED EOSINOPHILS [1/1]	0.014 ± 0.002	0.014 ± 0.002	0.017 ± 0.002	0.017 ± 0.002
BASOPHILS [1/1]	0.005 ± 0.002	0.005 ± 0.002	0.005 ± 0.002	0.005 ± 0.002
TOTAL [1/1]	0.398 ± 0.011	0.397 ± 0.008	0.414 ± 0.005	0.411 ± 0.015
Leukocyte system/erythrocyte system quantitative ratio	1.013 ± 0.043	1.016 ± 0.049	1.064 ± 0.010	1.069 ± 0.064

n - number of test animals

No statistical analyses were conducted due to insufficient data

 

Table 11. Results of haematological studies – bone marrow examination. Different cells - treatment group (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0/0		4/37.5		1/75		2/150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=10	Male n=9	Female n=7
LYMPHOCYTES [1/1]	0.177 ± 0.013	0.182 ± 0.011	0.176 ± 0.008	0.175 ± 0.011	0.170 ± 0.011	0.170 ± 0.010*	0.163 ± 0.009*	0.160 ± 0.011*
MONOCYTES [1/1]	0.006 ± 0.003	0.007 ± 0.003	0.006 ± 0.002	0.007 ± 0.003	0.006 ± 0.002	0.007 ± 0.002	0.007 ± 0.003	0.007 ± 0.002
PLASMOCYTES [1/1]	0.005 ± 0.002	0.006 ± 0.002	0.005 ± 0.002	0.006 ± 0.002	0.006 ± 0.002	0.005 ± 0.002	0.005 ± 0.002	0.005 ± 0.002
MEGAKARYOCYTES [1/1]	0.005 ± 0.002	0.004 ± 0.001	0.006 ± 0.002	0.006 ± 0.002	0.004 ± 0.001	0.005 ± 0.002	0.005 ± 0.002	0.006 ± 0.002
OTHER CELLS [1/1]	0.015 ± 0.003	0.015 ± 0.003	0.014 ± 0.003	0.013 ± 0.004	0.015 ± 0.004	0.015 ± 0.003	0.015 ± 0.003	0.018 ± 0.002
TOTAL [1/1]	0.208 ± 0.014	0.215 ± 0.014	0.206 ± 0.008	0.207 ± 0.010	0.201 ± 0.010	0.202 ± 0.010	0.194 ± 0.011*	0.195 ± 0.013*

n - number of test animals

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

 

Table 12. Results of haematological studies – bone marrow examination. Different cells - satellite groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0SAT / 0		2SAT / 150
	Male n=5	Female n=5	Male n=4	Female n=4
LYMPHOCYTES [1/1]	0.177 ± 0.021	0.178 ± 0.017	0.165 ± 0.007	0.173 ± 0.008
MONOCYTES [1/1]	0.006 ± 0.002	0.007 ± 0.003	0.007 ± 0.002	0.006 ± 0.002
PLASMOCYTES [1/1]	0.006 ± 0.002	0.005 ± 0.002	0.005 ± 0.002	0.006 ± 0.002
MEGAKARYOCYTES [1/1]	0.006 ± 0.002	0.006 ± 0.002	0.005 ± 0.002	0.005 ± 0.002
OTHER CELLS [1/1]	0.014 ± 0.002	0.016 ± 0.004	0.015 ± 0.002	0.014 ± 0.007
TOTAL [1/1]	0.208 ± 0.020	0.212 ± 0.016	0.197 ± 0.007	0.204 ± 0.014

n - number of test animals

No statistical analyses were conducted due to insufficient data

 

Table 13. Results of blood serum biochemical examination – treatment groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0/0		4/37.5		1/75		2/150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=10	Male n=9	Female n=7
TOTAL PROTEIN [g/L]	62.60 ± 3.77	64.76 ± 2.10	60.72 ± 3.66	64.40 ± 2.59	62.37 ± 2.30	63.43 ± 2.49	59.79 ± 3.21	65.90 ± 3.24
ALBUMIN [g/L]	32.86 ± 1.82	35.72 ± 1.33	32.32 ± 1.29	35.51 ± 1.76	31.67 ± 2.52	34.88 ± 1.51	29.53 ± 1.28*	34.56 ± 1.66
GLOBULIN [g/L]	29.74 ± 2.15	29.04 ± 1.16	28.40 ± 2.49	28.89 ± 1.35	30.70 ± 1.55	28.55 ± 1.21	30.26 ± 2.94	31.34 ± 2.36*
A/G RATIO	1.11 ± 0.05	1.23 ± 0.05	1.14 ± 0.07	1.23 ± 0.07	1.04 ± 0.12	1.22 ± 0.04	0.98 ± 0.10*	1.11 ± 0.08*
GLUCOSE [mmol/L]	8.42 ± 1.86	6.63 ± 1.13	8.71 ± 1.60	6.26 ± 0.88	8.59 ± 1.48	7.31 ± 1.07	8.17 ± 1.04	6.19 ± 0.79
CHOLESTEROL [mmol/L]	1.40 ± 0.27	1.06 ± 0.21	1.40 ± 0.21	1.15 ± 0.22	1.56 ± 0.37	1.16 ± 0.18	1.26 ± 0.19	1.13 ± 0.14
UREA NITROGEN [mmol/L]	5.43 ± 0.91	6.04 ± 0.83	5.47 ± 0.88	5.47 ± 0.58	4.91 ± 0.74	6.12 ± 1.26	5.33 ± 1.08	7.14 ± 1.06
CREATININE [μmol/L]	26.80 ± 2.82	31.80 ± 4.10	29.90 ± 2.69	29.90 ± 2.51	26.10 ± 3.98	31.50 ± 2.92	24.22 ± 3.11	27.71 ± 4.35
BILE ACIDS [μmol/L]	14.05 ± 13.87	12.09 ± 4.99	13.29 ± 11.33	15.25 ± 10.32	10.50 ± 7.77	15.78 ± 7.17	8.80 ± 2.85	13.13 ± 3.58
BILIRUBIN [μmol/L]	4.29 ± 1.04	4.00 ± 0.88	4.35 ± 1.53	4.38 ± 0.70	6.27 ± 5.57	5.41 ± 2.96	4.08 ± 0.28	5.36 ± 2.33
SODIUM [mmol/L]	144.80 ± 1.99	143.30 ± 2.11	144.00 ± 1.76	144.80 ± 1.32	146.10 ± 1.91	145.40 ± 1.26*	146.33 ± 1.58	145.00 ± 0.82
POTASSIUM [mmol/L]	4.17 ± 0.23	3.61 ± 0.25	3.99 ± 0.25	3.62 ± 0.24	4.09 ± 0.21	3.94 ± 0.67	4.01 ± 0.20	3.86 ± 0.22
CHLORIDES [mmol/L]	105.10 ± 1.66	106.90 ± 1.73	106.10 ± 1.37	109.60 ± 1.35*	106.60 ± 1.17	109.20 ± 1.14*	106.89 ± 1.83	107.86 ± 1.46
CALCIUM [mmol/L]	2.41 ± 0.09	2.45 ± 0.07	2.36 ± 0.05	2.42 ± 0.09	2.45 ± 0.08	2.53 ± 0.09	2.46 ± 0.09	2.56 ± 0.08

n - number of test animals

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

Table 14. Results of blood serum biochemical examination – satellite groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0SAT / 0	0SAT / 0	2SAT / 150	2SAT / 150
	Male n=5	Female n=5	Male n=4	Female n=4
TOTAL PROTEIN [g/L]	63.68 ± 1.57	63.98 ± 1.70	63.35 ± 4.97	67.17 ± 1.38
ALBUMIN [g/L]	33.46 ± 1.18	34.38 ± 0.68	31.60 ± 2.77	34.75 ± 1.98
GLOBULIN [g/L]	30.22 ± 0.99	29.60 ± 1.29	31.75 ± 3.01	32.42 ± 0.82
A/G RATIO	1.11 ± 0.05	1.16 ± 0.04	1.00 ± 0.09	1.07 ± 0.08
GLUCOSE [mmol/L]	9.48 ± 1.63	5.74 ± 0.57	7.25 ± 1.18	5.45 ± 0.47
CHOLESTEROL [mmol/L]	1.72 ± 0.29	1.24 ± 0.27	1.25 ± 0.33	1.08 ± 0.34
UREA NITROGEN [mmol/L]	5.60 ± 0.76	6.72 ± 1.01	6.42 ± 1.50	7.20 ± 1.39
CREATININE [μmol/L]	25.80 ± 5.97	33.40 ± 2.61	31.50 ± 8.70	30.00 ± 3.83
BILE ACIDS [μmol/L]	9.18 ± 6.49	24.16 ± 9.27	7.55 ± 4.58	28.18 ± 17.37
BILIRUBIN [μmol/L]	3.56 ± 0.38	3.96 ± 0.71	4.55 ± 1.61	4.93 ± 1.72
SODIUM [mmol/L]	146.40 ± 0.89	146.60 ± 0.89	144.00 ± 0.82	147.00 ± 1.63
POTASSIUM [mmol/L]	4.06 ± 0.35	3.96 ± 0.51	4.50 ± 0.77	3.83 ± 0.13
CHLORIDES [mmol/L]	105.80 ± 1.92	108.80 ± 0.84	107.00 ± 0.82	107.50 ± 0.58
CALCIUM [mmol/L]	2.44 ± 0.05	2.46 ± 0.09	2.38 ± 0.13	2.50 ± 0.08

n - number of test animals

No statistical analyses were conducted due to insufficient data

 

Table 15. Results of blood serum enzymatic examination – treatment group (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0 / 0		4 / 37.5		1 / 75		2 / 150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=10	Male n=9	Female n=7
AST [IU]	158.30 ± 80.67	111.40 ± 13.25	150.70 ± 64.76	114.10 ± 17.74	134.00 ± 26.73	177.50 ± 143.95	112.56 ± 17.26	121.14 ± 30.52
ALT [IU]	57.10 ± 46.87	21.90 ± 4.65	44.80 ± 18.80	27.10 ± 7.98	39.80 ± 14.60	48.20 ± 48.00	47.89 ± 14.86	33.43 ± 9.69
AP [IU]	111.20 ± 18.72	66.30 ± 15.12	120.50 ± 24.83	68.00 ± 20.83	117.90 ± 34.22	66.60 ± 16.91	103.56 ± 19.05	60.00 ± 22.78

n - number of test animals

 

Table 16. Results of blood serum enzymatic examination - satellite groups (mean ± SD)

Parameter	GROUP / DOSE [mg/kg bw]
	0SAT / 0	0SAT / 0	2SAT / 150	2SAT / 150
	Male n=5	Female n=5	Male n=4	Female n=4
AST [IU]	131.20 ± 41.58	107.60 ± 7.83	198.75 ± 140.32	120.50 ± 9.61
ALT [IU]	58.40 ± 53.07	26.40 ± 8.85	52.00 ± 22.02	30.00 ± 9.97
AP [IU]	82.00 ± 13.10	47.80 ± 13.16	88.75 ± 14.20	50.75 ± 23.67

n - number of test animals

No statistical analyses were conducted due to insufficient data

 

Table 17. Absolute weight of internal organs [mg] – treatment groups (mean ± SD)

Examined organ	GROUP/ DOSE [mg/kg bw]/ SEX /number of tested animals
	0 /0		4 / 37.5		1/ 75		2 /150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=10	Male n=9	Female n=7
Brain	2025.000 ±  99.839	1903.000 ±  73.332	2007.200 ±  81.584	1920.000 ±   103.756	2020.000 ±  64.132	1896.400 ±  56.473	1957.000 ±  62.062	1859.429 ±  52.233
Pituitary gland	9.400 ± 1.350	13.600 ± 1.350	9.600 ± 1.506	12.900 ± 1.101	9.200 ± 1.033	13.400 ± 2.221	9.111 ± 1.269	13.714 ± 2.215
Thyroid	22.400 ± 2.875	18.900 ± 1.663	25.500 ± 4.223	20.300 ± 3.592	22.500 ± 2.635	19.500 ± 3.274	25.222 ± 1.787	22.143 ± 4.220
Thymus	270.900 ±  34.511	268.400 ±  53.550	238.800 ±  36.715	313.500 ±  77.979	238.300 ±  64.147	233.100 ±  49.629	207.889 ±  46.115	287.000 ±  32.578
Heart	1048.800 ±   110.728	682.500 ±  82.288	1027.600 ±   161.094	692.300 ±  55.662	1013.800 ±  72.879	672.200 ±  33.416	989.889 ±  91.069	712.571 ±  63.364
Liver	12524.700 ±    1631.383	6840.600 ±   713.833	11329.900 ±    1914.870	7207.000 ±   459.416	11510.600 ±    1529.824	7019.100 ±   624.310	10475.556 ±    1230.242	7764.429 ±    1514.421
Spleen	665.800 ±  66.471	518.600 ±  78.983	628.900 ±  72.931	541.400 ±  72.749	660.700 ±  77.589	537.500 ±  60.508	643.667 ±  93.711	601.571 ±  90.286
Kidneys	2687.500 ±   210.247	1671.700 ±   148.110	2502.900 ±   321.112	1662.600 ±  77.188	2533.800 ±   205.871	1645.000 ±   103.522	2484.444 ±   133.587	1716.857 ±   131.868
Adrenal glands	64.100 ± 7.724	83.900 ±  12.369	66.800 ± 9.271	80.800 ± 7.671	68.300 ±  10.914	89.900 ±  15.808	66.222 ±  10.895	95.714 ±  11.898
Testicles	3514.400 ±   245.596	-	3545.600 ±   411851	-	3527.300 ±   139.547	-	3590.889 ±   119.913	-
Epididymides	1289.900 ±  90.225	-	1387.900 ±  97.870	-	1301.400 ±  98.766	-	1334.222 ±   108.059	-
Prostate with seminal vesicles and coagulating glands	1958.000 ±   292.765	-	1957.800 ±   285.079	-	1971.300 ±   282.732	-	1768.889 ±   257.981	-
Ovaries	-	111.400 ±  15.608	-	107.400 ± 9.548	-	112.500 ±  19.580	-	120.429 ±  20.411

 

Table 18. Relative weight of internal organs [%] – treatment groups (mean ± SD)

Examined organ	GROUP/ DOSE [mg/kg bw]/ SEX /number of tested animals
	0 /0		4 / 37.5		1/ 75		2 /150
	Male n=10	Female n=10	Male n=10	Female n=10	Male n=10	Female n=9	Male n=9	Female n=7
Brain	0.476 ± 0.023	0.746 ± 0.044	0.498 ± 0.054	0.746 ± 0.063	0.503 ± 0.051	0.768 ± 0.036	0.527 ± 0.027	0.752 ± 0.054
Pituitary gland	0.0005 ±  0.0001	0.005 ± 0.001	0.0005 ±  0.0001	0.005 ± 0.001	0.0005 ±  0.0001	0.005 ± 0.001	0.0005 ±  0.0001	0.006 ± 0.001
Thyroid	0.005 ± 0.001	0.007 ± 0.001	0.006 ± 0.001	0.008 ± 0.002	0.006 ± 0.001	0.008 ± 0.001	0.007* ± 0.001	0.009 ± 0.002
Thymus	0.064 ± 0.011	0.105 ± 0.021	0.059 ± 0.010	0.121 ± 0.028	0.060 ± 0.018	0.094 ± 0.019	0.056 ± 0.012	0.116 ± 0.012
Heart	0.246 ± 0.014	0.267 ± 0.032	0.252 ± 0.016	0.268 ± 0.015	0.251 ± 0.015	0.272 ± 0.014	0.266 ± 0.018	0.288 ± 0.033
Liver	2.932 ± 0.253	2.674 ± 0.232	2.775 ± 0.296	2.799 ± 0.250	20847 ± 0.309	2.839 ± 0.213	2.813 ± 0.277	3.162 ± 0.788
Spleen	0.158 ± 0.027	0.203 ± 0.033	0.156 ± 0.022	0.210 ± 0.033	0.165 ± 0.031	0.218 ± 0.025	0.173 ± 0.023	0.243 ± 0.039
Kidneys	0.633 ± 0.062	0.653 ± 0.041	0.615 ± 0.043	0.646 ± 0.054	0.628 ± 0.051	0.666 ± 0.041	0.668 ± 0.034	0.695 ± 0.077
Adrenal glands	0.015 ± 0.001	0.033 ± 0.006	0.017 ± 0.002	0.031 ± 0.003	0.017 ± 0.003	0.036 ± 0.006	0.018 ± 0.003	0.039 ± 0.007
Testicles	0.827 ± 0.072	-	0.872 ± 0.063	-	0.878 ± 0.091	-	0.968* ± 0.071	-
Epididymides	0.303 ± 0.022	-	0.344 ± 0.045	-	0.324 ± 0.040	-	0.360* ± 0.041	-
Prostate with seminal vesicles and coagulating glands	0.463 ± 0.089	-	0.481 ± 0.049	-	0.491 ± 0.089	-	0.478 ± 0.081	-
Ovaries	-	0.044 ± 0.007	-	0.042 ± 0.004	-	0.045 ± 0.007	-	0.049 ± 0.011

* – statistically significant difference at p≤0.05, Dunnett's test

 

Table 19. Absolute weight of internal organs [mg] - satellite groups (mean ± SD)

Examined organ	GROUP/ DOSE [mg/kg bw]/ SEX /number of tested animals
	0SAT/ 0		2SAT / 150
	Male n=5	Female n=5	Male n=4	Female n=4
Brain	2009.400 ± 126.342	1904.600 ± 82.327	2043.250 ± 105.019	1858.750 ± 65.250
Pituitary gland	8.800 ± 1.304	12.800 ± 1.789	10.250 ± 0.500	14.500 ± 1.915
Thyroid	26.200 ± 5.070	18.600 ± 1.342	28.000 ± 2.828	18.500 ± 2.517
Thymus	179.400 ± 28.086	264.000 ± 27.111	231.750 ± 16.500	292.750 ± 33.100
Heart	1084.800 ± 47.146	681.400 ± 54.784	1076.750 ± 53.637	652.750 ± 32.408
Liver	13039.200 ± 1844.809	6499.200 ± 885.599	11457.250 ± 856.439	6848.750 ± 369.244
Spleen	743.600 ± 69.650	540.600 ± 96.389	834.250 ± 40.036	607.000 ± 109.554
Kidneys	2667.200 ± 374.006	1584.000 ± 178.674	2726.000 ± 159.673	1745.750 ± 45.828
Adrenal glands	56.600 ± 10.991	80.000 ± 8.888	67.250 ± 5.909	98.250 ± 8.421
Testicles	3343.600 ± 555.634	-	3376.250 ± 220.135	-
Epididymides	1383.400 ± 183.877	-	1432.250 ± 51.136	-
Prostate with seminal vesicles and coagulating glands	2095.000 ± 344.400	-	2238.750 ± 565.526	-
Ovaries	-	111.000 ± 13.820	-	136.750 ± 19.380

No statistical analyses were conducted due to insufficient data

 

Table 20. Relative weight of internal organs [mg] - satellite groups (mean ± SD)

Examined organ	GROUP/ DOSE [mg/kg bw]/ SEX /number of tested animals
	0SAT/ 0		2SAT / 150
	Male n=5	Female n=5	Male n=4	Female n=4
Brain	0.466 ± 0.026	0.783 ± 0.023	0.513 ± 0.024	0.770 ± 0.020
Pituitary gland	0.0004 ± 0.0001	0.005 ± 0.001	0.0005 ± 0.00004	0.006 ± 0.001
Thyroid	0.006 ± 0.001	0.008 ± 0.0004	0.007 ± 0.001	0.008 ± 0.001
Thymus	0.041 ± 0.005	0.109 ± 0.012	0.058 ± 0.005	0.121 ± 0.013
Heart	0.252 ± 0.019	0.280 ± 0.018	0.270 ± 0.014	0.271 ± 0.018
Liver	3.017 ± 0.356	2.667 ± 0.316	2.877 ± 0.238	2.840 ± 0.167
Spleen	0.172 ± 0.017	0.221 ± 0.033	0.209 ± 0.009	0.252 ± 0.050
Kidneys	0.615 ± 0.048	0.651 ± 0.067	0.684 ± 0.035	0.724 ± 0.026
Adrenal glands	0.013 ± 0.003	0.033 ± 0.003	0.017 ± 0.001	0.041 ± 0.003
Testicles	0.771 ± 0.093	-	0.847 ± 0.046	-
Epididymides	0.320 ± 0.035	-	0.359 ± 0.012	-
Prostate with seminal vesicles and coagulating glands	0.485 ± 0.077	-	0.561 ± 0.136	-
Ovaries	-	0.045 ± 0.004	-	0.057 ± 0.007

No statistical analyses were conducted due to insufficient data

 

 

Conclusions:

In this GLP compliant study, performed accoring to OECD 408, the potential toxicity of Rokopol RF-151 was evaluated in a 90 day gavage study in rats. On the basis of the results of the clinical, clinical-chemical, and post-mortem examinations, the No-Observed-Adverse-Effect-Level (NOAEL) of the test item was determined to be 37.5 mg/kg bw/day for females and males.

Executive summary:

In this GLP compliant OECD 408 study, the potential toxicity of Rokopol RF-151 following oral administration to rats for 90 consecutive days was evaluated and reversibility of any effect was assessed at the end of 21-day recovery period. In total, 55 males and 55 females of Wistar (Cmdb: WI; outbred) rats were used in the experiment. At first the test item was administered at three different doses: 75 mg/kg bw (group 1), 150 mg/kg bw (group 2) and 300 mg/kg bw (group 3). Due to severe toxicity in the high dose group, this group was terminated for humanitarian reasons and a dose group of 37.5 mg/kg bw (group 4) was introduced. Each dose group, including control group, consisted of 10 animals per sex. Furthermore, two satellite groups were used, 0 SAT, and 2 SAT, receiving 0 and 150 mg/kb bw, respectively. Each satellite group consisted of 5 animals/sex/group and were treated for 90-days and after that, were observed for 21 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects.

During the whole experiment, the animals were observed for mortality and symptoms of toxicity of the test item. Ophthalmological examinations were conducted. Moreover, body weights and food consumption were measured and behavioural studies (open field observations, the evaluation of responses to stimuli and locomotor activity, and fore- and hindlimb grip strength measurements) were performed. After the 90-day experiment in groups 0, 4, 1 and 2 and after the 21-day additional observation period in groups 0 SAT and 2 SAT, all survived animals were anaesthetised to collect blood needed for the purpose of clinical-chemical examinations. Then, they were euthanised and subjected to post-mortem examinations. At the end of the experiment, all survived males and females from each group were subjected to clinical chemical examinations that included haematological examinations of peripheral blood and bone marrow, coagulation examinations, biochemical and enzymatic examinations of serum, general urinalyses and examinations of urine sediment. Post-mortem examinations involved gross examinations, the determination of absolute and relative weights of organs and histopathological examinations of tissues and organs.

There was no mortality in group 0, 0 SAT, 4 and 1. At the highest dose of 150 mg/kg bw 6 animals died during the course of the experiment, these were: 4 females (3 from group 2 and 1 from group 2 SAT) and 2 males (1 from group 2 and 1 from group 2 SAT). There were no significant differences in appearance and behaviour between the animals of treated groups, i.e. 4, 1, 2 and 2 SAT and the control groups (0 and 0 SAT). During the whole experiment clinical signs were noticed in several animals. However, they should not be associated with the action of the test item. The ophthalmic examinations did not reveal any changes both in the eyes of the control and the treated animals. The administration of the test item did not influence food intake in the treated groups, but it probably influenced body weights at the dose of 150 mg/kg bw. The behavioural studies showed that the nervous system functioned properly. On the basis of the detailed clinical observations, made during and after the treatment, and the open field observations, no muscarinic, nicotinic, or central symptoms were noticed. Delayed neurotoxicity was not observed. The evaluation of responses to stimuli showed no adverse effects of the test item. On the basis of the fore- and hindlimb grip strength measurement, the detailed clinical observations, the open field observations, it can be concluded that the test item did not cause any miotoxic changes but it probably affected locomotor activity measurement. Statistical analyses of the clinical-chemical examinations results showed several statistically significant changes which were perceived as caused by the test item. These changes may indicate inflammation in treated animals. Other statistically significant changes were considered as not related with treatment. Changes were observed also in satellite groups, after additional 21-days of observation, in the results of haematological examinations (increase in the number of leukocytes) and changes in the percentage content of leukocytes (increase in the number of neutrocytes, decrease in the number of lymphocytes).

At the doses of 75 and 150 mg/kg bw, there were gross and histopathological lesions stated, which probably may state for the adverse effect of the test item on the treated animals, i.e: in mesenteric lymph nodes from group 1, 2 and 2 SAT atrophic lymphoid tissue, increased histiocytes, necrosis, enlargement, fibrosis with accompanying in some cases angiogenesis and/or shrinking and/or plasma cell hyperplasia and/or cell degeneration and vacuolation; in spleen in females of group 2, white pulp hyperplasia, due to increase in the number of animals as well as severity degree in comparison with control group; erosions in stomach, due to a slightly higher intensity of occurrence in connection with necrosis of mucosa (singular cases in groups with the highest dose), there is a possibility of influence of test item on stomach mucosa in the highest dose.

On the basis of the results of the clinical, clinical-chemical, and post-mortem examinations which were parts of this study, it may be concluded that the test item related changes at the mid, i.e. 75 mg/kg bw and the highest dose, i.e. 150 mg/kg bw/day were observed in males and females. The No-Observed-Adverse-Effect-Level (NOAEL) of the test item was determined to be 37.5 mg/kg bw/day for females and males.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

37.5 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The study available is a GLP-compliant guideline study, fully adequate for assessment.

System:

haematopoietic

Organ:

mesenteric lymph node

spleen

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

Additional information

OECD 408

In this GLP compliant OECD 408 study, the potential toxicity of Rokopol RF-151 following oral administration to rats for 90 consecutive days was evaluated and reversibility of any effect was assessed at the end of 21-day recovery period. In total, 55 males and 55 females of Wistar (Cmdb: WI; outbred) rats were used in the experiment (Institute of Industrial Organic Chemistry 2018). At first the test item was administered at three different doses: 75 mg/kg bw (group 1), 150 mg/kg bw (group 2) and 300 mg/kg bw (group 3). Due to severe toxicity in the high dose group, this group was terminated for humanitarian reasons and a dose group of 37.5 mg/kg bw (group 4) was introduced. Each dose group, including control group, consisted of 10 animals per sex. Furthermore, two satellite groups were used, 0 SAT, and 2 SAT, receiving 0 and 150 mg/kb bw, respectively. Each satellite group consisted of 5 animals/sex/group and were treated for 90-days and after that, were observed for 21 days to evaluate the reversibility, stability, or delay in the onset of possible harmful effects.

Clinical studies During the whole experiment, the animals were observed for mortality and symptoms of toxicity of the test item. Ophthalmological examinations were conducted. Moreover, body weights and food consumption were measured and behavioural studies (open field observations, the evaluation of responses to stimuli and locomotor activity, and fore- and hindlimb grip strength measurements) were performed. After the 90-day experiment in groups 0, 4, 1 and 2 and after the 21-day additional observation period in groups 0 SAT and 2 SAT, all survived animals were anesthetized to collect blood needed for the purpose of clinical-chemical examinations. Then, they were euthanized and subjected to post-mortem examinations. Clinical-chemical examinations At the end of the experiment, all survived males and females from each group were subjected to clinical chemical examinations that included haematological examinations of peripheral blood and bone marrow, coagulation examinations, biochemical and enzymatic examinations of serum, general urinalyses and examinations of urine sediment. Post-mortem examinations All animals after the collection of blood for the clinical-chemical examinations were euthanized and subjected to post-mortem examinations. Post-mortem examinations involved gross examinations, the determination of absolute and relative weights of organs and histopathological examinations of tissues and organs.

There was no mortality in group 0, 0 SAT, 4 and 1. At the highest dose of 150 mg/kg bw 6 animals died during the course of the experiment, these were: 4 females (3 from group 2 and 1 form group 2SAT) and 2 males (1 from group 2 and 1 from group 2SAT). There were no significant differences in appearance and behaviour between the animals of treated groups, i.e. 4, 1, 2 and 2 SAT and the control groups (0 and 0 SAT). During the whole experiment clinical signs were noticed in several animals. However, they should not be associated with the action of the test item. The ophthalmic examinations did not reveal any changes both in the eyes of the control and the treated animals. The administration of the test item did not influence food intake in the treated groups, but it probably influenced body weights at the dose of 150 mg/kg bw. The behavioural studies showed that the nervous system functioned properly. On the basis of the detailed clinical observations, made during and after the treatment, and the open field observations, no muscarinic, nicotinic, or central symptoms were noticed. Delayed neurotoxicity was not observed. The evaluation of responses to stimuli showed no adverse effects of the test item. On the basis of the fore- and hindlimb grip strength measurement, the detailed clinical observations, the open field observations, it can be concluded that the test item did not cause any miotoxic changes but it probably affected locomotor activity measurement. Statistical analyses of the clinical-chemical examinations results showed several statistically significant changes which were perceived as caused by the test item. These changes may indicate inflammation in treated animals. Other statistically significant changes were considered as not related with treatment. Changes were observed also in satellite groups, after additional 21-days of observation, in the results of haematological examinations (increase in the number of leukocytes) and changes in the percentage content of leukocytes (increase in the number of neutrocytes, decrease in the number of lymphocytes). Post-mortem examinations At the doses of 75 and 150 mg/kg bw, there were gross and histopathological lesions stated, which probably may state for the adverse effect of the test item on the treated animals, i.e: in mesenteric lymph nodes from group 1, 2 and 2 SAT atrophic lymphoid tissue, increased histiocytes, necrosis, enlargement, fibrosis with accompanying in some cases angiogenesis and/or shrinking and/or plasma cell hyperplasia and/or cell degeneration and vacuolation; in spleen in females of group 2, white pulp hyperplasia, due to increase in the number of animals as well as severity degree in comparison with control group; erosions in stomach, due to a slightly higher intensity of occurrence in connection with necrosis of mucosa (singular cases in groups with the highest dose), there is a possibility of influence of test item on stomach mucosa in the highest dose.

On the basis of the results of the clinical, clinical-chemical, and post-mortem examinations which were parts of this study, it may be concluded that the test item related changes at the mid, i.e. 75 mg/kg bw and the highest dose, i.e. 150 mg/kg bw/day were observed in males and females. The No-Observed-Adverse-Effect-Level (NOAEL) of the test item was determined to be 37.5 mg/kg bw/day for females and males.

OECD 422

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, four groups of 12 Wistar rats per sex were administered the test substance by gavage once a day at 0 (sunflower oil), 500, 250 and 100 mg/kg bw/day (TOXI-COOP Zrt ,2014). Stability, homogeneity and correct concentration of the substance in the vehicle were confirmed by analysis. All animals of the parent generation received test item or vehicle prior to mating (14 days) and throughout the mating period. Test item or vehicle was administered to male animals until day before the necropsy post mating (altogether for 44 days). For females, test item or vehicle was administered through the gestation period and up to lactation days 3 - 7, i.e. up to the day before the necropsy (altogether for 44 - 59 days). Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of pups. Five dams and males cohabited with were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, organ weighing and histopathology. The dams were allowed to litter, and rear their young up to termination on day 4 postpartum. Offspring were weighed and observed for possible abnormalities and were euthanized on postnatal day 4. Two male and two female animals were found dead during the study due to the toxic effect of 500 mg/kg bw/day dose of the test item. Clinical signs (decreased activity, diarrhea, dyspnea. hunched back, incoordination, narrow eye aperture, noisy breathing, piloerection, salivation, swollen abdomen), necropsy observations (atrophy of spleen and thymus, gas content in the intestines and stomach, dark red liver, reddish colored duodenum, undernourishment, yellowish gelatinous intestinal content) and histopathology findings (histiocytic infiltration in the mucous membrane of small intestines, bile duct hyperplasia in the liver, multifocal tubulonephrosis in the kidney, alveolar edema in the lungs, lymphoid atrophy in the spleen and thymus) referred to test item effect and explain the fatal death of these animals. In the surviving animals, test item related clinical signs (decreased body tone, diarrhea, salivation, noisy breathing, nuzzling up the bedding material, piloerection, prone position and sanguineous fur around nose) were observed in male or female animals of 500 mg/kg bw/day group. Salivation (male and female) and noisy breathing (male) were detected in 250 mg/kg bw/day group. At the detailed weekly observations, most of the above listed signs appeared in male (decreased body tone, diarrhea, noisy breathing, nuzzling up the bedding material, piloerection, prone position or sanguineous fur around nose) or female (diarrhea or piloerection) animals of 500 mg/kg bw/day group mostly during the first half of the treatment period. Noisy breathing was recorded also for two male rats of 250 mg/kg bw/day group at the weekly observations. Functional observations did not demonstrate any test item related changes in the behavior, physical condition and reactions to different type of stimuli of animals selected for examination (500, 250 or 100 mg/kg bw/day, control) at the end of the treatment period. The body weight gain was reduced in male animals of 500 and 250 mg/kg bw/day group during the entire treatment period and in some female animals of 500 mg/kg bw/day during the first week of the treatment. The mean daily food consumption was less in full correspondence with the body weight development of male animals in 500 and 250 mg/kg bw/day groups during the first two weeks of the treatment period and in female animals of 500 mg/kg bw/day group between Days 0 and 7. Test item related haematological changes were observed in a higher percentage of neutrophil granulocytes (NEU) and in a lower percentage of lymphocytes (LYM) at 500 mg/kg bw/day (male and female) and at 250 mg/kg bw/day (male). In female rats, a statistically significant increase of ~75 and ~240% in total number of white blood cells (WBC) were observed in the 250 and 500 mg/kg bw dose group, respectively. Specific pathologic alterations related to the test item were not detected at the evaluation of red blood cell parameters, in clinical chemistry or blood coagulation parameters. Gross pathology examination did not reveal test item related macroscopic changes in the organs or tissues of surviving animals subjected to necropsy at any dose level (500, 250 and 100 mg/kg bw/day). The mean spleen weights relative to body and brain weights of female animals dosed with 500 mg/kg bw/day slightly exceeded the control value and although no related histopathological effect was noted, it cannot be excluded that these findings are related to a test item influence on the splenic function. Test item related lesions were observed in the intestines (histiocytic infiltration in the mucous membrane of small intestines; male and female) and liver (minimal or mild bile duct hyperplasia; male) of surviving animals of 500 mg/kg bw/day group. There were no differences between the control and test item treated groups in the reproductive ability of male and female animals and in the delivery data of dams. A test item effect on the offspring development was observed in the lower mean litter weight gain between postnatal days 0 and 4 and also on the offspring’s mean weight at the birth, on postnatal day 4 and mean offspring’s weight gain in 500 mg/kg bw/day group. Under the conditions of the present study, the test substance caused death, clinical signs, changes in body weight and body weight gain, food consumption, hematological parameters (white blood cells (WBC, NEU, LYM), accompanied with histological alterations in the small intestines (histiocytic infiltration) and in the liver (bile duct hyperplasia), in dead animals renal tubulonephrosis, pulmonary alveolar edema and lymphoid atrophy in the spleen and thymus after repeated dose oral administration to male or female rats at 500 mg/kg bw/day dose. At 250 mg/kg bw/day, clinical signs (male and female), slight changes in the body weight gain, food consumption and hematological parameters (NEU, LYM in male animals, WBC in female animals) were detected. At 100 mg/kg bw/day, there were no test item related effects. The male and female reproductive performance (gonad function, mating behavior, conception) was normal in parental male and female animals. Dam’s delivery data was not affected by the test item at any dose level. The offspring’s body weight development was depressed at 500 mg/kg bw/day between postnatal days 0 and 4. Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined for general toxicity, fertility and developmental toxicity to be 100, 500, and 250 mg/kg bw, respectively.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study available is a GLP-compliant guideline study, fully adequate for assessment.

Justification for classification or non-classification

Based on the results of the combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening test in rats with the test substance, classification according to Directive 67/548/EEC (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 is not warranted.