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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020-12-02 to 2021-05-30
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
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)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-benzenesulfonamidophenyl)-3-phenylurea
EC Number:
806-543-7
Cas Number:
215917-77-4
Molecular formula:
C19H17N3O3S
IUPAC Name:
1-(2-benzenesulfonamidophenyl)-3-phenylurea

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Han:WIST
Details on species / strain selection:
The rat is regarded as suitable species for toxicity studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain of rat in toxicity studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 57-61 days; females: 57-61 days
- Weight at study initiation: males: 270-319 g; females: 175-217 g
- Fasting period before study: no
- Housing: 2 or 3 animals of the same gender per Type III polypropylene/polycarbonate cage
- Diet: ad libitum, ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water: ad libitum, tap water
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The drinking water was periodically analyzed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
1%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (Methylcellulose (1 %)) at concentrations of 20 mg/mL, 60 mg/mL and 200 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand for not longer than 2 days and stored in a refrigerator (5±3 °C) until use. The test item was administered in a single dose by oral gavage on a
7 days/week basis, every day at a similar time (+/- 2 hours). Concurrent control animals were handled in an identical manner to the test groups but received vehicle instead of test item (5 mL/kg bw).
VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water. Therefore, Methylcellulose (1 %) was used for preparing formulations appropriate for oral administration. Methylcellulose (1 %) was a suitable vehicle for the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical examinations of dosing solutions (control of concentration) revealed appropriate concentrations of the test item in the dosing formulations (102-109 %) in comparison to the nominal values at each analytical occasion.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 (+5 in control and highest dose group for recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose setting with 100, 300 and 1000 mg/kg bw/day is based on findings obtained in a previous repeated dose toxicity study according to OECD 422 with NKK-1304 in the rat.
- Fasting period before blood sampling for clinical biochemistry: yes
- Post-exposure recovery period: Animals assigned to the recovery groups (ctr and highest dose group) were treated identically up to and including Day 89 or 90, afterwards they were observed without administration for four weeks.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma (treatment and recovery periods).
Sensory reactivity to different types of stimuli (e.g. auditory, visual and proprioceptive), grip strength and motor activity were assessed in all animals (including recovery animals) in the control and high dose groups during the last exposure week. General physical condition and behavior of animals were tested. A modified Irwin test was performed (see References). There were no test item related changes in the examined parameters in high dose administered animals at the end of the treatment period, therefore this kind of examinations were not performed in the animals of the of the low and mid dose groups at termination of the treatment and in animals of the recovery group towards the end of the recovery period.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed in the treatment period with accuracy of 1 g on Day 0, then weekly during the course of the treatment period and once weekly during the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The food consumption was determined with an accuracy of 1 g by weighing of the given and remaining food by weekly interval during the treatment phase and recovery period.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- During the acclimation period, the eyes of all rats being considered for study were examined by focal illumination, indirect ophthalmoscopy.
Mydriatic eye drops were administered prior to ophthalmoscopy and the eyes were examined in subdued light. Subdued light was maintained in the animal’s room for the remainder of the day. These procedures were repeated on all control and high dose test animals prior to test termination.
Ophthalmological examinations were not needed in animals of the low and middle dose groups at the termination of the treatment or in animals assigned for post-treatment observation at the end of the recovery period because treatment related changes were not seen in animals of the high dose.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of the treatment (i.e. one day after the last treatment) on Days 90 and 91 (male and female, respectively) and on recovery animals at the end of recovery period
- Anaesthetic used for blood collection: Yes, Isofluran CP® anesthesia
- Animals fasted: Yes
- How many animals: all
- Parameters checked: White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin , Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Mean platelet volume, Reticulocytes, Differential white blood cell count, Activated partial Thromboplastin Time, Prothrombin Time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of the treatment (i.e. one day after the last treatment) on Days 90 and 91 (male and female, respectively) and on recovery animals at the end of recovery period
- Animals fasted: Yes
- How many animals: all
- Parameters checked: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Gamma Glutamyltransferase activity, Alkaline Phosphatase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Blood urea nitrogen, Glucose concentration, Cholesterol concentration, High-density lipoproteins, Low-density lipoproteins, Inorganic phosphate concentration, Calcium concentration, Sodium concentration, Potassium concentration, Chloride concentration, Albumin concentration, Total Protein concentration, Albumin/globulin ratio

SERUM HORMONES: Yes (TSH, T3, T4)
- Time of blood sample collection: on the day of necropsy
- Animals fasted: Yes
- How many animals: all

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
All animals were necropsied one day after the last treatment (main groups) or after the four weeks post treatment observation period (recovery groups). Animals were euthanized by exsanguination after verification of deep narcosis by Isoflurane CP®

GROSS PATHOLOGY: Yes
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of the location, color, shape and size.
After organ weight determination, the following organs/tissues were preserved in 4 % buffered formaldehyde solution except testes and epididymides, which were fixed in modified Davidson solution.
Adrenal glands, Aorta, Bone with marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Esophagus, Eyes (lachrymal gland with Harderian glands), Heart, Kidneys, Large intestines (caecum, colon, rectum), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes (submandibular, mesenteric), Mammary gland (male and female), Muscle (quadriceps), Pancreas, Pituitary, Salivary glands (submandibular), Sciatic nerve, testes, epididymides, prostate, seminal vesicle with coagulating gland, ovaries, uterus with cervix and oviduct, vagina, Skin, Small intestines (duodenum, ileum, jejunum including Peyer’s patches), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder

HISTOPATHOLOGY: Yes
Full histopathology was performed on the preserved organs or tissues of the animals of the control (Group 1) and high dose (Group 4) groups including recovery groups. The representative samples of fixed organs and tissues were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by a light microscope.
Optional endpoint(s):
Estrous cycle examination
Statistics:
The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences.
Where significant heterogeneity was found, we examined the normal distribution of data by Kolmogorov-Smirnov test. In case of not normal distribution, we applied the non-parametric method of Kruskal-Wallis One-Way analysis of variance. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.
For the evaluation of data in the recovery group, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No adverse signs of systemic toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily clinical observations.
Treatment period:
The behavior and physical condition of control male and female animals were considered to be normal at control, 100, 300 or 1000 mg/kg bw/day dose groups during the entire treatment period.
Recovery period:
Clinical signs were not detected in animals of the control or 1000 mg/kg bw/day (male or female) dose group during the recovery period. The behavior and physical condition of animals were considered to be normal
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the course of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period:
The mean body weight was similar to their control in male animals treated with 100, 300 or 1000 mg/kg bw/day and female animals treated with 100 and 300 mg/kg bw/day during the entire treatment period.
Some statistically significant difference with respect to the control was detected at the mean body weight gain in male animals at 100 mg/kg bw/day between Days 14 and 21 (lower), Days 84 and 89 (higher), at 1000 mg/kg bw/day between Days 14 and 21 (lower), Days 56-63 (higher), and between Days 63-70 (lower).

In the female animals of the high dose group the mean body weight was statistically significantly lower compared to the control on day 70 of treatment period.
Some statistically significant difference with respect to the control was detected at the mean body weight gain in female animals at 300 mg/kg bw/day between Days 63 and 70 (lower), at 1000 mg/kg bw/day between Days 63 and 70 (lower), Days 77-84 (lower), and at 100 mg/kg bw/day between Days 77-84 (lower). In 1000 mg/kg bw/day dose group the total body weight gain was statistically significantly lower compared to the control.
These variations were minor, often balanced by increased/decreased body weight gain the weeks beforehand or thereafter and, therefore, they were considered to be fortuitous and without relation to treatment.
Recovery period:
The mean body weight and body weight gain was similar to the control in female animals and the mean body weight in male animals at 1000 mg/kg bw/day during the recovery period.

In the male animals at 1000 mg/kg bw/day the mean body weight gain was statistically significantly lower between Days 112 and 118 compared to the control. However, the mean body weight of these male animals was nearly similar to the mean body weight of the control animals, therefore the slight changes in the mean body weight gain were toxicologically not relevant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period:
The mean daily food consumption was comparable with the control in male animals at 100, 300 and 1000 mg/kg bw/day during the entire treatment period.

Statistically significant differences with respect to the control were observed for the slightly lower (-11 and -13 %) mean daily food consumption in female animals at 300 and 1000 mg/kg bw/day on week 10.
The minor differences were considered to be indicative of the biological variation and not related to the test item in the mean food consumption of female animals.
Recovery period:
During the course of the recovery period, the mean daily food consumption of male and female animals was nearly similar in the control and high dose group
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 eyes were without any abnormalities in all animals before the treatment and in animals in the control and high dose groups at termination of the treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period:
The examined hematological and blood coagulation parameters were similar to the control in female animals at 100 mg/kg bw/day.
In male animals at 300 and 1000 mg/kg bw/day statistically significant increase in the mean percentage of neutrophil (NEU), mean platelet volume (MPV) and statistically significant decrease in the mean percentage of lymphocytes (LYM) was detected compared to the results of the control.
In male animals at 300 mg/kg bw/day statistically significant decrease in the mean white blood cell (leukocyte) count (WBC) and in animals at 100 mg/kg bw/day statistically significant increase in the mean platelet volume (MPV) was detected compared to the results of the control.
In female animals at 300 and 1000 mg/kg bw/day statistically significant increase in the mean percentage of neutrophil (NEU) and statistically significant decrease in the mean percentage of lymphocytes (LYM) was detected compared to the results of the control.
In female animals at 1000 mg/kg bw/day statistically significant decrease in the mean white blood cell (leukocyte) count (WBC) and statistically significant increase in the mean platelet volume (MPV) was detected compared to the results of the control.
All other examined hematological parameters were comparable with their control in male and female animals at 100, 300 and 1000 mg/kg bw/day.
Recovery period:
The mean percentage of neutrophil (NEU) was statistically significantly higher and the mean percentage of lymphocytes (LYM) was statistically significantly lower than in the control group in male animals in 1000 mg/kg bw/day group at the end of the post-treatment period.
No statistically significant alteration was found in the female animals at 1000 mg/kg bw/day compared to the control group.
The changes in hematology parameters were judged to be toxicologically not significant as there were no supporting findings (histopathology, related biochemical parameters).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period:
In male animals at 100 mg/kg bw/day statistically significant decrease of inorganic phosphorous concentration (Pi) and statistically significant increase of mean concentration of chloride (Cl-) was detected with respect to the control.
In the male animals of 300 mg/kg bw/day, statistical differences with respect to the control were detected at the lower mean aspartate aminotransferase activity (AST), lower mean concentrations of urea, blood urea nitrogen (BUN), inorganic phosphorous (Pi) and potassium (K+). In this dose group statistically higher mean chloride (Cl-) concentration appeared compared to the control. In the male animals treated with 1000 mg/kg bw/day, elevated mean concentration of total protein (TPROT) was revealed in comparison with the control group.
The examined clinical chemistry parameters were comparable in the female animals in control and 100 mg/kg bw/day groups. In the female animals of 300 mg/kg bw/day, statistical differences with respect to the control were detected at the lower mean alkaline phosphatase activity (ALP), lower mean concentration of glucose (GLUC) and higher concentration of mean albumin (ALB). The mean concentration of glucose (GLUC) was lower with respect to the control in the female animals at 1000 mg/kg bw/day groups.

Recovery period:
Statistical significance with respect to the control was observed at the slightly lower mean alanine aminotransferase activity (ALT), mean aspartate aminotransferase activity (AST), lower mean concentrations of total bilirubin (TBIL), low-density lipoprotein (LDL), and higher mean chloride (Cl-) concentration in male animals in 1000 mg/kg bw/day group at the end of the recovery period.
In the female animals of 1000 mg/kg bw/day, the mean concentrations of high-density lipoprotein (HDL) was statistically significantly higher compared to the control. All other examined parameters were comparable with the relevant control at the end of recovery period.
All these changes were with minor degree and not related to doses (ALP, AST, UREA, BUN, GLUC, Pi, K+, Cl-, TPROT, GLUC, ALB) were judged to be toxicologically not significant as there were no supporting findings (hematology, histopathology, related biochemical parameters).
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period:
In all male dose groups the mean concentrations of FT3 and in male animals at 300 and 1000 mg/kg bw/day the concentrations of FT4 was statistically significantly higher compared to the control. However, the mean concentration of TSH was not decreased in these groups. These differences with respect to the control were considered to be indicative of the biological variation and not related to the test item effect. There were no significant differences with respect to the control in FT3, FT4 or TSH levels at 100 and 300 mg/kg bw/day in female animals. The FT3 concentration exceeded the control value in female animals at 1000 mg/kg bw/day.
Recovery period:
In male and female animals at 1000 mg/kg bw/day the level of FT3 (free T3) and FT4 (free T4) thyroid and TSH hormones were normal.

The structure and the cell morphology of the endocrine glands was the same in histopathological examination in the control and treated animals.
A test item effect on the estrous cycle was not detected at any dose level.
No morphological evidence of acute or subacute test item related injury (degeneration, inflammation, necrosis etc.) of the male and female reproductive system was observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The functional observation battery did not demonstrate any test item related differences with respect to the controls in the behavior or in reactions to different type of stimuli at 1000 mg/kg bw/day (male and female).
The behavior and reactions to different type of stimuli or manipulations of animals were judged to be normal in the control and high dose group at the end of the treatment period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day.
Treatment period:
Statistically significant differences with respect to the control were noted for the slightly lower mean adrenal weights (absolute) in male animals in 1000 mg/kg bw/day dose groups at termination of the treatment period. In the female animals, the mean weights of the thymus (absolute and relative to body and brain weights) were slightly higher than in the control group at 100 mg/kg bw/day. In female animals in 300 and 1000 mg/kg bw/day, the mean weight of uterus (relative to body weights) was higher compared to the control.
Recovery period:
The weights of the examined organs were similar in the female animals in control and 1000 mg/kg bw/day groups at the end of the recovery period. In the male animals at 1000 mg/kg bw/day, statistical significances were revealed for the lower mean thymus and adrenal weights (absolute and relative to body weights).
All these statistically significant differences during treatment and recovery period, with respect to the appropriate control, were of small degree and no related findings were detected at the histopathological examinations.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period.
Treatment period:
In the male animals, one or both sided pyelectasia was observed in the control and high dose groups at the terminal necropsy as follows: 1/10 control; 1/10 at 1000 mg/kg bw/day. In the female animals, both sided pyelectasia was found in one animal at 100 and 300 mg/kg bw/day.
Slight to marked hydrometra appeared in all groups at the terminal necropsy: control; 2/10, 100 mg/kg bw/day; 3/10, 300 mg/kg bw/day; 5/10 and 1000 mg/kg bw/day; 3/10.
Recovery period:
In the male animals, one or both sided pyelectasia was observed in control (1/5) and in high dose (3/5) group at the end of the recovery period. In the female control animals, one or both sided pyelectasia was observed in two cases and in other two animals appeared slight to moderate hydrometra at the end of the recovery period. In one animal of high dose was found moderate hydrometra.
The organs and tissues were judged to be normal in the male and female animals in 1000 mg/kg bw/day recovery group. These findings are common in untreated experimental rats and were not related to the test item. Hydrometra is indicative of female sexual cycle. Histological findings were in accordance with macroscopic observation in the uterus. Pyelectasia is a common finding in experimental rats of this strain with similar age. Pyelectasia occurred in each group of male animals independently from doses.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The pyelectasia in the kidney (one or both side) without other histopathological lesions (degeneration, inflammation, fibrosis etc.) is a common slight individual disorder in laboratory rats, without toxicological significance.
The CPN (chronic progressive nephropathy) is an important spontaneous renal disease of the used strain of laboratory rat and it is a serious confounding factor in experimental pathology and toxicology studies. Compared to female rats, there is a distinct male predisposition to CPN with respect to onset, incidence and severity progression. CPN is not grossly apparent until the advance stages of the disease. The earliest stage of CPN in H-E-stained reactions, identifiable as early as 2-3 month of age in male rats, is a focal tubule change in the cortex characterized by cytoplasm basophilia with crowded nuclei indicative of cellular regeneration. The early cortical focus in shortly followed by development of an eosinophilic hyaline cast in the descending limb of Henle of the same nephron. The lesions progress in the cortical and medullary regions. The primary factors modifying CPN severity are physiological, being diet related and hormonal. Restriction of caloric intake is the greatest influence for minimizing the disease, and increasing dietary protein is a primary factor for enhancing CPN. CPN severity can be enhanced by administration of testosterone, thyroxin and adrenal hormones but decreased by thymectomy.In this study the initial phase of disease was observed in two control male animals, without toxicological significance. No lesions, typical on CPN were detectable in the treated (male and female 1000 mg/kg bw/day) groups.
The dilatation of the uterine horns in some female animals is a slight neuro-hormonal phenomenon in connection with the sexual function -prooestrus phase - of the inner genital organs.
No morphological evidence of acute or subacute test item related injury (degeneration, inflammation, necrosis etc.) of the gastrointestinal tract, the liver, the pancreas, the respiratory system, the cardiovascular system, the urinary system, the lymphoid system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system, the eyes, the lachrymal glands, and the integumentary system, was observed.
In conclusion it can be stated that the 100, 300 and 1000 mg /kg/bw/day doses of test item did not cause any toxic or other test item related lesions detectable by histological examination of investigated organs of the experimental animals.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present study, the substance did not cause adverse effects in male or female Han: WIST rats after consecutive 90/91-day oral (by gavage) administration at 100, 300 or 1000 mg/kg bw/day. Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as 1000 mg/kg bw/day for male and female Han: WIST rats.
Executive summary:

The purpose of this study was to obtain information on the possible health hazards likely to arise from repeated exposure with the test item at three dose levels over a prolonged period of time (90 days) followed by four weeks recovery period in order to assess persistence or delayed occurrence of potential toxicological effects. The test item was administered orally (by gavage) to male and female Han: WIST rats (n=15 animals/sex in the control and high dose groups, n= 10 animals/sex in the low and middle dose groups) once a day at 0 (vehicle control), 100, 300 and 1000 mg/kg bw/day doses corresponding to concentrations of 0, 20, 60 and 200 mg/mL, applied in a dose volume of 5 mL/kg bw for 90 or 91 days. Five animals/ sex in the control and high dose groups assigned to the recovery groups were treated identically up to Day 89 or Day 90 then they were observed without administration for four weeks (recovery observations). The suitability of the chosen vehicle Methylcellulose (1 %) for the test item was analytically proven up front. Concentrations of the test item in the dosing formulations varied between ranges of 102 % and 109 % of nominal concentrations at each analytical occasion confirming proper dosing.


Animals were observed for mortality twice a day during the course of the study. Daily general clinical observations and weekly detailed clinical observations were performed. A functional observation battery was conducted at the end of the treatment. The body weight was determined weekly during the course of the treatment and recovery periods. Food consumption was measured and evaluated weekly. The estrous cycle of each female animal was examined at the end of treatment and recovery periods, before necropsy. Clinical pathology examinations (including hematology, blood coagulation and clinical chemistry) and gross pathology were conducted one day after the last treatment and at the end of the recovery period. The absolute and relative weights of selected organs were measured. Full histopathology was performed on the preserved organs or tissues of the animals of the control and high dose groups including recovery groups. Additionally, organs showing macroscopic changes were processed and evaluated histologically in the low and mid dose groups.


The results of the study were interpreted comparing test item treated groups with controls, which were administered concurrently with vehicle only.


There was no test item related mortality at any dose level (100, 300 or 1000 mg/kg bw/day).


No signs of toxicity related to the test item were detected at any dose level (100, 300 or 1000 mg/kg bw/day) at the daily or detailed weekly clinical observations or during the course of the functional observation battery. The behavior and physical condition of animals were normal during the entire observation period.


There were no clinical signs during the recovery period.


The body weight development was unaffected by the test item at 100, 300 and 1000 mg/kg bw/day (male and female animals) during the 3-month treatment period. The mean body weight was similar to the control in male and female animals at 1000 mg/kg bw/day during the recovery period.


The mean daily food consumption was comparable in animals of the control and test item treated groups (100, 300 and 1000 mg/kg bw/day).


There were no abnormalities in the eyes of animals in the high dose group at termination of the treatment (male and female at 1000 mg/kg bw/day).


A test item related influence on the estrous cycle was not detected (100, 300 and 1000 mg/kg bw/day).


Hematological investigations did not reveal test item or treatment related changes in the examined parameters in male or female animals at any dose level (100, 300 and 1000 mg/kg bw/day).


No pathologic test item effect was detected at the evaluation of clinical chemistry parameters (100, 300 and 1000 mg/kg bw/day).


FT3, FT4 and TSH levels were not affected by the test item at 100, 300 or 1000 mg/kg bw/day (male and female) at the end of the treatment and recovery periods.


Test item induced macroscopic changes were not detected during the necropsy at termination of the treatment period or at the end of the recovery period.


Test item related changes were not detected in the examined organ weights in male or female animals at 100, 300 or 1000 mg/kg bw/day.


Histological examination did not reveal severe test item related lesions in the organs or tissues of animals administered with 1000 mg/kg bw/day dose at termination of the treatment or at the end of the recovery period.


Under the conditions of the present study, the substance did not cause adverse effects in male or female Han:WIST rats after 90/91-day consecutive oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses.


There were no toxic changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry, serum levels of thyroid hormones, estrous cycle, necropsy findings, organ weights or histopathological findings).


Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as 1000 mg/kg bw/day for male and female Han: WIST rats.