Registration Dossier

Administrative data

Description of key information

NOAEL (subacute) (rat, male and female): 350 mg/kg bw/day, systemic toxicity

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 25 to July 14, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Reason / purpose:
reference to other study
Remarks:
validated analytical method
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females: nulliparous, non-pregnant females.
- Age at study initiation: male animals 90 – 97 days; female animals 90 – 97 days.
- Weight at study initiation: male animals 359 – 438 g; female animals 200 – 248 g. The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing:
before mating 2 animals of the same sex/cage; during mating 1 male and 1 female per cage; pregnant females individually housed; males after mating 2 animals per cage. Cage type was type III polypropylene/polycarbonate (22 x 32 x 19 cm).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. Food was changed at weekly intervals. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
- Water: animals received tap water from watering bottles, as for human consumption, ad libitum. Fresh drinking water was given daily. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).
- Acclimation period:
27 days.
- Animal health: only healthy animals were used for the study. Healthy status was certified by the breeder.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
TREATMENT VOLUME
A constant treatment volume of 5 ml/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.

FORMULATION
Test item was formulated in the vehicle (distilled water) in concentrations of 8, 24 and 70 mg/ml (corresponding to an active substance content of ca. 7.4, 22 and 64 mg/ml). Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days and stored at 5 ± 3 °C before the administration.

VEHICLE
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Recovery of test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/ml and 90 % at ca. 200 mg/ml).
A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical study.Test item proved to be stable in the vehicle in a refrigerator (at 5 ± 3 °C) for three days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical control of dosing formulations (control of concentration) was performed in the analytical laboratory of test facility twice during the study. Five aliquots of 5 ml of each formulation (8, 24 and 70 mg/ml) and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis.
Concentration of the test item in the dosing formulations varied between the range of 97 % and 103 % in comparison to the nominal values.
Duration of treatment / exposure:
Males: 42 days
Females: 51 – 65 days
Frequency of treatment:
Daily, 7 days/week
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Remarks:
Refer to formulation
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Remarks:
Refer to formulation
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
Refer to formulation
No. of animals per sex per dose:
12 animals/sex in the control and dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
Dosing of both sexes begun after 27 days acclimatization – including 14 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy.
The mating phase started after 14-days treatment (pre-mating) period.
Male animals were dosed for 42 days (14 days pre-mating and 1-16 days mating in animals plus 11-27 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-16 days mating period and throughout pregnancy and at least up to and including day 13 post-partum or the day before sacrifice.
Observations and examinations performed and frequency:
MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time. Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. 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.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

BODY WEIGHT
All parental animals were weighed with an accuracy of 1 g. Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not valuated statistically. Body weight was measured on day of necropsy for female animals subjected to organ weighing (selected for further examinations).
Body weight data were reported individually for adult animals. Individual body weight change was calculated.

FOOD CONSUMPTION
The food consumption was determined weekly by reweighing the non-consumed diet with a precision of 1 g during the treatment period except mating phase (pre-mating days 7, 13, and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

WATER CONSUMPTION

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY
Hematology examination was conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia.
Blood samples for hematology measurements were collected in tubes containing K3EDTA and tubes were filled up to the final volume (marked on the tubes). Blood were stored at 2-8 °C until analysis.
Parameters: 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, Reticulocytes, Differential white blood cell count.

Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.8 %. Tubes were filled up to the final volume (marked on the tubes). Blood were centrifuged at 2500 rpm for 15 minutes within 20-30 minutes after the sampling. Supernatant plasma samples were stored at 2-8 °C and measured.
Parameters: Activated partial Thromboplastin Time and Prothrombin Time.

CLINICAL CHEMISTRY
clinical chemistry examination was conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia.
Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A (no anticoagulant). At least 1.0 ml blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. Serum samples were stored at 2-8 °C and measured.
Parameters: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Sodium concentration, Potassium concentration, Albumin concentration, Total Protein concentration.

THYROID HORMONE
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH).
Blood samples were collected from animals as follows: from 2-7 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter); from all dams and 5-7 pups per litter on post-partal/post-natal day 13; from all parent male animals at termination on Day 42.
Parameters: Thyroxine – free Tetra-iodothyronine and Thyroid-stimulating hormone.
Blood samples from the day 13adult males were assessed for serum levels of thyroid hormones (T4 and TSH). Further assessment of T4 and TSH in blood samples from the dams were not performed because there were no relevant changes in the examined samples (based on observations in postnatal day 13 offspring and parental male animals).
The quantitative determination of thyroid hormones (T4 and TSH) in serum samples were performed by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.
Sacrifice and pathology:
GROSS NECROPSY
Gross necropsy was performed on each animal.
One male animal at 350 mg/kg bw/day died and was subjected to necropsy immediately after founding dead on Day 22 and one female animal at 120 mg/kg bw/day died and was subjected to necropsy immediately after founding on Day 49.
All other animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® and were subjected to gross necropsy as follows: parental male animals after the optionally extended post-mating period on Day 42; dams on post-partum day 13 or shortly thereafter (Days 51, 54, 55, 57 or 65).
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 including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system; the number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate and seminal vesicles with coagulating glands, adrenal glands and pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides) for five male and five female animals randomly selected from each group.

ORGAN WEIGHT
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. Absolute organ weight was recorded. Relative organ weight (to body and brain weight) were calculated and reported.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination was also performed on the testes, epididymides, prostate and seminal vesicles with coagulating gland in one not mated male in the mid dose group.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (350 mg/kg bw/day) groups and in dead animals – male no. 403 at 350 mg/kg bw/day and female no. 328 at 120 mg/kg bw/day.
In addition, thymus of one male animal (no. 304 in 120 mg/kg bw/day group), kidneys of one male animal (no. 209 in 40 mg/kg bw/day group) and uterus of three female animals (no. 223 in 40 mg/kg bw/day group, no. 322 and 323 in 120 mg/kg bw/day group) were processed and examined due to macroscopic findings (hemorrhage in the thymus, pyelectasia in the kidney, hydrometra in the uterus).
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs in any group, i.e. the surviving parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 40, 120 or 350 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
Piloerection and decreased activity were observed in single male animal (1/12) at 120 mg/kg bw/day group between Days 24 and 30. Alopecia on the abdomen (1 cm in diameter) was detected in another male animal (1/12) of this group.
These clinical signs - piloerection and decreased activity on Day 27 (1/12) and alopecia on the abdomen on Days 20, 27, 34 and 41 (1/12) – were also detected at the weekly detailed clinical observations.
These observations were considered to be individual signs, only detected in single animals of the mid dose group bot not in animals at 350 mg/kg bw/day.
Dark stool was detected in the bedding material in each cage of the male and female animals at 120 and 350 mg/kg bw/day groups from Day 8 until termination. The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was no test item related mortality at 40, 120 or 350 mg/kg bw/day groups during the course of study (male and female).
One male animal (no. 403) at 350 mg/kg bw/day was found dead on Day 22. There were no preceding clinical signs or body weigh change in this animal. Therefore, the cause of death was judged to be unknown, probably an individual lesion.
One female animal (no. 328) at 120 mg/kg bw/day was also found dead on lactation day 10 (Day 49). Individual clinical signs – abnormal head position on the right side, lateral body position on the right side and catalepsy – was noted for this dam on lactation days 8 or 9.
Dark stool was detected in the bedding material in the cages from Day 8 until the death in both cases.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight development was undisturbed in male and female animals at 40, 120 or 350 mg/kg bw/day during the entire treatment period.
The mean body weight was comparable in the control and at 40, 120 and 350 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating periods and in female animals during the pre-mating, gestation and lactation periods.
Statistical significance with respect to the control was only detected in the female animals at 350 mg/kg bw/day at the slightly higher mean body weight gain on the first week of treatment. This minor change was considered to be toxicologically not relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related changes in the mean daily food consumption of male or female animals at 40, 120 or 350 mg/kg bw/day.
The mean daily food consumption was comparable in the control and test item treated animals at 40, 120 or 350 mg/kg bw/day during pre-mating and post mating periods in male animals and during the course of the pre-mating, gestation and lactation periods in female animals.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 40, 120 or 350 mg/kg bw/day.
All examined hematological and blood coagulation parameters were comparable with the control in male animals at 40 mg/kg bw/day.
At 120 mg/kg bw/day, the mean percentage of reticulocytes (RET) was higher than in the control group in male animals.
In the male animals at 350 mg/kg bw/day, the mean red blood cell (erythrocyte) count (RBC), the mean hemoglobin concentration (HGB), the mean hematocrit value (HCT) and the mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) were statistically significantly lower while the mean percentage of reticulocytes (RET) was higher compared to their control.
The examined hematological and blood coagulation parameters were comparable in female animals in the control and 40, 120 and 350 mg/kg bw/day groups.
The elevated mean percentage of reticulocytes in male animals at 120 and 350 mg/kg bw/day (dose related) and in female animals – non-statistically significant – at 350 mg/kg bw/day was not accompanied by histopathological changes in the high dose treated animals. Slightly lower mean of red blood cell parameters (HGB, HCT) in male animals at 350 mg/kg bw/day corresponded well to the historical control value; RBC values in male control animals were slightly lower than the historical control.
In two female animals at 350 mg/kg bw/day, the individual RBC values were slightly above the historical control, although the mean at 350 mg/kg bw/day resulted to be non-statistically significantly higher than in the control group; no changes were observed in the red blood cell parameters.
The elevated mean percentage of reticulocytes in male animals at 120 and 350 mg/kg bw/day and in female animals at 350 mg/kg bw/day might be in connection with a slight stimulator effect on the release of erythroid cells from the cell -store tissues but it was not accompanied by histological changes (typical on hematopoietic cell hyperplasia) in the high dose treated animals.
Therefore, these findings were considered to have little or no toxicological relevance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item related adverse effects on the examined clinical chemistry parameters at 40, 120 or 350 mg/kg bw/day (male or female).
In male animals at 40 mg/kg bw/day, statistical significance with respect to the control was observed at the lower mean activity of alanine aminotransferase (ALT) and at the higher mean creatinine concentration (CREA).
The mean concentration of potassium (K+) was slightly below the control value in male animals at 120 mg/kg bw/day.
Compared to their control, the mean activity of alanine aminotransferase was lower in male animals at 350 mg/kg bw/day.
All examined clinical chemistry parameters were comparable in female animals in the control and in all the test item treated groups.
The statistically significant differences in ALT, K+ and CREA with respect to their controls in male animals were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 40, 120 or 350 mg/kg bw/day groups at the end of the treatment period (on Day 41).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Alopecia on the abdomen was noted for one male animal (1/5 at 120 mg/kg bw/day) at the functional observations.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no statistically significant differences between the control and test item treated male or female animals at any dose level (40, 120 and 350 mg/kg bw/day) in the examined organ weights.
Minor changes were observed in the weights of the thymus and spleen in male animals at 40, 120 and 350 mg/kg bw/day and in the thymus weights in female animals at 350 mg/kg bw/day when compared to the control. However, the differences were not related to doses in male animals and were not statistically significant either in male or female animals. There were no supporting histopathological lesions therefore changes in the spleen weight were considered to be of little or no biological relevance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Specific macroscopic alterations indicative of test item effect were not detected in the organs or tissues at any dose levels 40, 120 or 350 mg/kg bw/day at the necropsy.
In dead male animal (1/12 at 350 mg/kg bw/day) necropsy observation revealed black colored hair around the nose and mouth, black colored esophagus and lungs on Day 22. The visceral organs were slightly autolyzed.
In dead female animal (1/12 at 120 mg/kg bw/day), necropsy observation revealed thymic hemorrhages, rounded edges of the liver, black content in the stomach and intestines, black colored uterus, ovaries, submandibular lymph nodes and mesenteric lymph nodes on lactation day 10 (Day 49). The visceral organs were autolyzed.
Black or grey colored organs (spleen, submandibular or mesenteric lymph nodes) and presence of the test item or its metabolite(s) in the stomach and/or intestines were detected in male animals belonging to the groups dosed at 120 and 350 mg/kg bw/day at the terminal necropsy. In females, black or grey staining was observed mainly in mesenteric lymph nodes in several animals dosed at 120 mg/kg bw/day and in all animals dosed at 350 mg/kg bw/day; the kidneys staining was observed in 2/12 animals dosed at 350 mg/kg bw/day.
In detail, there were no macroscopic findings in the organs or tissues in male animal/s in the control (12/12).
At 40 mg/kg bw/day, pyelectasia was observed in one animal (1/12).
At 120 mg/kg bw/day, thymic hemorrhage was noted for one male animal (1/12).
In surviving male animals at 350 mg/kg bw/day, dark colored spleen (3/11) and black colored mesenteric lymph nodes (4/11) as well as enlarged and black colored submandibular lymph nodes (1/11) were observed.
There were no macroscopic findings in the organs or tissues in female animals in the control (12/12).
Marked hydrometra was observed in one dam at 40 mg/kg bw/day (1/12).
In female animals at 120 mg/kg bw/day grey or black colored mesenteric lymph nodes (6/11) and slight or moderate hydrometra was observed (2/11).
Grey or black colored mesenteric lymph nodes (12/12), marked hydrometra (1/12), enlarged spleen (1/12), grey colored (2/12) or pale (1/12) kidneys and pyelectasia (2/12) were observed in female animals treated with 350 mg/kg bw/day.
Hemorrhage in the thymus was due to circulatory disturbance developed during exsanguination procedure.
Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats.
In the lack of related histopathological alterations (inflammatory or other pathological lesion) the above-mentioned findings were judged to be toxicologically not relevant.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological examinations revealed test item related centrilobular vacuolation in the hepatocytes in the liver at 350 mg/kg bw/day in male animals: mild centrilobular vacuolation was observed in the hepatocytes in three male animals at 350 mg/kg bw/day (3/5).
Centrilobular vacuolation is indicative of hepatic lipidosis and was probably due to the test item in this study. Hepatic lipidosis is considered as a slight reversible liver injury in connection with a disturbance of energy metabolism of affected hepatocytes.
In females, the histopathological examinations did not reveal any adverse test item related finding.
There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 350 mg/kg bw/day).

In dead male animal (1/12) at 350 mg/kg bw/day, histological investigations revealed marked purulent inflammation in the lungs and mild centrilobular vacuolation in the hepatocytes in the liver.
In dead female animal (1/12) at 120 mg/kg bw/day, severe congestion and moderate alveolar emphysema were observed in the lungs.
In details, in animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in the control, 1/5 male and 1/5 female at 350 mg/kg bw/day), acute hemorrhage in the thymus (1/1 male at 120 mg/kg bw/day) were detected sporadically. The pulmonary emphysema and hemorrhage in the thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 control male) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Pyelectasia (one side) was observed in some animal (1/1 male at 40 mg/kg bw/day and 2/5 female at 350 mg/kg bw/day). This finding without other pathological lesion (degeneration, inflammation, fibrosis) is a common finding in Wistar rats without toxicological significance.
Subacute lymphocytic pyelitis accompanied with pyelectasia - as a sporadic individual lesion – was noted for one male animal at 40 mg/kg bw/day (1/1) and for one female at 350 mg/kg bw/day (1/5).
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the pancreas, the cardiovascular system, the immune system, the hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system in the animals.
The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.
The macroscopically visible grey-black discoloration of some organs (kidneys, spleen, mesenteric and submandibular lymph nodes) and the intestinal content could not be associated with histologically detectable pathological findings in the affected organs.
In the male animals belonging to the treated and control groups (12/12 control; 11/11 at 350 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 12/12 at 350 mg/kg bw/day), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was observed in four dams: 1/1 at 40 mg/kg bw/day, 2/2 at 120 mg/kg bw/day and 1/12 at 350 mg/kg bw/day. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONE
The thyroid hormone (free T4 and TSH) levels were similar in the control and test item treated parental male animals (40, 120 and 350 mg/kg bw/day).
Dose descriptor:
NOAEL
Effect level:
>= 350 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
NOAEL (rat, male and female): 350 mg/kg bw/day, systemic toxicity
Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted to obtain information on the toxic potential of test item, following the testing method and testing procedures outlined into the OECD guideline 422.

The substance was administered to rats orally (by gavage) once daily at 0 (vehicle only), 40, 120 and 350 mg/kg bw/day doses. The concentration of the test item in the dosing formulations was checked two times during the study and the concentrations varied within the range of 97 and 103 % in comparison to the nominal values.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 14-16, i.e. up to the day before necropsy (altogether for 51-65 days).

There was no test item related mortality at any dose level. One male animal at 350 mg/kg bw/day and one female animal at 120 mg/kg bw/day were found dead on Day 22 and on lactation day 10 (on Day 49), respectively. Based on clinical observation and necropsy findings, the cause of the death was independent from the test item.

Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The body weight development was not adversely affected by the test item in male or in female animals, as well as the mean daily food consumption.

A test item influence on the estrous cycle was not found at any dose level and there were no toxicologically significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams.

Hematological and clinical chemistry evaluations did not reveal adverse test item related changes in the examined parameters. There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring).

Macroscopic findings related to the effect of the test item were not found in male and female animals. Black or grey colored organs (spleen, submandibular or mesenteric lymph nodes) and presence of the test item or its metabolite(s) in the stomach and/or intestines were detected in male animals belonging to the groups dosed at 120 and 350 mg/kg bw/day at the terminal necropsy. In females, black or grey staining was observed mainly in mesenteric lymph nodes in several animals dosed at 120 mg/kg bw/day and in all animals dosed at 350 mg/kg bw/day; the kidneys staining was observed in 2/12 animals dosed at 350 mg/kg bw/day.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level. Non-statistically significant changes in spleen weights were observed in male animals at 40, 120 and 350 mg/kg bw/day and in female animals at 350 mg/kg bw/day; they were considered to be of little or no biological relevance. The examined organ weights of animals selected for toxicity examinations were comparable in the control and 40, 120 and 350 mg/kg bw/day groups at the end of the treatment period.

Histopathological examinations revealed test item related minimal centrilobular vacuolation in the hepatocytes – sign of hepatic lipidosis – in male animals at 350 mg/kg bw/day. Hepatic lipidosis is considered as a slight reversible liver injury in connection with a disturbance of energy metabolism of affected hepatocytes. There were no histological patterns of hematopoietic (erythroid or myeloid) hyperplasia in the splenic tissues or in the livers of animals in accordance with organ weight changes.

Conclusion

Under the conditions of the study, no signs of systemic toxicity in male or female animals was recorded; at 350 mg/kg bw/day, test item related minimal centrilobular vacuolation in the hepatocytes was observed in some male animals (3/5). Centrilobular vacuolation in the hepatocytes is considered as sign of hepatic lipidosis, which is a slight reversible liver injury.

NOAEL (rat, male and female): 350 mg/kg bw/day, systemic toxicity

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

The effects of repeated oral exposure to the substance have been investigated in rats, over a period of 28 days, according to the OECD Guideline 407 and EU Method B.7. Groups of 5 male and 5 female rats were dosed orally by gavage with 50, 200 or 1000 mg/kg bw/day of the substance for 28 days. A further control group received vehicle only. Duplicate groups of control and 1000 mg/kg bw/day animals were maintained for a further 2 weeks without treatment before necropsy.

Two females died incidentally after terminal blood sampling immediately prior to scheduled necropsy. No toxicologically or statistically significant effects were observed after the treatment with test item on absolute or relative food consumption and body weights. All animals dosed at 200 and 1000 mg/kg bw/day showed black discoloration of faeces from treatment day 8 to the end of the treatment period; animals of the recovery group (i.e. 1000 mg/kg bw/day) returned to normal until the end of the treatment-free recovery period.

Based on the test results, the "no-observed-adverse-effect level" of test item was indicated to be lower than 50 mg/kg bw/day for male and female rats when administered orally for a period of 28 days. The classification based on the fact that slight methemoglobinemia was observed in animals of the low-dose group treated at 50 mg/kg bw/day. In addition, in haematology a dose-related haemolytic anaemia was observed in animals of groups treated at 200 and 1000 mg/kg bw/day, respectively. The findings observed in group treated at 1000 mg/kg bw/day had regressed almost in the recovery animals. The absolute and relative increase in spleen weights compared to controls additionally indicates an effect to the treatment with the test item; this correlates with the observed dose-related increase in splenic haematopoiesis in animals of groups treated at 200 and 1000 mg/kg bw/day. The observed splenic and focal hepatic haematopoiesis identified the spleen and the liver as potential target organs.

In order to evaluate the study outcomes and to trace an appropriate hazardous profile of the substance, a deep review of the testing results has been performed.

Considering the recovery degree of the animals treated at 1000 mg/kg bw/day, it is likely that animals dosed at lower levels could completely recover the haematological effects caused by test-item treatment. In addition, although of minimal grade, a high incidence of splenic haematopoiesis was found also in control group, i.e. 3 males and 5 females out of 10 total animals, and in the control recovery group, i.e. 2 males and 4 females out of 10 total animals.

Therefore, in order to evaluate the study outcomes and to trace an appropriate hazardous profile of the substance, a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted, giving particular attention to the critical effects observed in the older study.

The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was conducted to obtain information on the toxic potential of test item, following the testing method and testing procedures outlined into the OECD guideline 422. The substance was administered to rats orally (by gavage) once daily at 0 (vehicle only), 40, 120 and 350 mg/kg bw/day doses. The concentration of the test item in the dosing formulations was checked two times during the study and the concentrations varied within the range of 97 and 103 % in comparison to the nominal values. All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 14-16, i.e. up to the day before necropsy (altogether for 51-65 days). There was no test item related mortality at any dose level. One male animal at 350 mg/kg bw/day and one female animal at 120 mg/kg bw/day were found dead on Day 22 and on lactation day 10 (on Day 49), respectively. Based on clinical observation and necropsy findings, the cause of the death was independent from the test item. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations or at the functional observations. The body weight development was not adversely affected by the test item in male or in female animals, as well as the mean daily food consumption. A test item influence on the oestrous cycle was not found at any dose level and there were no toxicologically significant differences between the control and test item treated male or female animals in the examined parameters of reproductive performance or in the delivery parameters of dams. Haematological and clinical chemistry evaluations did not reveal adverse test item related changes in the examined parameters. There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose (parental male or 13-day offspring). Macroscopic findings related to the effect of the test item were not found in male and female animals.

Black or grey coloured organs (spleen, submandibular or mesenteric lymph nodes) and presence of the test item or its metabolite(s) in the stomach and/or intestines were detected in male animals belonging to the groups dosed at 120 and 350 mg/kg bw/day at the terminal necropsy. In females, black or grey staining was observed mainly in mesenteric lymph nodes in several animals dosed at 120 mg/kg bw/day and in all animals dosed at 350 mg/kg bw/day; the kidneys staining was observed in 2/12 animals dosed at 350 mg/kg bw/day.

There were no test item related changes in the weights (absolute and relative to body or brain weights) of brain, testes and epididymides of male animals at any dose level. Non-statistically significant changes in spleen weights were observed in male animals at 40, 120 and 350 mg/kg bw/day and in female animals at 350 mg/kg bw/day; they were considered to be of little or no biological relevance. The examined organ weights of animals selected for toxicity examinations were comparable in the control and 40, 120 and 350 mg/kg bw/day groups at the end of the treatment period. Histopathological examinations revealed test item related minimal centrilobular vacuolation in the hepatocytes – sign of hepatic lipidosis – in male animals at 350 mg/kg bw/day. Hepatic lipidosis is considered as a slight reversible liver injury in connection with a disturbance of energy metabolism of affected hepatocytes. There were no histological patterns of hematopoietic (erythroid or myeloid) hyperplasia in the splenic tissues or in the livers of animals in accordance with organ weight changes.

No signs of hemolytic anaemia were observed, as well as, no signs of splenic or hepatic haematpoiesis were recorded in the new ca 50 day-study, OECD 422; the absence of any sign that could be related to the effects observed in the previously conducted 28 -day-study, legitimises the uncertainty of the obtained results and conclusions.

Based on the new study outcomes, the dose level of 350 mg/kg bw/day can be considered as an appropriate No Observed Adverse Effect Level.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values, which take into account the duration of exposure and the dose/concentration, which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. Nevertheless, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by-case basis). For example, for 28-day study the guidance values are increased by a factor of three, thus the limit for sub-acute studies should be 300 mg/kg bw/day.

Based on results obtained in subacute Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test performed on rats dosed by oral route, the substance does not produce significant effects up to the dosage of 350 mg/kg bw/day, which can be considered as the No Observed Adverse Effect Level.

In conclusion, the substance does not meet the criteria to be classified for specific target organ toxicity after repeated exposure, according to the CLP Regulation (EC) 1272/2008.