[No public or meaningful name is available]

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November the 15th, 2017 to January the 19th, 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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November the 15th, 2017 to January the 19th, 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 for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Remarks:

preliminary test

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Budapest.
- Females: nulliparous, non-pregnant females.
- Age at study initiation: males 85 - 95 days, females 85 – 95 days.
- Weight at study initiation: males 324 – 376 g, females 207 – 248 g.
- Housing: before mating 2 animals of the same sex/cage; during mating 1 male and 1 female per cage; pregnant females individually, while males 2 animals per cage. Type III polypropylene/polycarbonate (size: 22 x 32 x 19 cm width x length x height).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum. Food was changed at weekly intervals.
- Water: tap water, as for human consumption, ad libitum. Fresh drinking water was given daily.
- Acclimation period: 20 days.
- Health check: only healthy animals were used for the study. Healthy status was certified by the breeder.

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 supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. 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.

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:

The test item was formulated in the vehicle (distilled water) in concentrations of 15, 30 and 60 mg/ml calculated by the active ingredient content (the corresponding non-corrected concentrations were 15.79, 31.58 and 63.16 mg/ml).
Formulations were prepared beforehand not longer than for four days and stored at 5 ± 3 °C until the administration.
A constant treatment volume of 10 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.

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 and five aliquots of 5 ml 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 95 and 99 % in comparison to the nominal values.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.

Recovery of test item from distilled water formulations was within the acceptance criteria (relative to nominal concentrations: 100 % at ca. 1 mg/ml and 95 % at ca. 100 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.
The substance proved to be stable in distilled water at room temperature for one day and in a refrigerator (5 ± 3 °C) for four days.

Duration of treatment / exposure:

Males: 53 days (during the pre-mating, mating and post-mating periods)
Females: 50 - 64 days; (during the pre-mating, mating and post-mating periods depending on mating success)

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

based on active ingredient (corresponding to the actual dose of 157.9 mg/kg bw/day)

Dose / conc.:

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

Remarks:

based on active ingredient (corresponding to the actual dose of 315.8 mg/kg bw/day)

Dose / conc.:

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

Remarks:

based on active ingredient (corresponding to the actual dose of 631.6 mg/kg bw/day)

No. of animals per sex per dose:

12 males and 12 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were chosen on the basis of the results of a preliminary toxicity screening test.

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).

DETAILED CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time.
More 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 on Day 50. 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, 41, 48 and 53 for male animals), pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals.

HAEMATOLOGY
Hematology 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.
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, Activated partial Thromboplastin Time and Prothrombin Time.

CLINICAL CHEMISTRY
Clinical chemistry 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.
In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4).
Parameters: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Bile acids, Sodium concentration, Potassium concentration, Albumin concentration and Total Protein concentration.
Tyroid hormone. blood samples were collected from animals as follows: from at least pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter); from all dams and at least two pups per litter on post-partal/postnatal day 13; from all parent male animals at termination on Days 54.

Sacrifice and pathology:

SACRIFICE
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”)

GROSS PATHOLOGY
Gross necropsy was performed on each animal.
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 corpora lutea and implantation sites was recorded.
Organs: Adrenal glands, Aorta, Bone with bone marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina), Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes, (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines (representative regions: duodenum, ileum, jejunum), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder.

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 with the exception of testes and epididymides of animal no.411 due to vestigial organs on one side.

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.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (600 mg/kg bw/day).
In addition, in the low dose group uterus of one female (no. 225) and caecum of one female (no.230) was processed and examined due to macroscopic findings (hydrometra and dilatation, respectively).
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, treatment-related

Description (incidence and severity):

The test item related clinical signs were not detected in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 150, 300 or 600 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
The only finding, attributable to the test item treatment, was reddish-brownish stool in the bedding material, in all male and female groups at 150, 300 and 600 mg/kg bw/day from Days 2-4 up to the termination of the treatment. Reddish-brownish color of the faeces was due to the elimination of test item or its metabolite by the gastrointestinal tract.

Alopecia was observed on the right side of the neck of one female animal in the 600 mg/kg bw/day group (1/12) from Day 7 until Day 14. This finding was considered to be individual change occurring commonly in experimental rats and not related to the test item. Similarly, this observation was detected at the weekly detailed clinical observations on Day 7 and 13.
A wound (~1 cm in diameter) on the chest was noted for one female animal in the control group (1/12) from lactation day 6 to 13 (day of termination). This finding was considered to be individual change with unknown origin. Similarly, this observation was also detected during the weekly detailed clinical observations on lactation day 13.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality at 150, 300 or 600 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not adversely affected by the test item in male or in female animals at 150, 300 or 600 mg/kg bw/day during the entire treatment period.
The mean body weight was similar in both male and female animals in the control, 150, 300 and 600 mg/kg bw/day groups.

Statistical significance with respect to the control was detected in male animals at 600 mg/kg bw/day at the slightly higher mean body weight gain between Days 27 and 34 and at the slightly lower mean body weight gain between Days 34 and 41.
The mean body weight was similar in female animals in the control, 150, 300 and 600 mg/kg bw/day groups during the course of premating, gestation and lactation periods.
These slight differences compared to their control in male and female animals were transient and with minor degree and did not result in significant changes in the mean body weight. Therefore these findings in the body weight gain were 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 or treatment related changes in the food consumption of male and female animals at any dose level (150, 300 and 600 mg/kg bw/day).
The food consumption was comparable in the control and test item treated animals, i.e. statistically or biologically significant differences were not seen in the mean daily food consumption of male or female animals at any dose level with respect to their control group during the entire observation period (pre-mating and post mating periods in male animals; pre-mating, gestation and lactation periods in female animals).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 150, 300 or 600 mg/kg bw/day.
In male animals, statistical significances were detected at the slightly lower mean corpuscular (erythrocyte) volume (MCV) and mean corpuscular (erythrocyte) hemoglobin (MCH) at 150 and 300 mg/kg bw/day, at the lower mean percentages of reticulocytes (RET) at 150 and 600 mg/kg bw/day, at the higher mean red blood cell (erythrocyte) count (RBC) at 150 mg/kg bw/day and at the slightly higher mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 150 and 300 mg/kg bw/day.
The examined hematological parameters were comparable in female animals in the control and in all test item treated groups (150, 300 or 600 mg/kg bw/day).
All these slight changes in male animals were considered to have no toxicological relevance in the lack of dose dependence and minor degree.
All values can be considered as indicative of biological variation and not related to the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related adverse alterations occurred in the examined clinical chemistry parameters in male or female animals at 150, 300 or 600 mg/kg bw/day.
In the male animals at 150 mg/kg bw/day statistical significance was noted for the higher mean concentration of bile acids (BAC) when compared to the control. This difference only in the low dose was considered to be toxicologically not relevant.
All examined clinical chemistry parameters were comparable in female animals in the control and in all test item treated groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any treatment-related alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, 150, 300 or 600 mg/kg bw/day groups, on Day 50).
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.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item related adverse effects on the examined organ weights in male or female animals at 150, 300 or 600 mg/kg bw/day.
There were no statistically or biologically significant differences between the control and the 150, 300 or 600 mg/kg bw/day groups (male or female) in the weights of the examined organs at the end of the treatment period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Specific macroscopic alterations indicative of test item effect were not observed in the organs or tissues at any dose levels 150, 300 or 600 mg/kg bw/day at the necropsy.
Orange colored content was detected in the stomach or in the intestine in male animals at 150, 300 and 600 mg/kg bw/day and in female animals at 600 mg/kg bw/day probably due to the presence of the test item or its metabolites.
In male animals, in the stomach and intestines orange colored content was observed at 150 (2/12 male), 300 (12/12 males) and 600 mg/kg bw/day (12/12 males). In female animals, the orange colored content in the digestive tract was also observed with lower incidence than in the male animals and only at the high dose: orange colored content was detected in the stomach (3/12) and intestines (2/12).

At 600 mg/kg bw/day, in one male animal (1/12), the left side testis and epididymis were smaller than normal. This was considered as individual disorder without toxicological significance.
Scar on the chest was observed in one control female animal (1/12) which could be in connection with mechanical injury (probably bite).
Enlargement of the cecum (approximately two-times bigger than normal), full with food, was observed in single female animals at 150 mg/kg bw/day (1/12) and 600 mg/kg bw/day (1/12). This finding without inflammatory lesion in the mucous membrane was considered to be an individual phenomenon.
A dense formation in the uterine horn (approximately 1cm in diameter) was noted for one female animal at 600 mg/kg bw/day (1/12). This was considered to be a sporadic incidental finding not related to the test item.
Moderate hydrometra was noted for one dam (1/12) of the low dose group. 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) this finding was judged to be toxicologically not relevant.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 600 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male or female animals at the highest dose (600 mg/kg bw/day).
In the male animals belonging to the test item treated and control groups, 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 (12/12 in control; 12/12 at 600 mg/kg bw/day including right side testis and epididymis in animal no. 411 at the high dose). 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 animal no.411 (1/12 at 600 mg/kg bw/day), lack of matured spermatozoa in the left side epididymis and decreased intensity of spermatogenesis in the left side testis were detected. These findings were considered as individual disorder without toxicological significance.
In the female animals belonging to the treated and control groups (12/12 in control; 11/12 at 600 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 was normal in all cases as well.
Fibroma in the uterus was noted for one dam at 600 mg/kg bw/day (1/12, animal no.423) in compliance with the macroscopic observation (dense formation in the left side uterine horn) of this dam. This finding is a common sporadic incidental finding in experimental rats.
Dilatation of uterine horns was observed in one female animal (1/1 dam at 150 mg/kg bw/day), which is a slight neuro-hormonal phenomenon in connection with the normal sexual cycle of uterus (proestrus phase) without pathological significance as there were no inflammatory or other pathological lesions.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (2/5 control male and 1/5 control female; 1/5 female at 600 mg/kg bw/day) occurred sporadically and these were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in control (1/5 male and 1/5 female) and 600 mg/kg bw/day (1/5 male) groups. Hyperplasia of BALT is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
The focal chronic dermatitis accompanied with fibrosis observed on one control female (1/12, animal no.121) could be in connection with mechanical injury.
Dilatation of caecum was observed in one female at 150 mg/kg bw/day (1/1, animal no.230) (without inflammatory lesion in the mucous membrane). This finding could be in connection with accumulation of intestinal content, as an individual phenomenon.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver (except hematoma), 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 surviving animals. The quantity and quality of hemopoietic cells in the bone marrow, and the cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

SERUM THYROID HORMONE
Statistically significant difference with respect to the control was noted for the slightly higher mean thyroid hormone (free T4) level in PN13 offspring at 600 mg/kg bw/day. This minor difference was considered to be toxicologically not relevant as most of the individual values were within the historical control range (mean: 2.39 ± 0.37 ng/dl; n=99; min: 1.73 ng/dl; max: 3.73 ng/dl). Additionally, in none of the animals the T4 value resulted to be higher than the maximum one for the mean of the historical control.

Dose descriptor:

NOAEL

Effect level:

>= 600 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 (subacute) (male and female) ≥ 600 mg/kg bw/day, systemic toxicity

Executive summary:

The objective of the study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally to rats; the experiment was conducted according to the OECD guideline 422.

The substance was administered, by gavage, once daily at 0 (vehicle only) at the doses of 150, 300 and 600 mg/kg body weight/day (mg/kg bw/day) to four groups of Hsd.Han: of Wistar rats (12 animals per sex per group); a group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study.

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 54 days). Females were additionally exposed through the gestation period and up to lactation days 13-18, i.e. up to the day before necropsy (altogether for 51-65 days).

No deaths occurred in none of the tested groups. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations nor at the functional observations. The only finding, attributable to the test item treatment, was reddish-brownish stool in the bedding material, in all male and female. Reddish-brownish color of the faeces was due to the elimination of test item or its metabolite by the gastrointestinal tract. The body weight development was not disturbed and it was comparable in the control and test item treated groups. The mean daily food consumption was not adversely affected at 150, 300 or 600 mg/kg bw/day in male animals or in female animals.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters at all the doses tested, as well as there were no test item related adverse effects on the examined clinical chemistry parameters at any dose (male or female).

No changes in the serum thyroid hormone (T4) levels were recorded at any dose (male or 13-day offspring).

Macroscopic findings related to the effect of the test item were not found in male and female animals at 150, 300 and 600 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; the examined organ weights of animals selected for toxicity examinations were comparable in the control and in all the treated goups at the end of the treatment period.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 600 mg/kg bw/day; no pathologic changes were found in the examined organs or tissues of randomly selected male or female animals in the control or in the 600 mg/kg bw/day group.

Conclusion

NOAEL (subacute) (male and female) ≥ 600 mg/kg bw/day, systemic toxicity

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Remarks:

preliminary test

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From August 04th to September 22nd, 2017

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 for cross-reference:

reference to other study

Remarks:

main test

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 October 2008

Deviations:

yes

Remarks:

procedures disigned for preliminary test

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is commonly used species for toxicological studies in accordance with international recommendations.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop ZRT. 1103 Budapest.
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females: nulliparous and non-pregnant animals.
- Age at study initiation: 67 – 72 days.
- Weight at study initiation: males 272 – 320 g; females 157 – 203 g.
- Fasting period before study: overnight food deprivation before the blood sampling.
- Housing: 5 animals of the same sex/ cage from days 0 to 13 animals of main and satellite groups, type IV polypropylene/ polycarbonate; individual caging from day 14 animals of satellite group, Type III polypropylene/ polycarbonate.
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, ad libitum.
- Water: tap water, as for human consumption, ad libitum.
- Acclimation period: 22 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 supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. 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).

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

Details on route of administration:

The route of application was selected in compliance with international guidelines.

Vehicle:

water

Details on oral exposure:

TREATMENT
- Volume: 10 ml/kg body weight

FORMULATION
Test item was dissolved in the vehicle (distilled water) in concentrations of 7.5, 15, 30 and 60 mg/ml calculated by the active ingredient content (corrected concentrations; respectively to uncorrected concentrations of 7.89, 15.79, 31.58 and 63.16).
Formulations were prepared in the formulation laboratory of Test Facility beforehand not longer than for three days and stored at 5 ± 3 °C until use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item concentrations in the samples varied within the range from 95 % to 99 % in comparison to the nominal values calculated by the active ingredient content. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 100 % at ca. 1 mg/ml and 95 % at ca. 100 mg/ml).
Test item proved to be stable in distilled water at the intended concentrations at room temperature for one day and in a refrigerator (5 ± 3 °C) for four days.

Duration of treatment / exposure:

Main group animals: 14 days
Satellite group: 38 days males and 41-48 days females

Frequency of treatment:

Daily (7 days per week)

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

Dose / conc.:

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

Remarks:

calculated by the active ingredient content

No. of animals per sex per dose:

5 animals/ sex/ groups – animals of main groups
5 animals/sex/ group (600 mg/kg bw/day) – animals of satellite group

Control animals:

yes, concurrent vehicle

Details on study design:

- Necropsy: one day after the last treatment, i.e. on the Day 15th in case of groups; Day 40th for male animals and Day 43rd-50th for female animals for satellite group. Animals were anesthetized with Isofluran CP® and were exsanguinated from the abdominal aorta after verification of deep narcosis.
- Satellite group: animals of the satellite group were administered with the highest dose up to and including the day before the necropsy (during the pre-mating, mating and post-mating periods for male animals; during the pre-mating, mating, gestation and lactation periods for female animals).
- Mating procedures (satellite group): mating begun 2 weeks after the initiation of treatment (for animals only selected for reproduction examinations) with one female and one male of the high dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred or 8 days have elapsed.

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).

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were conducted once a day, after treatment at approximately the same time. 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.

BODY WEIGHT
Individual body weights were recorded on Day 0 (prior to study start) and once weekly (on Days 7, and 13) with a precision of 1 g. Individual body weight changes were calculated according to the days of measurements and for the study overall.
The animals were also weighed immediately prior to sacrifice (on Day 14; fasted body weight) in order to calculate organ weight to body weight ratio.
Male animals of satellite group were weighed on the first day of dosing (Day 0) and weekly thereafter and at termination.
Female animals of satellite group were weighed on the first day of dosing (Day 0) then weekly (on Days 7 and 13), on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition) and 4 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes.

FOOD CONSUMPTION
Food consumption was determined with the measurement of non-consumed diet with a precision of 1 g weekly for main group’s animals (given food on Days 0 and 7; remained food on Days 7 and 13).
For satellite animals, food consumption was determined weekly except mating period (pre-mating days 0, 7, 13, and by weekly interval during post-mating period for male animals; pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0 and 4).

HAEMATOLOGY
Clinical pathology examinations including hematology and blood coagulation were conducted in main group animals at termination of the treatment (i.e. one day after the last treatment).
Animals were food deprived overnight (for approximately 16 hours) prior to blood collection. Blood samples were harvested from the retro orbital venous plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for hematology.
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 (i.e. percentage of Neutrophil granulocytes, Lymphocytes, Eosinophil granulocytes, Monocytes, Basophil granulocytes), Activated Partial Thromboplastin Time and Prothrombin Time.

HAEMATOLOGY AND CLINICAL CHEMISTRY
Clinical chemistry were conducted in main group animals at termination of the treatment (i.e. one day after the last treatment).
One for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry.
Parameters: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Gamma Glutamyltransferase activity, Alkaline Phosphatase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Inorganic phosphate concentration, Calcium concentration, Sodium concentration, Potassium concentration, Chloride concentration, Albumin concentration, Total Protein concentration and Albumin/globulin ratio.

OBSERVATION OF REPRODUCTIVE PERFORMANCE

MATING PROCEDURE
Vaginal smears were examined for the presence of vaginal plug or sperm daily. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy). Sperm positive females were caged individually.
Mating pairs were clearly identified in the raw data.

PLACENTAL SIGN
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If negative on day 13, the examination was repeated on day 14 of gestation.

DELIVERY PROCESS
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestational day 21 onwards. All observations were recorded. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partal day 5, all animals (dams and sires) were subjected to necropsy.

OBSERVATION OF OFFSPRING
Each litter was examined as soon as possible after delivery (within 24 h of parturition), to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
On the day of birth, pup found dead was subjected to a lung flotation test to differentiate pup died intrauterine (stillborn; negative lung flotation test) from pup died after the birth (dead pups; positive lung flotation test).
All litters were checked and recorded daily for the number of viable and dead pups.

Sacrifice and pathology:

GROSS PATHOLOGY
Gross pathology was performed on every experimental animal at termination of the treatment i.e. one day after the last treatment.
The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.
Special attention was paid to the organs of the reproductive system. The number of corpora lutea and implantation sites were recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Dead pups and pups euthanized at day 5 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
Organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.

ORGAN WEIGHT
The following organs were weighed and recorded in main group animals. Paired organs were weighed together.
With precision of 0.01g: liver, kidneys, testes, epididymides, seminal vesicles with coagulating gland and prostate as a whole, uterus with fallopian tubes, thymus, spleen, brain and heart.
With precision of 0.001g: adrenal glands.

HISTOPATHOLOGY
Histopathological examinations were not performed.

Clinical signs:

no effects observed

Description (incidence and severity):

Test item caused no clinical signs in male or female animals at 75, 150, 300 and 600 mg/kg bw/day. The behavior and physical condition of animals were normal during the14-day treatment period. Yellowish color of stool was recorded in the bedding material at 75, 150, 300 and 600 mg/kg bw/day from Day 3 up to the end of the treatment period (including mating, post mating, gestation and lactation periods of satellite animals). Yellowish color of stool was due to the presence of test item or its metabolite in the faces.

Mortality:

no mortality observed

Description (incidence):

There was no mortality in the control, 75, 150, 300 or 600 mg/kg bw/day groups during the treatment period (male and female).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight development was not influenced in male and female animals at 75, 150, 300 and 600 mg/kg bw/day during the treatment period.
There were no statistically significant differences between the control and test item treated male or female animals at 75, 150, 300 and 600 mg/kg bw/day in the mean body weight or body weight gain during the 14-day observation period. The mean body weight gain of female animals at 300 and 600 mg/kg bw/day was slightly lower than in the control group between Days 0 and 7. This lower body weight gain did not result in significant changes in the mean body weight value of female animals at 300 and 600 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption was comparable in male and female animals at 75, 150, 300 and 600 mg/kg bw/day during the 14-day observation period.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematological evaluation did not reveal test item related changes in the examined parameters at 75, 150, 300 and 600 mg/kg bw/day.
The examined hematological parameters were comparable in the male control and test item treated groups. In the female animals, statistically significant difference with respect to the control was observed at the slightly higher mean percentage of neutrophil granulocytes (NEU) at 75 mg/kg bw/day. All other examined parameters were similar in the control and test item treated groups (150, 300 and 600 mg/kg bw/day).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the investigated clinical chemistry parameters of male or female animals at any dose level (75, 150, 300 and 600 mg/kg bw/day).
Some statistically significant difference were detected between the control and test item treated groups (ALT, AST, CHOL, Na+, K+, TPROT, A/G), although these differences were with small degree and all values remained well within the historical control ranges for these parameters. Some statistical significance was originated from the relative lower or higher value of the control group with respect to the historical control (TPROT and A/G in male animals; AST, ALT, K+ in female animals). In the lack of any dose relevance these findings were not considered to be toxicologically significant.
At 75 mg/kg bw/day, there was no statistically significant difference with respect to their control in the examined clinical chemistry parameters in male animals of the low dose group.
In the female animals, statistical significance was observed at the slightly higher mean activity of alanine amino transferase (ALT) compared to their control.
At 150 mg/kg bw/day, elevated mean activity of aspartate amino transferase (AST), lower mean total protein concentration (TPROT) and higher mean albumin:globulin ratio (A/G) were detected in male animals with respect to their control. In the female animals, statistical significances were observed at the higher mean activity of alanine amino transferase and aspartate amino transferase and lower mean potassium concentration (K+) with respect to their control.
At 300 mg/kg bw/day, in the male animals, the mean total protein concentration was lower and albumin: globulin ratio was higher than in the control group.
In the female animals, statistical significances with respect to the control were noted for the higher mean activity of alanine amino transferase, higher mean cholesterol concentration, lower mean potassium concentration and elevated A/G value.
At 600 mg/kg bw/day, the mean concentration of sodium (Na+) and total protein was lower and mean A/G was higher than in the control group in male animals of the high dose group.
In the female animals, the mean activity of alanine amino transferase slightly exceeded the control while the mean potassium concentration was lower than in the control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test item related effects were not detected in the weights of examined organs of male or female animals administered with 75, 150, 300 or 600 mg/kg bw/day doses.
The weights of examined organs (absolute and relative to body and brain weights) were comparable with their control in male and female animals of each dose group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related adverse macroscopic changes were not detected at the necropsy. Yellowish content in the stomach was detected at 150 mg/kg bw/day (female), 300 and 600 mg/kg bw/day (male and female) probably due to the presence of the test item or its metabolite in the stomach.
Control group: yellowish gray-foci in the caudal part of epididymides were observed in one control male animal (1/5). In the female control group, slight or moderate hydrometra (2/5) were seen at the necropsy.
75 mg/kg bw/day: in on male animal administered with the low dose, atrophy of one side horn of the seminal vesicle (1/5) and thymic hemorrhages (1/5) were detected. In one female animal of the low dose, slight hydrometra was observed (1/5).
150 mg/kg bw/day: there were no macroscopic findings in male animals (5/5). Yellowish content in the stomach (1/5) and slight or moderate hydrometra (2/5) were noted for some female animal.
300 mg/kg bw/day: the content of the stomach was yellowish colored in most of the male animals (4/5). In the female animals yellowish content of the stomach (3/5) and moderate hydrometra (3/5) were observed.
600 mg/kg bw/day: the content of the stomach was yellowish colored in male (3/5) and female (2/5) animals.
Yellowish gray-foci in the caudal part of epididymides and atrophy of one side horn of the seminal vesicle are common findings of this strain of experimental rats with similar age. These occurred in single animals of control and low dose group therefore were judged to be toxicologically not relevant.
Thymic hemorrhage is frequently observed in experimental rats – was observed in one low dose male animal only – and was considered to be related to the exsanguination procedure i.e. it was consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
Hydrometra, related to the female sexual cycle, is also a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs hydrometra was judged to be toxicologically not relevant.

Description (incidence and severity):

REPRODUCTION
The reproduction performance was not impaired during the course of this study. All pairs mated successfully within eight days and all females became pregnant. The mean number of corpora lutea, implantation site and live born pups was slightly below the historical control. A test item influence might be supposed however the difference with respect to the historical control might be originated from the low number of animals in the satellite group.
There was no test item effect on the mortality and body weight or body weight gain of offspring. The mean litter weight and litter weight gain was slightly lower than in the historical control but the mean body weight of pups was above the historical control value.

Dose descriptor:

dose level:

Effect level:

600 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:

There were no toxicologically significant changes in the examined parameters (clinical signs, body weight and body weight gain, food consumption, hematology, blood coagulation and clinical chemistry, necropsy findings, organ weights) after the 14-day oral (by gavage) administration of 75, 150, 300 or 600 mg/kg bw/day doses.
The examined reproductive parameters were not impaired at 600 mg/kg bw/day.

Executive summary:

The objective of the study was to obtain first information on the toxic potential of test item in rats at three dose levels to allow a dose-setting for a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat (main study).

For obtaining first information on the reproductive performance of female animals (corpus luteum deficiency), an additional (satellite) group administered with the highest dose (600 mg/kg bw/day) was examined (two weeks pre-mating, mating, pregnancy and lactation days).

Five groups of Hsd.Han: of Wistar rats (n=5 animals/sex/ group in main groups; n=5 animals/ sex in the satellite group) were administered orally (by gavage) once daily at 0 (vehicle only) 75, 150, 300 and 600 mg/kg bw/day doses in nominal concentrations of 0, 7.5, 15, 30 and 60 mg/ml corresponding to a 10 ml/kg bw dosing volume. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control.

Doses and concentrations were calculated by the active ingredient content as follows [corrected (uncorrected) dose concentration]: 75 (78.9), 150 (157.9), 300 (315.8) and 600 (631.6) mg/kg bw/day doses.

The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front.

The substance concentrations in the samples used for administration of animals varied within the range from 95 % to 99 % in comparison to the nominal values, thereby confirming proper dosing.

For main group animals, detailed clinical observations were performed once daily after the treatment. Body weights were recorded once weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted on all animals one day after the last treatment (on Day 14). Selected organs were weighed.

All animals of the satellite group were dosed with 600 mg/kg bw/day prior to mating (14 days) and throughout mating. In addition, males received the test item after mating up to the day before necropsy (altogether for 39 days). Females were additionally exposed through the gestation period and up to lactation day 4 (altogether for 42-49 days), i.e. up to the day before necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, lactation as well as development of offspring. The dams were allowed to litter, and rear their offspring up to day 4. All parental animals were subjected to gross pathology one day after the last treatment. The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands were preserved. Corpora lutea and implantation site were counted.

There was no mortality during the course of the study (control, 75, 150, 300 and 600 mg/kg bw/day). Test item caused no clinical signs at 75, 150, 300 and 600 mg/kg bw/day in male or female animals during the treatment period. The behavior and physical condition of all animals were normal (main and satellite groups). Yellowish color of stool was observed at 150, 300 and 600 mg/kg bw/day (male and female) from Day 3 up to the end of the treatment and was indicative of the presence of test item or its metabolite on the faeces.

The body weight development was undisturbed at 75, 150, 300 and 600 mg/kg bw/day (male and female) during the 14-day observation period.

The mean daily food consumption was comparable in the control and test item treated groups (75, 150, 300 and 600 mg/kg bw/day; male and female).

The examined reproduction parameters (copulatory and fertility indices, corpora lutea, implantation sites, pre-implantation mortality, post-implantation mortality Intrauterine mortality) were not adversely affected by 600 mg/kg bw/day. The development of offspring (mortality, body weigh) was undisturbed up to post-natal day 5.

Hematological evaluation did not reveal test item related changes in the examined hematological parameters.

Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male or female animals at 75, 150, 300 and 600 mg/kg bw/day.

Test item related pathologic findings were not seen in male or female animals at 75, 150, 300 or 600 mg/kg bw/day at the necropsy. There were no differences between the control and test item treated groups in the weights of examined organs. The substance did not induce adverse effects in male or female Hsd.Han: Wistar rats after the consecutive 14-day oral (by gavage) administration at 75, 150, 300 or 600 mg/kg bw/day.

Conclusion

There were no toxicologically significant changes in the examined parameters (clinical signs, body weight and body weight gain, food consumption, hematology, blood coagulation and clinical chemistry, necropsy findings, organ weights) after the 14-day oral (by gavage) administration of 75, 150, 300 or 600 mg/kg bw/day doses.

The examined reproductive parameters were not impaired at 600 mg/kg bw/day.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Remarks:

validated analytical method

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. Budapest.
- Females: nulliparous, non-pregnant females.
- Age at study initiation: males 85 - 95 days, females 85 – 95 days.
- Weight at study initiation: males 324 – 376 g, females 207 – 248 g.
- Housing: before mating 2 animals of the same sex/cage; during mating 1 male and 1 female per cage; pregnant females individually, while males 2 animals per cage. Type III polypropylene/polycarbonate (size: 22 x 32 x 19 cm width x length x height).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum. Food was changed at weekly intervals.
- Water: tap water, as for human consumption, ad libitum. Fresh drinking water was given daily.
- Acclimation period: 20 days.
- Health check: only healthy animals were used for the study. Healthy status was certified by the breeder.

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 supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. 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.

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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was formulated in the vehicle (distilled water) in concentrations of 15, 30 and 60 mg/ml calculated by the active ingredient content (the corresponding non-corrected concentrations were 15.79, 31.58 and 63.16 mg/ml).
Formulations were prepared beforehand not longer than for four days and stored at 5 ± 3 °C until the administration.
A constant treatment volume of 10 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.

Details on mating procedure:

- Mating: begun 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating), placed in a single cage.
- After successful mating each pregnant female was caged: individually.
- Pairing replacement: females remained with the same male until copulation occurred; only for one female animal in the 600 mg/kg bw/day group (no.431), male pair was replaced by a proven male (no.412).
- Proof of pregnancy: vaginal smears were examined for the presence of vaginal plug or sperm. Presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 422).
- Post-partum: the day of birth (viz. when parturition is complete) was defined as day 0 post-partum.

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 and five aliquots of 5 ml 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 95 and 99 % in comparison to the nominal values.
The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.

Recovery of test item from distilled water formulations was within the acceptance criteria (relative to nominal concentrations: 100 % at ca. 1 mg/ml and 95 % at ca. 100 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.
The substance proved to be stable in distilled water at room temperature for one day and in a refrigerator (5 ± 3 °C) for four days.

Duration of treatment / exposure:

Males: 53 days (during the pre-mating, mating and post-mating periods)
Females: 50 - 64 days; (during the pre-mating, mating and post-mating periods depending on mating success)

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

based on active ingredient (corresponding to the actual dose of 157.9 mg/kg bw/day)

Doses / concentrations 2

Dose / conc.:

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

Remarks:

based on active ingredient (corresponding to the actual dose of 315.8 mg/kg bw/day)

Doses / concentrations 3

Dose / conc.:

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

Remarks:

based on active ingredient (corresponding to the actual dose of 631.6 mg/kg bw/day)

No. of animals per sex per dose:

12 males and 12 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were chosen on the basis of the results of a preliminary toxicity screening test.

Examinations

Parental animals: Observations and examinations:

MORTALITY
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day).

DETAILED CLINICAL OBSERVATIONS
General clinical observations were made on parental animals once a day, after the administration at approximately the same time.
More 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 on Day 50. 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, 41, 48 and 53 for male animals), pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals.

HAEMATOLOGY
Hematology 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.
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, Activated partial Thromboplastin Time and Prothrombin Time.

CLINICAL CHEMISTRY
Clinical chemistry 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.
In addition, blood samples were collected for possible determination of serum levels of thyroid hormones (T4).
Parameters: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Bile acids, Sodium concentration, Potassium concentration, Albumin concentration and Total Protein concentration.
Tyroid hormone. blood samples were collected from animals as follows: from at least pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter); from all dams and at least two pups per litter on post-partal/postnatal day 13; from all parent male animals at termination on Days 54.

PLACENTAL SIGN
All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on the gestational day 13. If the test was negative on day 13, the examination was repeated on day 14 of gestation.

DELIVERY PROCESS
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible from gestation day 21 onwards. All observations and any evidence of abnormal deliveries were considered. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their new-born or not. The sucking success was observed by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it was feasible. Extra pups were eliminated by a random selection.
Partial adjustment of litter size was performed if the number of male and female pups did not allow having four of each sex per litter (for example, five males and three females).

Oestrous cyclicity (parental animals):

Estrous cycle was monitored by examining vaginal smears each day before the treatment started from each animal being considered for study for two weeks. Estrous cycle was evaluated and considered at randomization.
Vaginal smears were also prepared and estrous cycle was monitored daily from the beginning of the treatment period (two weeks pre-mating period) and during the mating period until evidence of mating.
Vaginal smears were also prepared on the day of the necropsy.
Vaginal smears were stained with 1 % aqueous methylene blue solution then were examined with a light microscope.

Litter observations:

Each litter were examined as soon as possible after delivery (within 24 h of parturition), to establish the number and gender of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the day after parturition is complete, day 0), and on day 13 post-partum with an accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate pups died intrauterine (stillborn) from pups died after the birth (dead pups).
All litters were checked and recorded daily for the number of viable and dead pups.
The anogenital distance of each pup was determined on postnatal day 4. The anogenital distance was normalized to the cube root of body weight. Therefore, individual body weight of pups was also determined with an accuracy of 0.01 g on postnatal day 4 and the litter weight was calculated for evaluation on postnatal day 4.
The number of nipples/areolae in male pups was counted on postnatal day 13.

Postmortem examinations (parental animals):

SACRIFICE
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP® (details are presented in paragraph “Characteristics of anesthetics”).

GROSS PATHOLOGY
Gross necropsy was performed on each animal.
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 corpora lutea and implantation sites was recorded.
Dead pups and pups euthanized at day 13 post-partum, or shortly thereafter, were carefully examined for gross abnormalities.
Organs: Adrenal glands, Aorta, Bone with bone marrow and joint (femur), Brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), Eyes (lachrymal gland with Harderian glands), Female mammary gland, Gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina), Heart, Kidneys, Large intestines (caecum, colon, rectum, including Peyer’s patches), Liver, Lungs (with main stem bronchi; inflation with fixative and then immersion), Lymph nodes, (submandibular, mesenteric), Muscle (quadriceps), Esophagus, Pancreas, Pituitary, Prostate, Salivary glands (submandibular), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, Small intestines (representative regions: duodenum, ileum, jejunum), Spinal cord (at three levels: cervical, mid-thoracic and lumbar), Spleen, Sternum, Stomach, Thymus, Thyroid + parathyroid, Trachea, Urinary bladder.

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 with the exception of testes and epididymides of animal no.411 due to vestigial organs on one side.

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.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (600 mg/kg bw/day).
In addition, in the low dose group uterus of one female (no. 225) and caecum of one female (no.230) was processed and examined due to macroscopic findings (hydrometra and dilatation, respectively).
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.

Postmortem examinations (offspring):

All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP®.
Dead pups found were subjected to necropsy by macroscopic examination. All observed abnormalities were recorded.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 150, 300 or 600 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
The only finding, attributable to the test item treatment, was reddish-brownish stool in the bedding material, in all male and female groups at 150, 300 and 600 mg/kg bw/day from Days 2-4 up to the termination of the treatment. Reddish-brownish color of the faeces was due to the elimination of test item or its metabolite by the gastrointestinal tract.

Alopecia was observed on the right side of the neck of one female animal in the 600 mg/kg bw/day group (1/12) from Day 7 until Day 14. This finding was considered to be individual change occurring commonly in experimental rats and not related to the test item. Similarly, this observation was detected at the weekly detailed clinical observations on Day 7 and 13.
A wound (~1 cm in diameter) on the chest was noted for one female animal in the control group (1/12) from lactation day 6 to 13 (day of termination). This finding was considered to be individual change with unknown origin. Similarly, this observation was also detected during the weekly detailed clinical observations on lactation day 13.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality at 150, 300 or 600 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight development was not adversely affected by the test item in male or in female animals at 150, 300 or 600 mg/kg bw/day during the entire treatment period.
The mean body weight was similar in both male and female animals in the control, 150, 300 and 600 mg/kg bw/day groups.

Statistical significance with respect to the control was detected in male animals at 600 mg/kg bw/day at the slightly higher mean body weight gain between Days 27 and 34 and at the slightly lower mean body weight gain between Days 34 and 41.
The mean body weight was similar in female animals in the control, 150, 300 and 600 mg/kg bw/day groups during the course of premating, gestation and lactation periods.
These slight differences compared to their control in male and female animals were transient and with minor degree and did not result in significant changes in the mean body weight. Therefore these findings in the body weight gain were 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 or treatment related changes in the food consumption of male and female animals at any dose level (150, 300 and 600 mg/kg bw/day).
The food consumption was comparable in the control and test item treated animals, i.e. statistically or biologically significant differences were not seen in the mean daily food consumption of male or femal e animals at any dose level with respect to their control group during the entire observation period (pre-mating and post mating periods in male animals; pre-mating, gestation and lactation periods in female animals).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related adverse changes in the examined hematological parameters in male or female animals at 150, 300 or 600 mg/kg bw/day.
In male animals, statistical significances were detected at the slightly lower mean corpuscular (erythrocyte) volume (MCV) and mean corpuscular (erythrocyte) hemoglobin (MCH) at 150 and 300 mg/kg bw/day, at the lower mean percentages of reticulocytes (RET) at 150 and 600 mg/kg bw/day, at the higher mean red blood cell (erythrocyte) count (RBC) at 150 mg/kg bw/day and at the slightly higher mean corpuscular (erythrocyte) hemoglobin concentration (MCHC) at 150 and 300 mg/kg bw/day.
The examined hematological parameters were comparable in female animals in the control and in all test item treated groups (150, 300 or 600 mg/kg bw/day).
All these slight changes in male animals were considered to have no toxicological relevance in the lack of dose dependence and minor degree.
All values can be considered as indicative of biological variation and not related to the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related adverse alterations occurred in the examined clinical chemistry parameters in male or female animals at 150, 300 or 600 mg/kg bw/day. In the male animals at 150 mg/kg bw/day statistical significance was noted for the higher mean concentration of bile acids (BAC) when compared to the control. This difference only in the low dose was considered to be toxicologically not relevant.
All examined clinical chemistry parameters were comparable in female animals in the control and in all test item treated groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any treatment-related alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, 150, 300 or 600 mg/kg bw/day groups, on Day 50).
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.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no pathologic changes in the examined reproductive organs or tissues of male or female animals (control and 600 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male or female animals at the highest dose (600 mg/kg bw/day).
In the male animals belonging to the test item treated and control groups, 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 (12/12 in control; 12/12 at 600 mg/kg bw/day including right side testis and epididymis in animal no. 411 at the high dose). 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 animal no.411 (1/12 at 600 mg/kg bw/day), lack of matured spermatozoa in the left side epididymis and decreased intensity of spermatogenesis in the left side testis were detected. These findings were considered as individual disorder without toxicological significance.
In the female animals belonging to the treated and control groups (12/12 in control; 11/12 at 600 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 was normal in all cases as well.
Fibroma in the uterus was noted for one dam at 600 mg/kg bw/day (1/12, animal no.423) in compliance with the macroscopic observation (dense formation in the left side uterine horn) of this dam. This finding is a common sporadic incidental finding in experimental rats.
Dilatation of uterine horns was observed in one female animal (1/1 dam at 150 mg/kg bw/day), which is a slight neuro-hormonal phenomenon in connection with the normal sexual cycle of uterus (proestrus phase) without pathological significance as there were no inflammatory or other pathological lesions.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (2/5 control male and 1/5 control female; 1/5 female at 600 mg/kg bw/day) occurred sporadically and these were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue (BALT) was observed in control (1/5 male and 1/5 female) and 600 mg/kg bw/day (1/5 male) groups. Hyperplasia of BALT is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
The focal chronic dermatitis accompanied with fibrosis observed on one control female (1/12, animal no.121) could be in connection with mechanical injury.
Dilatation of caecum was observed in one female at 150 mg/kg bw/day (1/1, animal no.230) (without inflammatory lesion in the mucous membrane). This finding could be in connection with accumulation of intestinal content, as an individual phenomenon.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver (except hematoma), 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 surviving animals. The quantity and quality of hemopoietic cells in the bone marrow, and the cyto-morphology of endocrine glands was the same in the control and high dose treated animals.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

A test item influence on the estrous cycle was not detected at any dose level (150, 300 or 600 mg/kg bw/day).
There were no significant differences between the control and treated groups in the number or percentage of animals with regular cycles, in the mean number of cycles, mean length of cycles, mean number of days in pro-estrous, estrous or diestrus during the pre-experimental or pre-mating period.

Reproductive performance:

no effects observed

Description (incidence and severity):

The examined parameters of reproductive performance were not affected by the treatment with the test item at 150, 300 or 600 mg/kg bw/day in male or female animals.
Statistical significance was observed at the slightly lower copulatory index of male animals at 600 mg/kg bw/day because one male animal (1/12) in this group failed to mate within 14 days. This minor change was considered to be not related to the test item because similar incidences also occur in non-treated experimental rats of this strain. There were no significant differences between the control and test item treated groups in the fertility index of male animals.
The percentage of pregnant females, non-pregnant females, and dams delivered, pregnants with liveborn(s) and the pre-coital interval and the mean number of conceiving days were comparable in female animals of control and test item treated animals. There were no significant differences between the control and test item treated groups in the copulatory, fertility and gestation indices in female animals.

Details on results (P0)

DELIVER - no effects observed
The delivery data of dams were not affected by the treatment with the test item at 150, 300 or 600 mg/kg bw/day.
The mean number of corpora lutea, implantation sites per dams, mean of pre-implantation loss, post-implantation loss and total intrauterine mortality were similar in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, live-borns, stillborns or viable pups on post-partal day 0, in the percentage of dams with viable offspring on pos-tpartal day 0 or in the live birth index.

SERUM THYROID HORMONE - no effects observed

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 600 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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected in the offspring between postnatal days 0 and 13.
The percentage of offspring with signs (cold, not suckled) at 150, 300 and 600 mg/kg bw/day was higher than in the control group on postnatal day 0. However, these findings were not considered to be toxicologically relevant as no dose relevance was observed and the sign were transient – detected shortly after the delivery. Moreover, these observations were not associated with any influence on the development of the offspring.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item related effect on offspring’s extra uterine mortality.
The mean number of dead offspring (including missing pups) per litter was comparable in the control and test item treated groups (150, 300 or 600 mg/kg bw/day).
There were no differences between the control and test item treated (150, 300 or 600 mg/kg bw/day) groups in the survival indices. The mean number of live pups per litter and the mean number of viable pups per litter were similar in all groups on postnatal days 0, 4 and 13.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and mean pup weights as well as the mean litter weight gains and mean pup weight gains were similar in the control and in all test item treated groups (150, 300 and 600 mg/kg bw/day) on postnatal days 0, 4 and 13.
Considering the offspring’s body weight in males and females, separately, statistically significant difference with respect to the control was detected at the higher mean weight of female pups at 150 mg/kg bw/day on postnatal day 4. The difference with respect to the control was minor and it was considered to be toxicologically not relevant.

Sexual maturation:

no effects observed

Description (incidence and severity):

The anogenital distances (in male and female offspring) or nipple retention (male) were not affected by the test item at 150, 300 and 600 mg/kg bw/day).
The anogenital distances, both absolute and normalized, were consistent with the control in male and female offspring in the all treated groups.
Nipples/areoles were not visible in any of the examined male offspring in the control or 150, 300 or 600 mg/kg bw/day groups on postnatal day 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.
Seven pups (one male in the control, one male at 150 mg/kg bw/day, one male and two females at 300 mg/kg bw/day, one male and one female at 600 mg/kg bw/day) had no macroscopic changes in the organs or tissues and the lung flotation test was negative referring to intrauterine death of these offspring subjected to necropsy on postnatal day 0.
In one female pup at 300 mg/kg bw/day, autolyzed visceral organs were detected on post-natal day 0 and one male pup at 600 mg/kg bw/day was partially cannibalized. In case of these two pups the determination of stillborn or liveborn was not feasible due to autolysis or cannibalism.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

SEX DISTRIBUTION - no effects observed
There were no test item related differences between the control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0, 4 or 13.

SERUM THYROID HORMONE - effects observed, non-treatment-related
Statistically significant difference with respect to the control was noted for the slightly higher mean thyroid hormone (free T4) level in PN13 offspring at 600 mg/kg bw/day. This minor difference was considered to be toxicologically not relevant as most of the individual values were within the historical control range (mean: 2.39 ± 0.37 ng/dl; n=99; min: 1.73 ng/dl; max: 3.73 ng/dl). Additionally, in none of the animals the T4 value resulted to be higher than the maximum one for the mean of the historical control.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 600 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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

NOAEL (male and female) ≥ 600 mg/kg bw/day, reproductive toxicity
NOAEL ≥ 600 mg/kg bw/day, developmental toxicity

Executive summary:

The objective of the study was to obtain initial information on the toxic potential of test item and on the possible effects of the test item on reproduction and development when repeatedly administered orally (by gavage) to rats at doses of 150, 300 and 600 mg/kg bw/day compared to control animals. The experiment was conducted according to the OECD guideline 422.

The substance was administered orally (by gavage) once daily at 0 (vehicle only) at the doses of 150, 300 and 600 mg/kg body weight/day (mg/kg bw/day) to four groups of Hsd.Han: of Wistar rats (12 animals per sex per group); a group of vehicle (distilled water) treated animals (n= 12/sex) served as a control.

The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study.

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 54 days). Females were additionally exposed through the gestation period and up to lactation days 13-18, i.e. up to the day before necropsy (altogether for 51-65 days).

No deaths occurred in none of the tested groups. Clinical signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor the detailed weekly clinical observations nor at the functional observations. The body weight and body weight gain were not affected by the treatment.

A test item influence on the estrous cycle was not found at any dose level (150, 300 and 600 mg/kg bw/day) and there were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

No changes in the serum thyroid hormone (T4) levels were recorded at any dose (male or 13-day offspring).

Macroscopic findings related to the effect of the test item were not found in male and female animals at 150, 300 and 600 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.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, pituitary, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes at 600 mg/kg bw/day; no pathologic changes were found in the examined organs or tissues of randomly selected male or female animals in the control or in the 600 mg/kg bw/day group.

No adverse effect on the mortality, clinical signs body weight development or necropsy findings were detected in the offspring terminated as scheduled. The anogenital distance (male and female) or nipple retention (male) were not affected.

Under the conditions of the present study, the test item administered at 150, 300 or 600 mg/kg bw/day by oral gavage did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, parturition) in parental male and female rats.

The development of the F1 offspring was not impaired from birth to post-natal day 13 at any dose level after repeated oral administration of dams.

Conclusion

NOAEL (male and female) ≥ 600 mg/kg bw/day, systemic toxicity

NOAEL (male and female) ≥ 600 mg/kg bw/day, reproductive toxicity

NOAEL ≥ 600 mg/kg bw/day, developmental toxicity