[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Not toxic to reproduction

Link to relevant study records

Reference

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

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 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

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.

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.

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: 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.

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

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

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.

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

Critical effects observed:

no

Reproductive effects observed:

no

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The possible effects of the test item on reproduction and development were investigated in rats, 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 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 oestrous cycle was not found at any dose level 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. 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; no pathologic changes were found in the examined organs or tissues of randomly selected male or female animals.

Under the conditions of the present study, the test item did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behaviour, conception, parturition) in parental male and female rats.

Effects on developmental toxicity

Description of key information

Not toxic to developmental

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The possible effects of the test item on reproduction and development were investigated in rats, 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 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 changes in the serum thyroid hormone (T4) levels were recorded at any dose (male or 13-day offspring). 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 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.

Justification for classification or non-classification

According to CLP Regulation (EC) No 1272/2008, 3.7 reproductive toxicity section, reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

 

Based on the available information, the substance does not meet the criteria to be classified for reproductive toxicity, according to CLP Regulation (EC) No 1272/2008.