Registration Dossier

Diss Factsheets

Administrative data

Description of key information

NOAEL (subacute, rat M/F) ≥ 750 mg/kg bw/day, based on systemic toxicity

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 30 to August 12, 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 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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
TEST ANIMALS
- Source:
Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary.
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 92 – 97 days parent males and females.
- Weight at study initiation: 365 – 418 g male animals and 208 – 246 g female animals. The weight variation did not exceed ± 20 per cent of the mean weight
- Animal health: only healthy animals were used for the study. Healthy status was certified by the breeder.
- Housing:
2 animals of the same sex/cage before mating; 1 male and 1 female / cage during the mating; individually pregnant females: individually; 2 animals / cage males after mating. Cage type III polypropylene/polycarbonate (22 x 32 x 19 cm).
- Diet: animals received ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany -, ad libitum. Food was changed at weekly intervals. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
- Water: animals received tap water from watering bottles, as for human consumption, ad libitum. Food was changed at weekly intervals. Fresh drinking water was given daily. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service. The quality control results are available at testing facility's archives.
- Acclimation period:
27 days

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.
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
TREATMENT
A constant treatment volume of 5 ml/kg body weight was administered in all groups. The individual volume of the treatment was based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on Day 0.

FORMULATION
The test item was formulated in the vehicle (distilled water) in concentrations of 16.6, 50 and 150 mg/ml calculated by the active ingredient content. Formulations were prepared beforehand not longer than for three days and stored at 5 ± 3 °C before the administration.FORMULATION
The test item was formulated in the vehicle (distilled water) in concentrations of 16.6, 50 and 150 mg/ml calculated by the active ingredient content. Formulations were prepared beforehand not longer than for three days and stored at 5 ± 3 °C before the administration.
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 or 10 ml of each formulation and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis.
Concentration of the test item in the dosing formulations varied between the range of 98 and 105 % 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 the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/ml and 100 % at ca. 200 mg/ml).
A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical study. The substance proved to be stable in the vehicle in a refrigerator (at 5 ± 3 °C) for three days.
Duration of treatment / exposure:
42-59 days treatment/observation period (depending on the effectiveness of mating)
Frequency of treatment:
Daily, 7 days/week
Dose / conc.:
83 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
based on active ingredient
No. of animals per sex per dose:
12 males and 12 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
The experimental period involved 27 days of acclimatization (including 14 days for examination of estrous cycle) and 42-59 days treatment/observation period (depending on the effectiveness of mating) and necropsy days.
The day of first treatment is considered as Day 0 of examination.
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.
Detailed examinations were made at the times of weekly weighing, prior to and during the mating and until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week but before the blood sampling. General physical condition and behavior of animals were tested. A modified Irwin test was performed.

BODY WEIGHT
The body weight of all parental animals was determined 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).

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

SERUM THYROID HORMONES
Clinical pathology examinations were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia.
Blood samples were collected for possible determination of serum levels of thyroid hormones (T4 and TSH).
Blood samples for determination of serum levels of thyroid hormones (T4 and TSH) were collected from animals as follows: from 2-9 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter); from all dams and 2-7 pups per litter on post-partal/post-natal day 13; from all parent male animals at termination on Day 42.
Parameters measured: Thyroxine – free Tetra-iodothyronine and Thyroid-stimulating hormone.

HAEMATOLOGY
Clinical pathology examinations were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia.
Blood samples for hematology measurements were collected in tubes containing K3EDTA and tubes were filled up to the final volume (marked on the tubes). Blood were stored at 2-8 °C until analysis by Siemens ADVIA120.
Parameters measured: White Blood Cell (leukocyte) count, Red Blood Cell (erythrocyte) count, Hemoglobin concentration, Hematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Hemoglobin, Mean Corpuscular (erythrocyte) Hemoglobin Concentration, Platelet (thrombocyte) count, Reticulocytes, Differential† white blood cell count.

BLOOD COAGULATION
Clinical pathology examinations were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia.
Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation 3.2 %. Tubes were filled up to the final volume (marked on the tubes). Blood were centrifuged at 2500 rpm for 15 minutes within 20-30 minutes after the sampling. Supernatant plasma samples were stored at 2-8 °C and measured.
Parameters measured: Activated partial Thromboplastin Time, Prothrombin Time.

CLINICAL CHEMISTRY
Clinical pathology examinations were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection. Blood samples were harvested from the retro-orbital venous plexus under Isofluran anesthesia.
Blood samples collected for clinical chemistry measurements were drawn in tubes Vacuette 2.5 ml Z Serum Sep C/A. At least 1.0 ml blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. Serum samples were stored at 2-8 °C and measured.
Parameters measured: Alanine Aminotransferase activity, Aspartate Aminotransferase activity, Total Bilirubin concentration, Creatinine concentration, Urea concentration, Glucose concentration, Cholesterol concentration, Sodium concentration, Potassium concentration, Albumin concentration, Total Protein concentration.

EXAMINATION OF 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.

OBSERVATION OF 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 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).
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”) and were subjected to gross necropsy as follows: parental male animals after the optionally extended post-mating period on Day 42; dams on post-partum day 13 or shortly thereafter (Days 51, 54, 55, 57, 58 or 59).

GROSS NECROPSY
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 implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate and seminal vesicles with coagulating glands, adrenal glands and pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.

ORGAN WEIGHTS
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.
In addition, for five males and females randomly selected from each group, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.
The thyroid weight will be determined – if relevant – after fixation.
Paired organs were weighed together.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (750 mg/kg bw/day).
In addition, kidneys of three male animals (no. 306, 311, 312 in the mid dose group), and uterus of three female animals (no. 229 in the low dose group, no. 322, 330 in the mid dose group) was processed and examined due to macroscopic findings (pyelectasia in the kidneys, hydrometra in the uterus).
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related clinical signs in any group, i.e. the parental animals (male and female) exhibited normal behavior and physical condition with no abnormalities in the control and at 83, 250 or 750 mg/kg bw/day at the daily or at the detailed weekly clinical observations.
Noisy breathing was noted for one male animal at 750 mg/kg bw/day (1/12) on Day 28. Piloerection and slightly decreased activity were observed in one male animal at 750 mg/kg bw/day (1/12) between Days 34-36 and 34-41, respectively. Signs of bites around the vaginal orifice were noted for one control female animal (1/12) between gestation days 0 and 6.
These observations were considered to be individual signs and some of these were detected at the weekly detailed clinical observations, too (piloerection: 1/12 on Day 34; decreased activity: 1/12 on Days 34 and 41).
Mortality:
no mortality observed
Description (incidence):
There was no mortality at 83, 250 or 750 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 undisturbed in male and female animals at 83, 250 or 750 mg/kg bw/day during the entire treatment period.
Statistically significant difference with respect to their control was detected at the higher mean body weight gain in male animals at 83 mg/kg bw/day between Days 13 and 20 and at 250 mg/kg bw/day between Days 34 and 41.
The mean body weight gain was lower than in the control group in male animals at 750 mg/kg bw/day during the entire observation period reaching statistical significance between Days 20 and 27 and for the study overall (between Days 0 and 41).
These slight changes in the body weight gain did not result in significant changes in the body weight of male animals in these groups, therefore were considered to be toxicologically not relevant.
In female animals at 250 mg/kg bw/day, statistically significantly higher mean body weight was detected when compared to the control on lactation day 0. This slight and transient change in the mean body weight was considered to be indicative of biological variation without toxicological relevance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related changes in the mean daily food consumption of male or female animals at 83, 250 or 750 mg/kg bw/day.
Statistical significance with respect to the control was detected in male animals administered with 750 mg/kg bw/day at the slightly lower mean daily food consumption during the first week of the treatment period.
The mean daily food consumption was comparable in the control and test item treated male animals at 83, 250 or 750 mg/kg bw/day during the post mating period and in female animals at 83, 250 or 750 mg/kg bw/day during the course of the pre-mating, gestation and lactation periods.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematological evaluation did not reveal adverse test item related changes in the examined parameters at 83, 250 or 750 mg/kg bw/day.
The examined hematological parameters were comparable in male and female animals in the control and 83 and 750 mg/kg bw/day groups.
In male animals at 250 mg/kg bw/day, statistical significance was noted for the slightly lower red blood cell (erythrocyte) count (RBC), mean concentration of hemoglobin (HGB) and mean hematocrit value (HCT); in female animals at 250 mg/kg bw/day, statistical significance was noted for the slightly lower mean prothrombin time (PT).
All these slight changes were considered to have no toxicological relevance in the lack of dose dependence and due to the minor degree.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related adverse effects on the examined clinical chemistry parameters at 83, 250 or 750 mg/kg bw/day (male or female).
Statistical significance with respect to the control was observed at the lower mean activity of aspartate aminotransferase (AST) in male animals at 83 and 250 mg/kg bw/day. Although the mean group value was comparable to the control, the activity of aspartate aminotransferase was slightly elevated in one male animal (1/5) at 750 mg/kg bw/day, however there were no related changes in the organ weight, necropsy or histopathology findings. Therefore, slight change in the AST of this animal was considered to be individual one
The mean concentration of creatinine (CREA) slightly exceeded the control value in male animals at 750 mg/kg bw/day group..
These statistically significant differences in male animals with respect to their controls were considered to have no toxicological importance because all values – mean and individual – remained within the historical control ranges (CREA, AST) or there was no dose relevance (AST). Moreover, there were no histological changes in related organs.
The examined clinical chemistry parameters were similar in female animals of the control and 83, 250 and 750 mg/kg bw/day groups.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation battery did not demonstrate any alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (selected male and female, control, 83, 250 or 750 mg/kg bw/day groups, on Day 42).
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant differences between the control and test item treated male or female animals at any dose level (83, 250 and 750 mg/kg bw/day) in the examined organ weights.
Compared to their control, statistical significance was detected at the slightly lower mean weight of seminal vesicles (absolute and relative to body weight) in male animals at 83 mg/kg bw/day.
The weights of the examined organs were comparable in male animals in the control and 250 mg/kg bw/day group.
Statistical significance was observed for the slightly lower mean fasted body weight and at the lower mean weight of seminal vesicles (absolute and relative to body and brain weights) in male animals at 750 mg/kg bw/day.
The differences with respect to the control in male animals were judged to be toxicologically not relevant as there was no dose relevance and the individual values were mostly within the historical control ranges (with the exception of the thymus of one animal).
In the female animals, there were no statistically or biologically significant differences between the control and the 83 or 250 mg/kg bw/day groups in the weights of the examined organs at the end of the treatment period.
Statistical significance was observed for the higher kidney weights (absolute and relative to body weight) in female animals at 750 mg/kg bw/day.
The difference of mean kidney weight in female animals in the high dose was considered to have no or little toxicological meaning as individual values were within the historical control range and it was not associated with any clinical pathological or histopathological findings.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Specific macroscopic alterations indicative of test item effect was not observed in the organs or tissues at any dose levels 83, 250 or 750 mg/kg bw/day at the necropsy.
There were no macroscopic findings in the organs or tissues in male animals in the control and 83 mg/kg bw/day groups (12/12, each).
Pyelectasia – one or both sides – was observed in three male animals (3/12) at 250 mg/kg bw/day. In one male animal at 750 mg/kg bw/day, pea-sized thymic hematoma and one side pyelectasia was noted (1/12).
Pyelectasia is a common macroscopic finding in experimental rats of this strain with similar age. It was without degeneration, inflammation or fibrosis therefore this finding was considered as slight individual lesion without pathological significance.
Hematoma in the thymus was probably due to circulatory disturbance developed during the exsanguination procedure.
Slight or moderate hydrometra was noted for four dams (1/12 in the control, 1/12 at 83 mg/kg bw/day and 2/12 at 250 mg/kg bw/day). There were no macroscopic findings in the organs or tissues in female animals at 750 mg/kg bw/day (12/12).
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) these findings were 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 750 mg/kg bw/day).
Histopathological examinations did not reveal any test item related alterations in the organs or tissues of selected male and female animals at the highest dose (750 mg/kg bw/day).
In the male animals belonging to the treated and control groups (12/12 at 750 mg/kg bw/day; 12/12 control), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 at 750 mg/kg bw/day; 12/12 control), the ovaries, uterus, cervix and vagina had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortical region of ovaries contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes, and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
Dilatation of uterine horns was observed in four dams as follows: 1/12 control; 1/1 at 83 mg/kg bw/day; 2/2 at 250 mg/kg bw/day. This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and is in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 control male; 1/5 male and 1/5 female at 750 mg/kg bw/day), acute hemorrhage in the lungs (1/5 male and 1/5 female in the control group; 1/5 male at 750 mg/kg bw/day) and thymic hemorrhage (1/5 male at 750 mg/kg bw/day) were detected sporadically. The pulmonary emphysema and hemorrhages in the lungs and thymus were considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (1/5 male and 1/5 female in the control group; 1/5 male at 750 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Slight pyelectasia (one side or both sides) was observed in three male animals (3/3 at 250 mg/kg bw/day and 1/12 at 750 mg/kg bw/day). This finding without degeneration, inflammation or fibrosis is considered as slight individual lesion without pathological significance.
There was no morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary system, the immune system, the hematopoietic system, the skeleton, the muscular system, the eyes, the integumentary system, the male and female reproductive system or the central, or peripheral nervous system in the animals.
The cyto-morphology of endocrine glands was the same in the control and high dose treated animals.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant differences with respect to the control in the TSH thyroid hormone levels in parental male animals or in offspring sampled on postnatal day 13 at any dose levels.
Statistically significant difference with respect to the control was noted for the lower thyroid hormone (free T4) level in parental male animal at 750 mg/kg bw/day. The individual values were within or marginal to the historical control range (2.72 ± 0.39 ng/dl; n = 96; min = 1.91 ng/dl; max = 3.86 ng/dl), therefore this difference was considered to be toxicologically not relevant. Moreover, the TSH levels revealed no differences when compared to the control and there were no findings in regards to histopathological examinations of respective organs, there is no indication that hypothalamic–pituitary–thyroid axis was affected by the test item.
Dose descriptor:
NOAEL
Effect level:
>= 750 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: systemic toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
NOAEL (subsatnce) (rat, male/female) ≥ 750 mg/kg bw/day, based on systemic toxicity
Executive summary:

The toxic potential of test item to cause effects on reproduction and development was investigated performing a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to the testing guideline OECD 422.

The substance was administered orally (by gavage) to rats at doses of 83, 250 and 750 mg/kg bw/day by active ingredient (corresponding to uncorrected dose of 92.7, 279.3 and 838 mg/kg bw/day, respectively). The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study and the concentrations in the dosing formulations were found to varie within the range of 98 and 105 % in comparison to the nominal values, confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-59 days). The dams were allowed to litter and rear their offspring up to day 13 post-partum.

No deaths occurred during the course of study (male and female). Clinical signs of systemic toxicity were not detected at any dose level and the behavior and physical condition of the animals was not impaired at each dose level.

Test item related changes in the body weight or body weight gain were not detected, as well as the body weight development.

Hematological evaluation did not reveal adverse test item related changes in the examined parameters, as well as the examined clinical chemistry parameters.

There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose.

Macroscopic findings related to the effect of the test item were not found in male and female animals at any dose level. The examined organ weights of animals selected for toxicity examinations were comparable to the control.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes; no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 750 mg/kg bw/day groups were observed.

Conclusion

Under the conditions of the study, the test item 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.

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.

NOAEL for systemic toxicity of male/female rats ≥ 750 mg/kg bw/day

NOAEL for reproductive performance of male/female rats ≥ 750 mg/kg bw/day

NOAEL for F1 Offspring ≥750 mg/kg bw/day

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

Additional information

The toxic potential of test item to cause effects on reproduction and development was investigated performing a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, according to the testing guideline OECD 422.

The substance was administered orally (by gavage) to rats at doses of 83, 250 and 750 mg/kg bw/day by active ingredient (corresponding to uncorrected dose of 92.7, 279.3 and 838 mg/kg bw/day, respectively). The concentration of the test item in the dosing formulations administered to the animals was checked two times during the study and the concentrations in the dosing formulations were found to varie within the range of 98 and 105 % in comparison to the nominal values, confirming the proper preparation of the dosing formulations.

All animals of the parent (P) generation were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before the necropsy (altogether for 42 days). Females were additionally exposed through the gestation period and up to lactation days 13-15, i.e. up to the day before necropsy (altogether for 51-59 days). The dams were allowed to litter and rear their offspring up to day 13 post-partum.

No deaths occurred during the course of study (male and female). Clinical signs of systemic toxicity were not detected at any dose level and the behaviour and physical condition of the animals was not impaired at each dose level.

Test item related changes in the body weight or body weight gain were not detected, as well as the body weight development.

Haematological evaluation did not reveal adverse test item related changes in the examined parameters, as well as the examined clinical chemistry parameters.

There were no test item related changes in the serum thyroid hormone (T4 and TSH) levels at any dose.

Macroscopic findings related to the effect of the test item were not found in male and female animals at any dose level. The examined organ weights of animals selected for toxicity examinations were comparable to the control.

Histopathological examinations of the selected organs (ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland) did not reveal any test item related changes; no pathologic changes in the examined organs or tissues of randomly selected male or female animals in the control or 750 mg/kg bw/day groups were observed.

Justification for classification or non-classification

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

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

In the specific case, the No Observed Effect Level was established to be equal/higher than 750 mg/kg bw/day, on the basis of the results from the subacute study on rats.

Therefore, the substance does not meet the criteria to be classified for repeated dose toxicity, according to the CLP Regulation (EC) No 1272/2008.