[No public or meaningful name is available]

Endpoint summary

Description of key information

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

The toxic potential of test item was investigated according to the OECD guideline 422.

In order to obtain first information on the toxic potential of test item in rats, a dose range-finding test was run at three dose levels to allow a dose-setting the dosages for the 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 Wistar rats were administered orally (by gavage) once daily at 0 (vehicle only) 75, 150, 300 and 600 mg/kg bw/day doses. 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. 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. Only colouration of stool was observed at 150, 300 and 600 mg/kg bw/day 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 examined reproductive parameters were not impaired at 600 mg/kg bw/day.

In the main test, the substance was administered orally (by gavage) to rats at doses of 150, 300 and 600 mg/kg bw/day. 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 only finding, attributable to the test item treatment, was reddish-brownish stool in the bedding material, in all male and female. Reddish-brownish colour 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.

Haematological 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 groups 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.

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 Adverse Effect Level was established as equal/higher than 600 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.