5',5'''-[ethylenebis(p-phenyleneazo)]bis[1',2'-dihydro-6'-hydroxy-4'-methyl-2'-oxo-1,3'-bipyridinium] dilactate

Endpoint summary

Description of key information

NOAEL (sub-acute, male, rat) = 83 mg/kg bw/day

NOAEL (sub-acute, female, rat) < 83 mg/kg bw/day; LOAEL = 83 mg/kg bw/day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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 August 29th to December 07th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

July 2000

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. 1103 Budapest.
- Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study. Only healthy animals were used for the study. Healthy status was certified by the breeder.
- Females: nulliparous and non-pregnant.
- Age at study initiation: 90 – 92 days.
- Weight at study initiation: males 345 – 433 g ; females 198 – 242 g. The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: before mating: 2 animals of the same sex/cage; during the mating 1 male and 1 female / cage. Pregnant females were individually house, while males after mating were housed 2 animals / cage. Type III polypropylene/polycarbonate (22 × 32 × 19 cm).
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice, ad libitum. Food was changed at weekly intervals.
- Water: tap water, ad libitum. Fresh drinking water was given daily.
- Acclimation period: 27 days.

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 172-174. Budapest, H-1138 Hungary).

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: above 10 air-exchanges/ hour by a central air-condition system.
- Photoperiod: artificial light, from 6 a.m. to 6 p.m.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

FORMULATION
Formulations were prepared beforehand not longer than for four days and stored at room temperature until use.

TREATMENT
- Volume: a constant treatment volume of 5 ml/kg body weight was administered in all groups.
- Administration: 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.

DOSES CHOICE
The dose levels were chosen on the basis of the results of a preliminary dose range finding study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations was performed twice during the study. Five aliquots 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 100 % and 106 % in comparison to the nominal values.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/ml and 98 % at ca. 200 mg/ml).
Test item proved to be stable in distilled water at the intended concentrations at room temperature for four days.

Duration of treatment / exposure:

42-65 days (depending on the effectiveness of mating)

Frequency of treatment:

7 days/week

Dose / conc.:

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

Remarks:

as active ingredient, corresponding to 100 mg/kg bw/day

Dose / conc.:

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

Remarks:

as active ingredient, corresponding to 300 mg/kg bw/day

Dose / conc.:

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

Remarks:

as active ingredient, corresponding to 1000 mg/kg bw/day

No. of animals per sex per dose:

12 animals/sex in the control and dose groups

Control animals:

yes, concurrent vehicle

Details on study design:

SCHEDULING
Dosing of both sexes begun after 27 days acclimatization – including 13 days pre-treatment estrous cycle examination – and was continued up to and including the day before the necropsy.
The mating phase started after 14-days treatment (pre-mating) period.
Male animals were dosed for 42 days (14 days pre-mating and 1-16 days mating plus 12-27 days of post-mating period; until the necessary number of pregnant female animals was evident); then they were sacrificed.
Females were dosed for 14 days pre-mating, through 1-16 days mating period and throughout pregnancy and at least up to and including day 12 post-partum or the day before sacrifice.
The day of birth (viz. when parturition is complete) was defined as day 0 post-partum.

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.

NEUROBEHAVIOURAL EXAMINATION
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.
Additionally, the body weight of female animals was determined daily because of body weight loss and for ensuring a propped dosing. These values were recorded bot not reported.
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 given and non-consumed diet with an accuracy 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 12 or 13 for female animals).

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 CP® anesthesia.
Parameters: Alanine Aminotransferase activity (ALT), Aspartate Aminotransferase activity (AST), Total Bilirubin concentration (TBIL), Creatinine concentration (CREA), Urea concentration (UREA), Glucose concentration (GLUC), Cholesterol concentration (CHOL), Sodium concentration, Potassium concentration, Albumin concentration (ALB), Total Protein concentration (TPROT).

THYROID HORMONES
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 CP® anesthesia.
Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH).
Blood samples were collected from all dams on post-partal/post-natal day 13 and from all parent male animals at termination on Day 42.
Parameters were measured: Thyroxine – free Tetra-iodothyronine (FT4), Thyroid-stimulating hormone (TSH).

HAEMATOLOGY
Blood samples were collected 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 CP® anesthesia.
Parameters: White Blood Cell (leukocyte) count (WBC), Red Blood Cell (erythrocyte) count (RBC), Hemoglobin concentration (HGB), Hematocrit (relative volume of erythrocytes, HCT), Mean Corpuscular (erythrocyte) Volume (MCV), Mean Corpuscular (erythrocyte) Hemoglobin (MCH), Mean Corpuscular (erythrocyte) Hemoglobin Concentration (MCHC), Platelet (thrombocyte) count (PLT), Reticulocytes (RET), Differential white blood cell count, Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT).

Sacrifice and pathology:

SACRIFICE
All animals were euthanized by exsanguination after verification of deep narcosis by Isofluran CP®. All animals were subjected to gross necropsy as follows: parental male animals after the post-mating period on Day 42; dams on post-partum day 13 or shortly thereafter (between Days 51 and 65); 2 dams at 834 mg/kg bw/day were euthanized on lactation day 6 as no living pups remained; non-pregnant and not mated females on Days 42 or 51.

GROSS PATHOLOGY
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 and oviduct, 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 and from one male and one female pup per litter for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4 % buffered formaldehyde solution (except testes and epididymides) for five male and five female animals randomly selected from each group: 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), 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), mammary gland, 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 and 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.

HISTOPATHOLOGY
Detailed histological examination was performed on the ovaries, uterus with cervix and oviduct, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Histological examination of these organs was performed in non-pregnant and not mated female animals and their mating partner in the low and mid dose groups (1/12 male at 83 mg/kg bw/day; 3/12 male and 2/12 female at 250 mg/kg bw/day).
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (834 mg/kg bw/day) groups.
Spleen and kidneys were processed and evaluated histologically in selected animals in the low and mid dose groups based on histology findings at 1000 mg/kg bw/day.
In addition, liver, spleen, kidneys, uterus with cervix and oviduct, vagina, skin and stomach were processed histologically in animals showing macroscopic findings in these organs at the necropsy: skin 2/12 male at 83 mg/kg bw/day; kidneys 1/12 female at 83 mg/kg bw/day; 1/12 male and 1/12 female at 250 mg/kg bw/day; liver 2/12 female at 250 mg/kg bw/day; spleen 3/12 female at 83 mg/kg bw/day, 1/12 male and 2/12 female at 250 mg/kg bw/day; stomach 1/12 male at 250 mg/kg bw/day; uterus 1/12 female at 250 mg/kg bw/day; sexual organs of one male and one female animal at 250 mg/kg bw/day were processed due to technical mistake.
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.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs of systemic toxicity related to the test item were detected in some animals at 250 mg/kg bw/day (piloerection, decreased activity and hunched back in single male) and at 834 mg/kg bw/day (piloerection, decreased activity in two male animals and in one dam, noisy breathing or diarrhea activity in one male and in one dam) dose levels at the daily and weekly clinical observations.
The stool was soft in each male and female animal at 83 mg/kg bw/day from Day 5 and paler than normal and soft in each male and female animal at 250 and 834 mg/kg bw/day during the entire observation period (from Day 1 up to and including last day of the treatment).
The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.
The control male animals exhibited normal behavior and physical condition with no abnormalities at the daily or at the detailed weekly clinical observations.

At 83 mg/kg bw/day, soft stool was observed in the bedding material for each male animal from Day 5 up to the last treatment day (Day 41). Alopecia was detected in two male animals at 83 mg/kg bw/day (2/12) on the forelimbs, scrotum and thigh (1/12) and between the ears and under the right ear (1/12) daily from Day 13 to Day 41 and at the weekly clinical observations.
At 250 mg/kg bw/day, decreased activity, piloerection and hunched back were observed in one male animal (1/12) daily from Day 23/27 to Day 33 or 41 and weekly at the detailed observations. Pale than normal and soft stool was seen in the bedding material of male animals (12/12) from Day 1 to termination of the treatment.
Pale than normal and soft stool (12/12) was observed in male animals at 834 mg/kg bw/day during the entire treatment period (From Day 1 to Day 41). Piloerection (2/12), decreased activity (2/12), noisy breathing (1/12) and diarrhea (1/12) were observed in two male animals at 834 mg/kg bw/day at the daily and at the weekly clinical observations.
There were no clinical signs in female control animas during the entire observation period (pre-mating, post-mating, gestation and lactation periods).
In the female animals, soft stool (12/12 at 83 mg/kg bw/day), paler than normal and soft stool (12/12 at 250 and 834 mg/kg bw/day) were observed from Day 1 and Day 5 respectively, up to the termination of the study (pre-mating, post-mating, gestation or lactation periods).

One non-pregnant female at 250 mg/kg bw/day showed piloerection and decreased activity daily between Days 27/28 and 37, and at the weekly clinical observations on Days 28 and 35.
One dam at 834 mg/kg bw/day showed piloerection and decreased activity daily during lactation days 2-5 and at the detailed weekly clinical observation on lactation day 4.
The color of stools was indicative of presence of the test item or its metabolite(s) in the gastro-intestinal tract.

Mortality:

no mortality observed

Description (incidence):

There was no test item related mortality at 83, 250 or 834 mg/kg bw/day groups during the course of study (male and female).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was depressed in male animals at 83, 250 and 834 mg/kg bw/day during the entire treatment period and in female animals at 83, 250 and 834 mg/kg bw/day during the first week of the treatment and during the gestation period. However, at 83 mg/kg bw/day the difference with respect to the control in the mean body weight was low (< 10 %) in male and female animals, thus, the toxicological relevance of the finding at this dose level is questionable.
Statistical significance with respect to the control was detected at the slightly lower mean body weight of male animals at 83 mg/kg bw/day on Days 20 and 41, at 250 and 834 mg/kg bw/day from Day 7 up to the termination the study (on Days 7, 13, 20, 27, 34 and 41).
The mean body weight gain was below the control value in male animals at 83, 250 and 834 mg/kg bw/day in the most cases reaching statistical significances and in several case during the treatment period: between Day 0-7, 7-13, 13-20 and between Days 0 and 41 at 83 mg/kg bw/day; between Days 0-7, 13-20, 20-27, 27-34 and between Days 0 and 41 at 250 mg/kg bw/day; between Days 0-7, 27-34 and between Day 0 and 41 at 834 mg/kg bw/day.
The mean body weight was comparable in the control and at 83 and 250 mg/kg bw/day groups in female animals during the pre-mating period; slightly lower mean body weight was observed in female animals at 834 mg/kg bw/day compared to the control on Day 7 during the pre-mating period when compared to the control.
During the gestation period, the mean body weight of dams was lower than in the control group reaching statistical significances at 83 mg/kg bw/day on gestation day 21, at 250 and 834 mg/kg bw/day on gestation days 7, 14 and 21.
The mean body weight of dams was also lower than in the control group at 83 mg/kg bw/day on lactation day 0 and at 250 and 834 mg/kg bw/day on lactation days 0, 4 and 13.
There were no statistical differences in the mean body weight gain between the control and 83 or 250 mg/kg bw/day groups of female animals during the pre-mating period. Statistical significance was observed at the slightly lower mean body weight gain (between Days 0 and 7) and at the higher mean body weight gain (between Days 7 and 13) of female animals administered with 834 mg/kg bw/day comparing to the control.
The mean body weight gain was lower than in the control group during the gestation period (between gestation day 0 ad 21) at 83 mg/kg bw/day, at 250 mg/kg bw/day (between gestation days 0-7, 7-14, 14-21, 0-21) and at 834 mg/kg bw/day (between gestation days 7-14, 14-21 and for the study overall).
During the lactation period, the mean body weight gain was similar in the control and test item administered animals at 83, 250 and 834 mg/kg bw/day groups. Although, the body weight of one dam significantly decreased after delivery at 834 mg/kg bw/day (between lactation days 0 and 4).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was significantly reduced in male and female animals at 250 and 834 mg/kg bw/day.
The mean daily food consumption was comparable in the control and at 83 mg/kg bw/day in male animals during the entire treatment period.
Statistically significant difference with respect to the control was detected at the lower mean food consumption in male animals at 250 mg/kg bw/day between Days 0 and 7 and at 834 mg/kg bw/day between Days 0 and 7 and between Days 27 and 34.
The mean daily food consumption of female animals was lower than in the control group in the most cases during the pre-mating and gestation period at 83, 250 and 834 mg/kg bw/day reaching statistical significances in several cases: at 83 mg/kg bw/day during the entire pre-mating period and between gestation days 7 -14, 14-21; at 250 mg/kg bw/day between pre-mating days 0-7, between gestation days 7-14, 14-21 and at 834 mg/kg bw/day between pre-mating days 0-7, between gestation days 14-21.
During the lactation period, the mean daily food consumption was similar to the control at 83 mg/kg bw/day and statistical significance was detected at the lower mean food consumption at 250 and 834 mg/kg bw/day between lactation days 4 and 13.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological evaluation revealed changes in percentage of reticulocytes in female animals at 83, 250 and 834 mg/kg bw/day along with changes in red blood cell parameters (red blood cell count, hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin content) at 834 mg/kg bw/day.
Slight elevation of platelet count in male animals at 834 mg/kg bw/day might be related to the test item effect. However, there were no accompanying changes in the investigated parameters (PT, APTT), therefore toxicological importance of this findings is questionable.

All examined hematological and blood coagulation parameters were comparable with the control in male animals at 83 and 250 mg/kg bw/day.
At 834 mg/kg bw/day, the mean platelet count (PLT) was slightly but statistically significantly higher than in the control group in male animals.
In the female animals at 83 mg/kg bw/day, statistically significances were observed at the slightly higher mean corpuscular volume (MCV), at the higher mean percentage of reticulocytes (RET) and at the slightly shorter mean prothrombin time (PT) when compared to the control.
At 250 mg/kg bw/day, higher mean percentage of lymphocytes (LYM), lower mean percentage of monocytes (MONO), lower mean corpuscular hemoglobin concentration (MCHC), shorter mean prothrombin time and significantly higher mean percentage of reticulocytes were detected with respect to their control in female animals.
In female animals at 834 mg/kg bw/day, with respect to their control, lower mean percentage of monocytes, mean red blood cell count (RBC), hemoglobin concentration (HGB) and mean corpuscular hemoglobin concentration as well as higher mean corpuscular volume, higher mean corpuscular hemoglobin content (MCH) and significantly elevated mean percentage of reticulocytes were observed.
Slight elevation of platelet count in male animals at 834 mg/kg bw/day was mainly due to the significantly higher number of platelet count in one animal (no. 411) along with slightly elevated white blood cell count (WBC) and percentage of neutrophil granulocytes and reduced percentage of lymphocytes. A test item influence cannot be excluded entirely, however, there were no accompanying changes in the investigated parameters (PT, APTT) as platelet maintain the hemostasis. Therefore, toxicological importance of this findings is questionable.

The elevated mean percentage of reticulocytes in female animals at 83, 250 and 834 mg/kg bw/day was in connection with the test item effect along with changes of some red blood cell parameters mainly at 834 mg/kg bw/day (MCV, RBC, HGB, MCH and MCHC).
Other changes in female animals at 83 and 250 mg/kg bw/day – with respect to their control – were with minor degree or not related to doses (LYM, MCV, MCHC, PT). Therefore, these findings in female animals were considered to be toxicologically not relevant.
Monocytopenia – detected at 250 and 834 mg/kg bw/day – usually has no diagnostic significance.

SERUM THYROID HORMONE LEVELS
Statistical significances with respect to the control was detected at the lower mean FT4 level in the parental male animals at 83, 250 and 834 mg/kg bw/day independently from doses. There were no changes in the TSH levels of these animals.
In the female animals, statistical significances were noted for the lower mean FT4 levels at 83.4 and 834 mg/kg bw/day when compared to the control. There were no changes in the TSH levels of these animals.

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 834 mg/kg bw/day (male or female).

In male animals, statistical significances with respect to the control were observed at the slightly lower mean sodium (Na+) concentration at 83 mg/kg bw/day and at the slightly lower mean concentration of glucose (GLUC) at 250 g/kg bw/day.
Slight changes in total bilirubin concentration in female animals at 834 mg/kg bw/day might be related to changes in red blood cell parameters. However, values were well within the historical control range.
There were no statistically significant differences between the control and 834 mg/kg bw/day treated male animals at the investigated clinical chemistry parameters. However, the total bilirubin (TBIL) level was markedly elevated in male animal showing changes in PLT, WBC, LYM and NEU at 834 mg/kg bw/day.
In the female animals at 83 mg/kg bw/day, the mean concentration of sodium was lower than in the control group.
In the female animals administered with 250 mg/kg bw/day, statistical significance with respect to the control was observed at the higher mean concentration of cholesterol (CHOL) the lower mean creatinine concentration (CREA).
The mean concentration of total bilirubin and cholesterol was above the control value in female animals at 834 mg/kg bw/day.

Changes in concentration of total bilirubin were related to doses in female animals at 83, 250 and 834 mg/kg bw/day, reaching statistical significance at the high dose only. However, the individual values remained well within the historical control ranges.
The statistically significant differences in Na+, GLUC, CREA, CHOL with respect to their controls were considered to have little or no toxicological importance because values – mean and individual – corresponded to the historical control values, there was no dose relevance or related histopathological alterations.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery did not demonstrate any alterations in the behavior or reactions to different type of stimuli of selected male or female animals in the control, 83, 250 or 834 mg/kg bw/day groups at the end of the treatment period (on Day 42 for male animals and on Day 48 for female animals).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.
Alopecia, was noted for one male animal (1/5 at 83 mg/kg bw/day) between ears and under right ear – approximately 2 cm in width – at the functional observations.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher mean weights of kidneys and spleen (absolute and relative to body and brain weights) were indicative of a test item influence in female animals at 834 mg/kg bw/day and were coinciding with changes in hematology parameters, necropsy and histopathology observations.
Similar changes with respect to their control were also detected at the weights of these organs at the lower dose groups. Additional histopathological evaluation could reveal the nature of these changes in organ weights at 83 and 250 mg/kg bw/day.
The statistically significant differences with respect to control in the absolute organ weights and in organ weights relative to the body weight were partially originated from the slightly lower fasted body weight of animals in the test item treated groups (brain, liver, testes, epididymides and adrenal glands) male and female.

In the male animals at 83 mg/kg bw/day, statistical significances were detected at the slightly lower mean body weight and mean weights of seminal vesicle with coagulating gland and prostate as a whole (absolute and relative to brain weight, both) and at the higher mean weight of liver, kidneys and testes, each relative to body weight.
At 250 mg/kg bw/day (mid dose group), the mean body weight, mean thymus weight and mean weight of seminal vesicle with coagulating gland and prostate as a whole (absolute and relative to brain weight, each) were lower than in the control, while the mean kidney weights (absolute and relative to body and brain weight) were significantly higher than in the control group in male animals. The mean weight of brain, liver, testes and epididymides each relative to body weight were higher in male animals in mid dose group when compared to the control.
In the male animals at 834 mg/kg bw/day, statistical significances with respect to the control were observed at the lower mean body weight, mean thymus weight and mean weight of seminal vesicle with coagulating gland and prostate as a whole (absolute and relative to brain weight, each); in addition, statistical significances were recorded at the higher mean kidney weights (relative to body and brain weight) as well as at the higher mean weight of brain, liver, testes, epididymides and adrenal glands, each relative to body weight.
In the female animals at 83 mg/kg w/day, higher mean weights of kidneys (relative to body and brain weights) and spleen (absolute and relative to body and brain weight) were detected in comparison with the control.
At 250 mg/kg bw/day, the mean body weight and heart weight (absolute and relative to brain weight) were lower, the mean kidney weight relative to body weight, the mean weight of spleen relative to body weight and brain weight relative to body weight were higher than in the control group in female animals.
In the female animals at 834 mg/kg bw/day, statistical significance was detected at the lower mean body weight (absolute and relative to brain weight), at the lower mean thymus weights (absolute and relative to body and brain weights), at the higher mean kidney and spleen weights (absolute and relative to body and brain weight).

Minor changes in the weights of some organs (kidneys, thymus, seminal vesicles with coagulating gland and prostate) in male animals were considered to be toxicologically not relevant. There were no related findings in the examined clinical pathology parameters, or at the necropsy or histopathological observations.
Changes in the spleen and kidneys weights were considered to be related to the treatment with the test item in female animals at 83, 250 or 834 mg/kg bw/day. Histological examinations of these organs are needed to reveal the nature of these findings in the organ weight in the low and mid dose groups. Slight changes in weights of heart or thymus have no or little biological significance in female animals as there were no supporting histopathological lesions in these organs at 834 mg/kg bw/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Specific macroscopic alterations indicative of test item effect were detected in female animals: enlarged or black colored spleen at 83, 250 or 834 mg/kg bw/day and uneven surface of the kidneys at 834 mg/kg bw/day were observed at the necropsy.

Yellowish colored content in the stomach or intestines in some male animals at 250 and 834 mg/kg bw/day was indicative of presence of the test item or its metabolite(s) in the gastrointestinal tract.
Right side pyelectasia was noted for one control male animal (1/12) and there were no macroscopic findings in the organs or tissues in other ones (11/12) in the control group.
At 83 mg/kg bw/day, alopecia was observed in two male animals (2/12) on the forelimbs, on the right side scrotum and thigh as well as between the ears.
At 250 mg/kg bw/day, undernourishment, uneven surface of kidneys, right side pyelectasia and dark colored spleen were noted for one male animal (1/12). Besides these, hemorrhage in the stomach (1/12) and yellowish content in the stomach and intestines (3/12) were observed in some male animals at 250 mg/kg bw/day.
One side pyelectasia (2/12, right side and left side) and yellowish content in the stomach (4/12) and intestines (3/12) were observed in some male animals at 834 mg/kg bw/day.
In one control dam, both sides pyelectasia (1/11) was seen and there were no macroscopic findings in the organs or tissues in other ones (10/11) and in non-pregnant female animal in the control group.
At 83 mg/kg bw/day, both sides pyelectasia (1/12) and larger than normal spleen (3/12) were observed in dams.
At 250 mg/kg bw/day, both sides pyelectasia (1/10), black colored and enlarged spleen (2/10), food filled stomach (1/10), slight or moderate hydrometra (2/10) and Hernia diaphragmatica including small part of liver (2/10) were observed in dams.
There were no macroscopic findings in the organs or tissues in not mated female animal (1/1) and food filled stomach was noted for non-pregnant female animal (1/1) at 250 mg/kg bw/day, both later.
In high dose (834 mg/kg bw/day) treated dams, uneven surface of the kidneys (4/11), enlarged and black colored spleen (11/11), undernourishment (1/11) and food filled stomach (4/11) were detected.
In non-pregnant female animal of the high dose group (1/1), the stomach was fulfilled with food, the surface of kidneys was uneven and renal pelvis was dilated (pyelectasia, both sides).

Macroscopic findings in spleen (enlargement, black colored) and kidneys (uneven surface) refer to the test item influence. Enlarged and black colored spleen occurred with high incidence in female animals at 834 mg/kg bw/day and some others at 83 and 250 mg/kg bw/day as well as in single male animal at 250 mg/kg bw/day. Uneven surface of the kidneys was observed in high dose treated female animals and in one male rat at 250 mg/kg bw/day. Considering the low incidence of these findings in male animals, test item effect cannot be proven with the results of this study.
Hemorrhage in the stomach occurred in one male animal at 250 mg/kg bw/day and was considered to be individual alteration in the stomach mucosa.
The food filled stomach is an unusual sign in experimental rats after overnight fasting, however it was observed in some dams at 250 and 834 mg/kg bw/day.
Pyelectasia is a common finding in this strain of experimental rats and was observed with similar incidence in animals of control and test item treated groups (male and female) in this study.
Alopecia is a species-specific dermal change in experimental rat and was present in two male animals at 83 mg/kg bw/day.
Hydrometra, related to the female sexual cycle and hernia diaphragmatica are also frequent observations in experimental rats.
In the lack of related histopathological alterations (inflammatory or other pathological lesion) the above-mentioned findings – pyelectasia, alopecia, hydrometra, hernia diaphragmatica – were judged to be toxicologically not relevant.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item related lesions detectable by histological examination of the investigated reproductive organs of male animals at 83, 250 or 834 mg/kg bw/day.
Histopathological examinations revealed test item related chronic progressive nephropathy (CPN) and hyperplasia in the spleen (hyperplasia of hematopoietic erythroid, myeloid and lymphoid cells) in female animals at 834 mg/kg bw/day and hyperplasia in the spleen (hyperplasia of hematopoietic erythroid, myeloid and lymphoid cells) in female animals at 83, 250 or 834 mg/kg bw/day.

In the male animals belonging to the control and treated groups (12/12 control; 1/1 at 83 mg/kg bw/day, 4/4 at 250 mg/kg bw/day, 12/12 at 834 mg/kg bw/day), the investigated organs of reproductive system (testes, epididymides, prostate seminal vesicles, coagulating glands) were histologically normal and characteristic on the sexually mature organism in all cases. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa); representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of investigated control and treated animals. The histological picture of prostate, epididymides, seminal vesicles, and coagulating glands was normal in all cases as well.
In the female animals belonging to the treated and control groups (12/12 control; 3/3 at 250 mg/kg bw/day, 12/12 at 834 mg/kg bw/day, including non-pregnant or not mated females), 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 one dam at 250 mg/kg bw/day (1/4). This finding – without inflammatory or other pathological lesions – is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
The histological structure and the cellularity of pituitary with special attention on the cytomorphology and proportion of acidophilic and basophilic cells in the adenohypophysis were the same in the control and treated male and female animals.
Histological examination revealed chronic progressive nephropathy (CPN) in several animals – 1/5 control female; 1/1 male at 250 mg/kg bw/day; 1/5 male and 6/8 female at 834 mg/kg bw/day – in different severity. CPN is an important spontaneous renal disease of the commonly used strains of laboratory rat because it is a serious confounding factor in experimental pathology and toxicology studies. In our study – regarding the higher incidence of CPN in the animals belonging to the 834 mg/kg bw/day treated female group – it cannot be excluded that the high dose of test item was a predisposing factor in the pathogenesis of this renal lesion. There are no details for the explanation of higher incidence of CPN in the female animals as compared to the males.
Hyperplasia – associated with enlargement – of spleen was detected in test item treated groups, mainly in female animals: 1/1 male at 834 mg/kg bw/day; 3/8 female at 83 mg/kg bw/day, 2/7 female at 250 mg/kg bw/day and 11/11 female 834 mg/kg.
The hyperplasia affected all the hematopoietic cells (erythroid, myeloid and lymphoid) in the spleen. In this study, hyperplasia affected only treated animals and was considered to be treatment related lesion. Histological results gave no explanation on the higher incidence in the female animals as compared with the male groups.
In animals selected for full histological examinations, minimal or mild alveolar emphysema in the lungs (1/5 male and 1/5 female in the control, 1/5 female at 834 mg/kg bw/day), were detected sporadically. The pulmonary emphysema is considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure.
Hyperplasia of bronchus associated lymphoid tissue (BALT) in the lungs (3/5 control male; 1/5 female at 834 mg/kg bw/day) is a physiological immune-morphological phenomenon, occurring also in not treated rats and has no toxicological significance.
Erosion in the stomach (1/1 male at 250 mg/kg bw/day) was considered an individual disease.
Focal fibrosis in the Glisson’s capsule of the liver (2/2 female at 250 mg/kg bw/day) may be the consequence of the mechanical irritation due to the diaphragmatic hernia.
Pyelectasia (one or both sides) was observed in several animal: 1/5 male and 2/5 female control; 1/1 female at 83 mg/kg bw/day; 1/1 male and 1/1 female at 250 mg/kg bw/day and 2/5 male and 3/8 female at 834 mg/kg bw/day. This finding without other pathological lesion (degeneration, inflammation, fibrosis) is a common finding in Wistar rats without toxicological significance.
Focal atrophy of hair follicles (reduced in size) in two male animals at 83 mg/kg bw/day (2/2) without inflammatory was in connection with the focal alopecia observed at the necropsy. Abnormal hair follicle atrophy might be due to inflammatory lesion, dermal ischemia or autoimmune conditions, as well.
No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the stomach, the small and large intestines, the liver, the cardiovascular system, the respiratory system, the hematopoietic system, the skeleton, the muscular system, the central, or peripheral nervous system, the eyes, the integumentary system, the reproductive system was observed.
The cytomorphology of endocrine glands were the same in the control and treated animals.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

female

Basis for effect level:

haematology

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Two dams at 834 mg/kg bw/day (no. 426 and 422), were subjected early necropsy on lactation day 6. Therefore, procedures and observations of in-life period for these dams were terminated on lactation day 5.

Two dams at 250 mg/kg bw/day (no. 329 and 331) had to be subjected to necropsy on lactation day 13, therefore, procedures and observations of in-life period for these dams were terminated on lactation day 12.

Conclusions:

NOAEL = 83 mg/kg bw/day (male)
LOAEL = 83 mg/kg bw/day (female)

At 834 mg/kg bw/day, clinical signs with low incidence (piloerection, decreased activity, noisy breathing or diarrhea), depressed body weight development, reduced food consumption were observed in male and female animals.
In the female animals at the same dose level, changes in hematological parameters (reticulocytes, red blood cell count, hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin content) and total bilirubin concentration, macroscopic and microscopic changes in the kidneys (uneven surface, elevated organ weight, chronic progressive nephropathy) and spleen (black color, enlargement, elevated organ weight, hyperplasia) were detected.
At 250 mg/kg bw/day, clinical signs with low incidence (piloerection, decreased activity, hunched back), depressed body weight development, reduced food consumption (male and female), elevated percentage of reticulocytes (female), macroscopic and microscopic changes in the spleen (black color, enlargement, elevated organ weight, hyperplasia) were observed.
83 mg/kg bw/day induced minor changes in the body weight (<10 %) in male and female animals, macroscopic and microscopic changes in the spleen (enlargement, elevated organ weight, hyperplasia) in female animals.

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 83, 250 and 834 mg/kg bw/day by active substance content (corresponding to uncorrected doses of ca 100, 300 and 1000 mg/kg bw/day).

The substance was administered to four groups of Han:WIST rats consisting of 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; the test item concentrations in the dosing formulations varied within the range of 100 % and 106 % in comparison to the nominal values and 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). Dams were additionally exposed through the gestation period and up to and including lactation days 5 (no living pups remained) or 12-15, i.e. up to the day before necropsy (females were administered altogether for 42-65 days).

Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process. Blood samples were collected for possible determination of serum levels of thyroid hormones (FT4 and TSH) from all dams and from 1-7 pups per litter (where it was feasible) at termination on post partum/post-natal day 13 and from all parent male animals at termination (Day 42).

All parental animals were subjected to gross pathology one day after the last treatment. Organs showing macroscopic findings at the necropsy were also processed and examined histologically in animals of the low and mid dose groups.

There was no test item related mortality at any dose level. Clinical signs of systemic toxicity related to the test item were detected with low incidence at 250 mg/kg bw/day and at 834 mg/kg bw/day dose levels in male or female animals at the daily and at the detailed weekly clinical observations. The functional observations did not reveal any test item influence on the behavior or neurological functions of selected animals. The stool was softer than normal in each test item treated animals and paler than normal at 250 and 834 mg/kg bw/day.

The body weight gain and body weight were depressed in male and female animals at 83, 250 and 834 mg/kg bw/day and the mean daily food consumption was reduced in male or female animals at 250 and 834 mg/kg bw/day during the entire study.

Hematological investigation revealed significant elevation in percentage of reticulocytes in female animals at 83, 250 and 834 mg/kg bw/day along with changes in red blood cell parameters (red blood cell count, hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin content) at 834 mg/kg bw/day.

There were no test item related adverse effects on the examined clinical chemistry parameters at any dose level. There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose level in parental male animals.

Enlarged or black colored spleen at 83, 250 or 834 mg/kg bw/day and uneven surface of the kidneys at 834 mg/kg bw/day were observed in female animals at the necropsy. Yellowish colored content in the stomach or intestines in some male animals at 250 and 834 mg/kg bw/day was indicative of presence of the test item or its metabolite(s) in the gastrointestinal tract.

Higher mean weights of kidneys and spleen (absolute and relative to body and brain weights) were indicative of a test item influence in female animals at 834 mg/kg bw/day and were coinciding with changes in hematology parameters, necropsy and histopathology observations. Similar changes with respect to their control were also detected at the weights of these organs (absolute or relative to body or brain weights) in female animals at the lower dose groups (83, 250 mg/kg bw/day).

Histopathological examinations revealed chronic progressive nephropathy (CPN) and hyperplasia in the spleen (hyperplasia of hematopoietic erythroid, myeloid and lymphoid cells) in female animals at 834 mg/kg bw/day. Macroscopic and microscopic changes in the spleen, such as enlargement, elevated organ weight, hyperplasia were seen mainly in female rats.

Conclusion

NOAEL for systemic toxicity of male rats: 83 mg/kg bw/day

NOAEL for systemic toxicity of female rats: < 83 mg/kg bw/day; LOAEL = 83 mg/kg bw/day

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

83 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

good quality and reliability

System:

haematopoietic

Organ:

spleen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test was run according to OECD guideline 422.

There was no test item related mortality at any dose level. Clinical signs of systemic toxicity related to the test item were detected with low incidence at 250 mg/kg bw/day and at 834 mg/kg bw/day dose levels in male or female animals at the daily and at the detailed weekly clinical observations. The functional observations did not reveal any test item influence on the behavior or neurological functions of selected animals. Stools was softer than normal in each test item treated animals and paler than normal at 250 and 834 mg/kg bw/day.

The body weight gain and body weight were depressed in male and female animals at 83, 250 and 834 mg/kg bw/day and the mean daily food consumption was reduced in male or female animals at 250 and 834 mg/kg bw/day during the entire study.

Hematological investigation revealed significant elevation in percentage of reticulocytes in female animals at 83, 250 and 834 mg/kg bw/day along with changes in red blood cell parameters (red blood cell count, hemoglobin concentration, mean corpuscular volume and mean corpuscular hemoglobin content) at 834 mg/kg bw/day.

There were no test item related adverse effects on the examined clinical chemistry parameters at any dose level. There were no test item related changes in the serum thyroid hormone (FT4 and TSH) levels at any dose level in parental male animals.

Enlarged or black colored spleen at 83, 250 or 834 mg/kg bw/day and uneven surface of the kidneys at 834 mg/kg bw/day were observed in female animals at the necropsy. Yellowish colored content in the stomach or intestines in some male animals at 250 and 834 mg/kg bw/day was indicative of presence of the test item or its metabolite(s) in the gastrointestinal tract.

Higher mean weights of kidneys and spleen (absolute and relative to body and brain weights) were indicative of a test item influence in female animals at 834 mg/kg bw/day and were coinciding with changes in hematology parameters, necropsy and histopathology observations. Similar changes with respect to their control were also detected at the weights of these organs (absolute or relative to body or brain weights) in female animals at the lower dose groups (83, 250 mg/kg bw/day).

Specific examinations revealed macroscopic and microscopic changes in the kidneys (uneven surface, elevated organ weight, chronic progressive nephropathy) and spleen (black color, enlargement, elevated organ weight, hyperplasia) in the female animals at 834 mg/kg bw/day; at 250 mg/kg bw/day, macroscopic and microscopic changes in the spleen (black color, enlargement, elevated organ weight, hyperplasia) were observed; at the dose of 83 mg/kg bw/day, macroscopic and microscopic changes in the spleen (enlargement, elevated organ weight, hyperplasia) were seen in female animals.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), substances are classified as specific target organ toxicants following repeated exposure, and are placed in one of two categories, depending on the nature and severity of the effects observed. 

Substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure, are classified in category 1 for target organ toxicity (repeated exposure). Classification in category 1 is applicable when significant toxic effects are observed in a 90-day repeated-dose animal study at concentrations < 10 mg/kg bw/day in oral studies.

Substances which can be presumed to have the potential to be harmful to human health following repeated exposure, based on evidence from animal studies, are classified in category 2. Classification in category 2 is applicable when significant toxic effects are observed in a 90-day repeated-dose animal study at generally concentrations between 10 to 100 mg/kg bw/day in oral studies.

By considering the study duration, i.e. 42 days at minimum, new threshold values for classification may be extrapolated using dose/exposure time extrapolation similar to Haber's rule: classification in category 1 is applicable at concentration < ca. 20 mg/kg bw/day; in category 2 at concentrations between ca. 20 to 200 mg/kg bw/day.

Overall, dose-related effects were seen on rats upon repeated dosing with test substance, mainly on kidney, spleen and hematopoietic system. Similar effectes were seen both in male and female rats, and female rats resulted as more sensitive, with adverse effects already seen at the low dose. In particular, dose-related adverse effects on spleen and reticulocytes count were seen in female rats starting at a dose of 83 mg/kg bw/day; thus this dose was identified as LOAEL. Similarly, dose-related effects were seen in males starting at a dose of 250 mg/kg bw/day.

On these bases, the substance is classified as a Category 2 repeated dose toxicant according to the CLP Regulation (EC 1272/2008).