Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity (28 days): Test method OECD 422. GLP study. The NOAEL after at least 28 days of oral exposure to test item was determined to be 250 mg/kg bw/day in rats (basis for effect: minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level) .

Repeated dose toxicity (90-days): Test method OECD 408. GLP study. The NOAEL after a 90 days of oral exposure was determined to be 1000 mg/kg bw/day in rats (basis on no overt adverse effects that could be ascribed to the test item administration).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 06, 2013 - March 24, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories, Research Models and Services, Germany GmbH- Age at study initiation: Approx. 10 weeks at starting and 12 weeks at mating.- Weight at study initiation: Males: 362–391 g, Females: 195-225 g - Housing: up to 5 animals per sex and cage type II and/or III polycarbonatem, with lignocel bedding.- Diet (e.g. ad libitum): Ad libitum- Water (e.g. ad libitum): Ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 18.5-23.6 ºC- Humidity (%): 33-68 %- Air changes (per hr): 15-20 air chainges per hour- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(distilled)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals. The amount of the dose volumes was formulated considering the density of the test item and then they were corrected with the purity of the test item in water solution.VEHICLE- Amount of vehicle (if gavage): 1.71 mL/kg bw- Lot/batch no. (if required): 3450611 / 4310612
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations for concentration and homogeneity was performed using validated spectrophotometric method (CiToxLAB study code 12/331-316AN). Control, top, middle and bottom duplicate samples were taken from test item formulations on 3 occasions, during the first and last weeks and approximately midway during the treatment, one set to analyse and the other for back-up. No test item was identified in the control samples. The test item formulation appeared to be homogenous and had actual concentrations of 99-105% of the nominal concentrations, within the 100±10% acceptable range. These results were considered suitable for the study purposes.
Duration of treatment / exposure:
2 weeks before mating, during the mating, and continued up to and including the day before the necropsy.
Frequency of treatment:
Daily, 7 days per week.
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based the previous repeated dose range finding study in the rat (CiToxLAB study code 12/331-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Test item administrated to Wistar rats at 62.5, 250 and 1000 mg/kg bw/day, daily for 7 consecutive days, was not associated with any overt adverse effects that could be clearly ascribed to test item administration other than a transient body weight and food intake effect in the first few days of treatment in males at the High dose. Based on the results, the dose levels selected for the main study were 0, 62.5, 250, 1000 mg/kg bw/day.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: Twice daily for morbidity and mortalityOnce daily for general clinical observations and behavious.DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once before the first exposure, and at least weekly.- Observations: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study. BODY WEIGHT: Yes- Time schedule for examinations: before treatment for randomization purposes, on Day 0, afterwards at least weekly and at termination.FOOD CONSUMPTION:- Animal food consumption was determined by re-weighing the non-consumed diet on Day 7 and then at least weekly.HAEMATOLOGY: Yes- Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Red Blood Cell (erythrocyte), White Blood Cell (leukocyte), Haemoglobin concentration, Haematocrit (relative volume of erythrocytes), Mean Corpuscular (erythrocyte) Volume, Mean Corpuscular (erythrocyte) Haemoglobin, Mean Corpuscular (erythrocyte) Haemoglobin Concentration, Red Cell (erythrocyte) volume, Platelet (thrombocyte) count, Mean Platelet Thrombocyte volume, Reticulocyte count, Neutrophil, Lymphocyte, Monocyte, Basophil, Eosinophil, Large Unstained Cells, Activated Partial Thromboplastin Time, Prothrombin Time.CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: Immediately prior to scheduled necropsy.- Anaesthetic used for blood collection: Yes, pentorbital anaesthesia.- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Blood sugar concentration, Total Bilirubin concentration, Urea concentration, Cholesterol concentration, Creatinine concentration, Phosphorus concentration, Sodium concentration, Potassium concentration, Calcium concentration, Chloride concentration, Total Protein concentration, Albumin concentration, Alb/glob ration, Alanine Aminotransferase activity, Alkaline. Phosphatase – activity, Gamma Glutamyltransferase -activity, Bile acids. URINALYSIS: Yes- Time schedule for collection of urine: Prior to scheduled necropsy- Metabolism cages used for collection of urine: Yes (16 hours)- Animals fasted: Yes- How many animals: 5 rats per sex and per dose (subgroup B)- Parameters checked: Leukocyte, Nitrite, pH, Protein, Glucose, Urobilinogen, Bilirubin, Ketones, ERY Blood, Erythrocytes, Specific Gravity, Sediment, Volume, Colour/Appearance.NEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: During the last exposure week (males on Day 24, females on PND 4).- Dose groups that were examined: 5 rats per sex and per dose (subgroup A)- Battery of functions tested: sensory activity / grip strength / motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: YesAll animals were necropsied. External appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.BODY WEIGHT AND ORGAN WEIGHTS: YesIn all animals, uterus (with and without cervix), vagina, testes, epididymides, prostate, seminal vesicles with coagulating glands, brain. ovaries and pituitary were measured. For 5 rats per sex and group (subgroup B), heart, kidneys, liver, spleen, thymus and adrenals were also weighted. Paired and individual organ weights were summarised. Relative organ weight (to body and brain weight) were calculated.HISTOPATHOLOGY: YesThe weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. For 5 rats per sex and group (subgroup B), the following organs and tissues were preserved: Gross findings, Adrenal glands, Animal identification, Aorta (thoracic and abdominal), Brain, Clitoral gland / Preputial gland, Epididymes, Eye with the optic nerve, Oesophagus, Femur with marrow incl. joint, Heart, Kidney, Large intestine, External lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph nodes, Larynx, Nasopharynx, Ovaries with oviduct, Pancreas, Pituitary, Prostate, Salivary glands, Sciatic nerve, Seminal vesicles (with coagulating glands), Skeletal muscle (quadriceps), Skin/subcutis with mammary, gland area, Small intestine, Spinal cord (cervical, lumbar, and thoracic levels), Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina.Detailed histological examination were performed in the control and high dose groups and all macroscopic findings from all animals.
Statistics:
The statistical evaluation 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) was 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 as feasible.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs related to treatment.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the pre-mating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Compared to control, lower food consumption values, attaining statistical significance were noted for males at 1000 mg/kg (High dose) during the first week of the treatment. These changes were generally correlated with the body weight and body weight gain values. In addition, slightly lower food consumption was observed during the mating period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant differences between the control and any of treated groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Minor changes at 1000 mg/kg bw/day. Higher bile acid concentration, attaining statistical significance were noted in High dose male and female animals. Compared to the control higher potassium and serum urea concentration (Urea) were observed in Mid and High dose female animals. No signs of kidney injury were observed during pathology evaluation.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Minor changes at 1000 mg/kg bw/day. Urobilinogen and urinary bilirubin, calcium oxalate crystals as well as dark yellow discoloration of the urine was presented in high dose males. In high dose females and in one from the mid dose, calcium oxalate crystals were also presented in the urine. Dark yellow discoloration of the urine was observed in one mid dose female. Semi quantitative measurement of protein in the urine showed a slight elevation in high dose, when compared to the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects on organ weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Single or multiple ulcers of the non-glandular mucosa of the stomach (4/12 High dose males and 4/12 High dose females) were observed as treatment-related macroscopic findings. Other minor changes were considered incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related ulcers of the non-glandular mucosa of the stomach were microscopically observed in 4/7 High dose males and 4/7 High dose females, and were in correlation with necropsy. There was no evidence of test item-related histological findings in the High dose animals or macroscopic observations from all groups in the reproductive organs.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Basis for effect level: minor changes in clinical chemistry and urinalysis parameters and ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Based on the minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level, the no adverse effect level (NOAEL) was determined to be 250 mg/kg bw/day in male and female rats after at least 28 days of test item treatment by oral gavage.
Executive summary:

This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECD Guideline 422 following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels, 62.5, 250 and 1000 mg/kg bw/day. The control animals were treated with the vehicle only (distilled water). 12 Male and female Wistar rats per group were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. At termination, necropsy with macroscopic examination was performed. Selected organs were subjected to histopathology examination. No mortality was observed during the study. There were no clinical signs related to treatment. There was no effect of treatment noted during evaluation of the functional observation battery, grip strength, foot splay or motor activity. In males treated at 1000 mg/kg, transient lower body weight and body weight gain values were observed during the pre-mating period. The mean body weight value at termination was comparable to the control. The mean body weight values of females in all test item treated groups were comparable to the control. Single or multiple ulcers of the non-glandular mucosa of the stomach (4/12 High dose males and 4/12 High dose females) were observed as treatment-related macroscopic findings. There was no effect of treatment on organ weights. Test item related microscopic findings were found at 1000 mg/kg bw/day (High dose). In the stomach, ulcers of the non-glandular mucosa were microscopically observed in 4 High dose males and 4 High dose females. There were no microscopic findings related to treatment at 62.5 or 250 mg/kg bw/day. Lower food consumption values were noted for males at 1000 mg/kg during the first week of the treatment. These changes generally correlated with the body weight and body weight gain values. There was no effect of treatment on haematology and blood clotting parameters. Increases were noted in potassium and/or urea and bile acids in high dose males and females. Calcium oxalate crystals were noted in high dose males and females, with increases in urobilinogen and bilirubin in males.

Based on the minor changes in clinical chemistry and urinalysis parameters and the ulcers of the non-glandular mucosa of the stomach at 1000 mg/kg bw/day dose level, the no adverse effect level (NOAEL) was determined to be 250 mg/kg bw/day in male and female rats after at least 28 days of test item treatment by oral gavage.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 14, 2016 - September 13, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: males: 230-257 g, females: 162-183 g
- Fasting period before study: no
- Housing: 2 animals/sex/cage
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 5/6 days

DETAILS OF FOOD AND WATER QUALITY:
- Food: ssniff® SM R/M-Z "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany. Batch No.: 278 5652. Expiry date: November 2016
- Water: from municipal supply, as for human consumption. The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36, Hungary)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 27.2 °C
- Humidity (%): 36 – 73 %
- Air changes (per hr): 15-20 air exchanges per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected. Formulations were prepared fresh prior to administration to animals. The amount of the dose volumes was formulated considering the density of the test item (1232 kg/m3) and the purity (45.4 % w/w water solution).

VEHICLE: distilled water
- Amount of vehicle (if gavage): 1.79 ml/kg bw
- Lot/batch no. (if required): 8090416 / 8160616
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of the formulations for concentration and homogeneity was performed using validated spectrophotometric method (CiToxLab study code 12/311/-316AN). To verify the concentration of the Test Item in formulations, representative samples were taken and analysed from each concentration four times during the study (on Week 1, 5, 9 and Week 13). One sample was taken in duplicate from the Control group solution for concentration measurements.
The UV absorbance of the samples was measured in a Hitachi U-2910 spectrophotometer:
- Limit of quantification: 2.315 (w/w)%
- Repeatibility: CV% ≤ 0.7
- Linearity: 2.315 – 11.575 (w/w)%
- Recovery from gavage formulation: 2.894 (w/w)%: 99% (RSD: 0.020%); 11.575 (w/w)%: 97% (RSD: 0.005%); 46.300 (w/w)%: 101% (RSD: 0.002%)
All formulations were found to be in the range of 97 to 104% of nominal concentrations and were considered suitable for the study purposes.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily on a 7 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control group
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals/group/sex
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on a previous Dose Range Finding study in the rat (12/331-220PE) and another Combined Repeated Dose Toxicity study with Reproduction/Developmental toxicity screening test in rats (12/331-220P).
At Dose Range Finding Study, test item were administrated to Wistar rats at 62.5, 250 and 1000 mg/kg bw/day, daily for 7 consecutive days. There was no association with any adverse effects that could be clearly ascribed to test item administration other than a transient body weight and food intake effect in the first few days of treatment in males at the High dose.
At 28-days study (62.5, 250 and 1000 mg/kg bw/day) minor changes in clinical chemistry and urinalysis parameters in males and females and ulcers of the non-glandular mucosa of the stomach were noted at 1000 mg/kg bw. No adverse effect was found on reproduction/developmental parameters at this dose level. No adverse effects or test item related histopathology findings were observed at the low or mid-dose levels (62.5 and 250 mg/kg bw/day).
On this basis, the dose levels selected were 100, 300 and 1000 mg/kg bw, with the aim of inducing toxic effects but no death or suffering at the highest dose (up to 1000 mg/kg bw/day) and a NOAEL at the lowest dose.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: morbidity and mortality twice daily (at the beginning and end of each working day)
- General clinical observation daily (except day 91), at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am). All animals were placed outside the home cage in a standard arena.
- Deatiled observations: skin, fur, eyes, eyeballs and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system (tremor, convulsion, muscular contractions, etc.), somatomotor activity and behaviour pattern (changes in exploratory behaviour, ordinary behaviour including changes in grooming, headshaking, gyration, etc., abnormal behaviour such as autophagia/self-mutilation, backward motion, abnormal vocalization, etc., aggression, etc.), motor coordination, ambulatory abnormalities, changes in body position and posture (hunchback posture, etc), gait, or response to handling and to environmental stimulation. Particular attention was directed to observations for tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to start of treatment, then at least weekly, including on Day 90 (last treatment day) and prior to necropsy (fasted, on Day 91).

FOOD CONSUMPTION: Yes
- Weekly food consumption was calculated by weighing the remaining, non-consumed food.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals were examined before treatment (Day -2/-1) and in the Control and High dose (1000 mg/kg bw/day) groups, during Week 13 (Day 83/84).
- Observation: Mydriasis was produced after instillation of eye drops "Humapent" (5 mg/mL cyclopentolate hydrochloride; Batch No.: 8680415, exp.: April 2017) into the conjunctival sac. The evaluation was performed using a Gowlland ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91(3 blood samples), by heart puncture
- Anaesthetic used for blood collection: Yes. Pentobarbital.
- Animals fasted: Yes. Overnight period.
- How many animals: all animals
- Parameters checked: Red Blood Cell (erythrocyte) count; Haemoglobin; Haematocrit; Mean Corpuscular (erythrocyte) Volume; Mean Corpuscular (erythrocyte) Haemoglobin; Mean Corpuscular (erythrocyte) Haemoglobin Concentration; Red Cell (erythrocyte) width; Platelet (thrombocyte) count; Mean Platelet Thrombocyte Volume; Reticulocyte count; White Blood Cell (leukocyte) count and Neutrophil, Lymphocite, Monocyte, Eosinophil, Basophil counts and Large Unstained cells. Activated Partial Thromboplastin Time and Prothrombin Time were measured too.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91, after an overnight period of food deprivation of animals.
- Animals fasted: Yes. Overnight period
- How many animals: all the animals
- Parameters checked: Blood sugar (Glucose), Total Bilirubin, Urea, Cholesterol, Triglycerides, Creatinine, Phosphorus, Sodium, Potassium, Calcium, Chloride, Total Protein, Albumin, Alb/glob ratio, Aspartate Aminotransferase activity, Alanine aminotransferase activity, Alkaline-phospatase activity, Gamma Glutamyltransferase activity and Bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: approximately 16 hours, during an overnight period of food deprivation of animals
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight
- Parameters checked: Leukocyte, Nitrite, pH, Protein, Glucose, Urobilinogen, Bilirubin, Ketones, Blood, Erytrhocytes, Specific Gravity, Sediment and Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period, during Week 12/13 (on Day 85, 86, 87, 88)
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, fore/hind grip strength and motor activity as well as general physical condition and behaviour of animals were tested. A modified Irwin test was performed.

OTHER:
- Examination of vaginal smears: for determining the oestrus cycle of all females.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were necropsied at the end of the treatment period (Day 91). External appearance of each rat was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as applicable. Bone marrow smears were collevted but not analysed.

ORGAN WEIGHTS: Yes (absolute and relative weight)
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Livers, Ovaries, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid with parathyroid glands, Pituitary, Prostate, Uterus including cervix

HISTOPATHOLOGY: Yes
The following tissues and organs were retained for all animals: Adrenals, Animal identification, Aorta thoracic and abdominal, Brain, Epididymis, Eye with the optic nerve, Oesophagus, Femur with marrow, Heart, Kidney, Large intestine, Extraorbital lachrymal gland, Harderian gland, Liver, Lungs with bronchi, Lymph node, Ovary, Oviduct, Pancreas, Pituitary, Prostate, Salivary gland (including mandibular, sublingual and parotid glands), Sciatic nerve, Seminal vesicle with coagulating gland, Skin, subcutis with mammary gland (inguinal), Skeletal muscle (quadriceps), Small intestine, Spinal cord (3 levels), Spleen, Sternum with marrow, Stomach, Testis, Thymus, Thyroid with parathyroid gland, Tongue, Trachea, Urinary bladder, Uterus and Vagina.
Full histopathology was performed in Control and High dose groups.
Statistics:
Statistical analysis was performed using SAS 9.2 (built in Provantis System). SAS 9.2 was used with the following decision tree automatically within the validated Provantis system for statistical evaluation of numeric data: the normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified).
Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result is positive, Dunnett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.

If either of the Shapiro-Wilk or Levene tests shows significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A clinical sign of red discharge from the right eye socket was observed from day 21 until the end of the study at concentrations of 100 mg/kg bw/day in one female animal; this was considered as incidental and not related to treatment.
Mortality:
no mortality observed
Description (incidence):
There was no mortality during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights of males and females were comparable to the control throughout the treatment period. The overall body weight gains (Days 0-90) of males and females were similar to the controls. Sporadic statistically significant lower weight gain was noted in males at 1000 mg/kg bw/day but the overall gain for the whole duration was not statistically different to control. There was a trend for lower weight gain, particularly in males, but in the absence of a consistent statistically significant difference, and the relatively small mean difference (<10%) there were no differences that could be clearly considered as an adverse effect.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption was transiently lower than control in the High Dose group of both sexes in the first week and only males on the third week, but the overall food intake was not affected by the test item administration. No toxicologically significant variations were recorded during the treatment period in any of the groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test item related changes compared to pre-treatment were noted.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment related statistically differences in the haematology data. All values were within the historical ranges, hence there were no differences considered to be an effect of the test item.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Evaluation of the clinical chemistry parameters (mean and individual results) in comparison with the Control data did not reveal any toxicological important test item related effect. All values were within the historic range no statistical differences were considered to reflect a treatment related effect.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically lower urine volume of the High dose females was detected at the end of the treatment period, on Day 91. This variation was unrelated to treatment and within the historic range. All other investigated parameters were comparable with the control.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in the animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or treated groups. During evaluation of motor activity, the total travelled distance was comparable to the control in both sexes. There was no effect of treatment noted during the assessment of landing foot splay, grip strength or motor activity. All results were within the expected control range.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Compared to controls, weight difference ataining statistical significance was noted in adrenal glands for females at Low and Mid dose. As the changes showed no consistent dose reponse and were not correlated with pathological findings, they were considered incidental and not related to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was no evidence of test item-related macroscopic findings at dose levels of 100, 300 and 1000 mg/kg bw/day. Uterine dilatation seen in 1/10, 2/10 and 3/10 females from Control, Low and High dose groups respectively, were considered a common background.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related microscopic findings were noted at a dose level of 1000 mg/kg bw/day. Proestrus of the cervix +body +horn in (correlated with dilatation observed at necropsy) in 1/10 Control, 2/2 Low and 3/10 High Dose females, slight reduced sperm in 1/10 High dose male and minimal inflammation of the epididymis in 1/10 Control male, minimal to slight inflammation of the prostate in 1/10 Control and 1/10 High Dose male, minimal inflammatory cell infiltrate of the Harderian glands in 1/10 High Dose female, minimal cardiomyocyte degeneration of the heart in 2/10 High Dose males, minimal tubule basophilia (1/10 Control male), minimal mineralization tubule (2/10 Control females), slight pelvic dilatation (1/10 High Dose female), and minimal/slight proteinaceous casts of the kidneys (3/10 Control males, 2/10 High Dose males, 1/10 Control and 1/10 High Dose females), minimal to slight extramedullary hematopoiesis of the spleen in 6/10 Control males, 5/10 High Dose males, 5/10 Control and 4/10 High Dose females, slight tubular vacuolation of testis in 1/10 Control male, slight tubular dilatation of testis in 1/10 High Dose male, minimal to slight multinucleated cells of the testes in 1/10 Control and 1/10 High Dose males, were observed without meaningful changes in the incidence and severity and therefore were considered to be incidental or a common background. In addition, the changes examined due to observed gross lesions at necropsy were regarded as a common background.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Examination of Vaginal Smears: no test item related changes in the animal oestrus cycle. The animals showed the normal distribution of the oestrus phases.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: No test item related adverse effects observed up to the highest dose tested
Key result
Critical effects observed:
no

Bodyweight (mean of body weights for dose and week)

MALES

 

 

 

 

 

 

 

 

 

 

 

 

 

 

dose

1

8

15

22

29

36

43

50

57

64

71

78

85

90

0

249.3

303.2

340.9

378.2

406.5

432.4

450.7

467.3

484.1

500.0

512.5

522.0

526.7

530.8

100

251.6

307.8

346.3

379.8

409.3

434.2

453.8

475.6

490.8

506.6

518.6

527.6

535.3

541.4

300

251.0

306.7

342.9

380.5

406.8

431.2

451.4

472.8

492.2

508.3

521.8

529.3

538.0

545.3

1000

249.8

300.1

335.3

365.1

387.2

411.7

429.5

446.6

460.9

473.5

483.4

492.6

494.4

506.6

FEMALES

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DOSE

1

8

15

22

29

36

43

50

57

64

71

78

85

90

0

182.5

211.9

231.7

244.1

254.5

268.4

277.6

284.8

291.7

298.7

302.4

310.1

316.7

315.2

100

182.3

213.3

237.4

254.3

262.0

271.5

283.0

292.1

301.2

305.5

313.1

321.0

320.3

322.8

300

182.8

210.0

235.1

248.8

262.3

275.4

288.2

294.0

299.2

309.0

313.3

316.4

321.1

323.4

1000

183.7

203.2

226.8

240.1

253.1

262.1

271.5

282.3

285.8

292.7

299.3

304.2

307.5

309.9

Analytical results for test item concentration

measured concentrations with 95% confidence interval

Sample code

Measured concentrations on 14 June 2016

Measured concentrations on 12 July 2016

Measured concentrations on 10 August 2016

Measured concentrations on 07 September 2016

% w/w

Percentatge of the nominal

% w/w

Percentatge of the nominal

% w/w

Percentatge of the nominal

% w/w

Percentatge of the nominal

Control

not detected

-

not detected

-

not detected

-

not detected

-

4.54 % w/w

4.73 ± 0.038

104

4.46 ± 0.012

98

4.66 ± 0.007

103

4.70 ± 0.007

103

13.63 % w/w

13.79 ± 0.053

101

13.22 ± 0.050

97

13.34 ± 0.892

98

13.47 ± 0.090

99

45.4 % w/w

46.2 ± 0.073

102

44.3 ± 0.208

98

46.5 ± 0.253

102

47.1 ± 0.445

104

Conclusions:
Based on the absence of any adverse effects on any of the doses of the test item, the No Adverse Effect Level (NOAEL) was determined to be 1000 mg/kg bw/day in male and females Wistar rats after 90 days of treatment by oral gavage.
Executive summary:

A 90-day Oral Gavage Toxicity Study in Wistar Rats, according to the OECD Guideline No. 408 was performed. (GLP Study).

Test item was administrated daily in distilled water at a dose volume of 1,79 ml/kg bw to male and female Wistar rats (10 male and 10 female per group) at 3 dose levels (100, 300 and 1000 mg/kg bw/day) for 90 days for oral gavage. A control group was treated concurrently with the vehicle only (distilled water).

Parameters measured during the study included signs of morbidity and mortality (twice daily), clinical signs, body weight, body weight gain (prior to treatment and then weekly), food consumption (weekly) and neurological assessment (towards the end of the treatment period) in all animals.Ophtalmology evaluation were performed before treatment and on week 13, only in control and high dose group. All animals were euthanized after 90 days, after an overnight period of food deprivation. Urinalysis, haematology and clinical chemistry parameters were analysed and vaginal smears were taken for all of them. Necropsy and macroscopic examination were performed. Selected organs were weighed and preserved. Full histopathology was performed in control and high dose group.

There was no test item related mortality, no signs of toxicity observed on body weights, body weigth gains or food consumption; no changes at ophtalmoscopy examination; no toxicologically signifcant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strenght or motor activity in the control or treated groups. There were no differences in haematology, blood clotting, urinalysis or clinical chemistry parametres, no changes in the animal oestrous cycle, no macroscopic findings were made at necropsy, no notable effects on organ weights and no microscopic changes at histopathology.

Based on these results, the test item was not associated with any overt adverse effects and the NOAEL was considered to be the high dose level of 1000 mg/kg bw/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Both studies are a GLP compliant and have Klimisch score 1.

Additional information

Repeated dose toxicity (28 -days): This Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed in accordance with OECD Guideline 422 following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels, 62.5, 250 and 1000 mg/kg bw/day. The control animals were treated with the vehicle only (distilled water). 12 Male and female Wistar rats per group were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period, up to and including postpartum/lactation Day PPD4. Single or multiple ulcers of the non-glandular mucosa of the stomach were observed as treatment-related macroscopic findings. Test item related microscopic findings were found at 1000 mg/kg bw/day (High dose). In the stomach, ulcers of the non-glandular mucosa were microscopically observed in High dose males and females. Increases were noted in potassium and/or urea and bile acids in high dose males and females. Calcium oxalate crystals were noted in high dose males and females, with increases in urobilinogen and bilirubin in males. Based on these results the NOAEL was determined to be 250 mg/kg bw/day in rats.

Repeated dose toxicity (90 -days): A 90-day Oral Gavage Toxicity Study in Wistar Rats, according to the OECD Guideline No. 408 was performed. (GLP Study). Test item was administrated daily by oral gavage to 10 male and 10 female Wistar rats per group at 3 dose levels: 100, 300 and 1000 mg/kg bw/day for 90 days. A control group was treated concurrently with the vehicle only (distilled water). No test item related adverse effects were observed in any of the parameters analyzed up to the highest dose tested. Based on these results, the NOAEL was considered to be the higher dose level of 1000 mg/kg bw/day.

Justification for classification or non-classification

Based on available data, the substance is not classified for repeated dose toxicity in accordance with CLP Regulation (EC) no. 1272/2008.