Registration Dossier

Administrative data

Description of key information

In a subchronic toxicity study with rats, adverse effects on the thyroid (minimal to slight follicular hypertrophy/ hyperplasia accompanied by minimal to slight colloid alteration) were observed in males. Adaptive, non-adverse changes in the liver were seen in females (significant increase of relative liver weight in females correlated with minimal hepatocellular centrilobular hypertrophy (5 out of 10 females). Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 50 mg/kg bw/d (39.5 mg/kg be/d corrected for the recovery values determined during concentration control analyses) for male and 1000 mg/kg bw/d for female Wistar rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 04 Feb 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient; protect against humidity
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 42 ± 1 days
- Weight at study initiation:
- Fasting period before study: no
- Housing: 5 animals per cage
- Diet: ground Kliba maintenance diet mouse/rat “GLP”, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
deionized water containing 0.5% sodium carboxymethyl cellulose and 5 mg/100 mL Tween® 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, deionized water containing 0.5% sodium carboxymethyl cellulose and 5 mg/100 mL Tween® 80 (SIGMA-ALDRICH), in order to achieve a better homogenous distribution of the test item in the vehicle, was filled up to the desired volume and subsequently homogenized with a homogenizer (Ultraturrax T25, Janke & Kunkel GmbH, Staufen, Germany). All test item formulations were prepared once weekly. The administration volume was 10 mL/kg body weight.

VEHICLE
- Concentration in vehicle: 0, 0.5, 2.0, 10.0 g/100 ml
- Amount of vehicle (if gavage): 10 mL/kg body weight.
Details on analytical verification of doses or concentrations:
The stability of the test article in drinking water containing 0.5% sodium carboxymethyl cellulose at room temperature for a period of 7 days was proven before the start of the administration period. Homogeneous distribution of the test item in the test substance preparations was performed in the highest and lowest concentration. Considering the low relative standard deviation in the homogeneity analyses, it can be concluded that the test item was distributed homogeneously in deionized water containing 0.5% sodium carboxymethyl cellulose and 5 mg/100 mL Tween® 80. Concentration control was performed in all concentrations at the beginning and towards the end of the administration period. The concentration control analyses of test group 3 (10.0 g/100 mL) revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 90-110% of the nominal concentrations at any time point. For test group 2 (2.0 g/100 mL) recoveries of 87-91% with reference to the nominal concentration were found. This deviation from the acceptance criterion is, however, considered acceptable for the analysis of test substances in complex matrices like deionized water containing 0.5% sodium carboxymethyl cellulose and 5 mg/100 mL Tween® 80. Taken together, the results demonstrate the correctness of the concentrations of the test item in the vehicle for test groups 1 and 2. The determined concentration for test group 1 (0.5 g/100 mL) was in some samples below 90% and, and, thus, did partly not meet the specification limit of 90-110% based on SOP’s of the test facility. Taken all recovery rates together an average recovery rate of 79% was found. Thus, the calculated dose level was 39.5 mg/kg bw/d instead of 50 mg/kg bw/d.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
actual dose 39.5 mg/kg
Dose / conc.:
200 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:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined:
Abnormal behavior when handled, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Impairment of gait, Lacrimation, Palpebral closure, Exophthalmus, Feces (appearance/ consistency), Urine, Pupil size

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period, the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION
Drinking water consumption was observed by daily visual inspection of the water bottles for any overt changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes
One day prior to the start of the administration period the eyes of all animals and on study day 90 the eyes of the control and high-dose animals were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Bausch & Lomb GmbH, Heidelberg, Germany).

HAEMATOLOGY: Yes
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer). The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units.
Parameters investigated: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA).
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Only evaluated blood smears will be archived.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Parameter investigated: Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
Parameters investigated: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL)

URINALYSIS: Yes
On the afternoon preceding the day fixed for urinalysis, the animals were transferred individually into metabolism cages (no food or drinking water provided). Urine was sampled overnight. On the following day, the samples were examined in a randomized sequence (the list of randomization instructions was compiled with a computer).
Parameters investigated: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.) [g/L], Sediment, Color, turbidity (COL, TURB), Volume (VOL) [mL]

NEUROBEHAVIOURAL EXAMINATION: Yes
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages type III supplied by TECNIPLAST (Hohenpeißenberg, Germany) with wire covers from Ehret (Emmendingen, Germany; floor area of about 800 cm2) and small amounts of bedding material. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable.  

- Home cage observations: The animals were observed in their closed home cages; during this period, any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the rats. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings

- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait, Activity/ arousal level, Feces excreted within 2 minutes (appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes, Other findings

- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), Touch sensitivity (touch response), Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (auditory startle response), Coordination of movements (righting response), Behavior during handling, Vocalization, Pain perception (tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings

- Motor activity assessment: Motor activity (MA) was also measured from 13:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats were placed in new clean polycarbonate cages. For motor activity (MA) measurements, the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST (Hohenpeißenberg, Germany) with wire covers from Ehret (Emmendingen, Germany; floor area of about 800 cm2) with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the rats during these measurements and the measurement room was darkened after the transfer of the last rat. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (terminal body weight), Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys, Liver, Ovaries (fixed), Spleen, Testes, Thymus (fixed), Thyroid glands (fixed), Uterus with cervix
All paired organs were weighted together (left and right).

HISTOPATHOLOGY: Yes
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Mammary gland (male and female), Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes , Thymus ,Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below.
Statistics:
- Body weight,body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
- Rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians. For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test is performed.
- Urine pH, volume and specific gravity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians.
- Weight parameters: on-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No test substance-related, adverse effects were obtained in any test group (50, 200 and 1000 mg/kg bw/d).
Salivation shortly after treatment was observed in 6 of 10 male and 5 of 10 female animals of test group 3 (1000 mg/kg bw/d) on several days of the application period. The finding occurred for the first time in male animal No. 34 on study day 34 and in female animal No. 72 on study day 49. From the temporary, short appearance immediately after dosing it was concluded that the finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations. A protruding right eyeball was observed in control female animal No. 42 of test group 0 (0 mg/kg bw/d) and female animal No. 71 of test group 3 (1000 mg/kg bw/d) between study days 14-93 and 34-93, respectively. This finding was considered to be incidental and not related to treatment. Note: On study day 86 (19 Jan 2018) clinical examinations between 2-5 hours after treatment were performed for all animals of test groups 0-3 but not documented in the electronic system. Therefore, the findings for this time period are not shown in the report. These missing records do not have an influence on the validity of the present study.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related, adverse effects on body weight parameters were obtained in any test group (50, 200 and 1000 mg/kg bw/d).
Mean body weights and body weight change values of male and female animals did not show any significant deviations to the control, except a significant increase in body weight change in females of test group 2 (200 mg/kg bw/d) on study day 7 and in females of test group 1 (50 mg/kg bw/d) on study 14. Because of their transient character and a missing dose-response relationship, the findings were assessed to be incidental and not related to treatment. No significant changes were observed in female animals of test group 3 (1000 mg/kg bw/d).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to food consumption were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related, adverse changes with regard to water consumption were observed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed. All other apparent findings were also assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period, in females of test group 3 (1000 mg/kg bw/d) mean corpuscular volume (MCV) was significantly decreased. However, this calculated parameter was within the historical control range (females, MCV 50.3-53.8 fL) and no measured red blood cell parameter (i.e., hemoglobin, hematocrit and RBC counts) was altered. In females of test group 2 (200 mg/kg bw/d) total white blood cell (WBC) counts, absolute lymphocyte, basophil and large unstained cell (LUC) counts were significantly lower compared to study controls. However, all mentioned parameters were not dose-dependently changed.
Therefore, all mentioned alterations among the hematology parameters were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period, in females of test group 3 (1000 mg/kg bw/d) total bilirubin values were significantly decreased. This was the only altered clinical chemistry parameter among these individuals and the mean was still within the historical control range (females, total bilirubin 1.41-2.74 µmol/L). Therefore, this change was regarded as incidental and not treatment-related. In females of test group 2 (200 mg/kg bw/d) aspartate aminotransferase (AST) activities were significantly lower whereas sodium levels were significantly higher compared to study controls. However, both parameters were not dose-dependently changed. Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.
Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with control group 0, the mean absolute organ weight of the liver was statistically significantly increased in males and females of the high dose group. All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
When compared with control group 0, the mean relative organ weight of the liver was statistically significantly increased in males and females of the mid and high dose group. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
In test group 3 (1000 mg/kg bw/d), the absolute (males: 10.06 g, females: 6.04 g) and relative (males: 2.63%, females: 2.87%) liver weights were minimally above the historical control range in both, i.e. males (absolute: 6.835-9.083 g; relative: 2.045-2.318%) and females (absolute: 4.722-5.577g; relative: 2.246-2.542%) and correlated with histopathological findings and therefore assessed as treatment-related. In test group 2, minimal increases of the relative liver weight in males (2.33%) and females (2.55%) were marginally above the maximal historical control values (male: 2.318%; female: 2.542%). Since a histopathological correlate was found in females but not in males of test group 2, the relative weight increase in females was considered to be treatment-related and in males assumed as possibly treatment-related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the liver of male and female animals and in the thyroid gland of males with incidences and grading according to the tables below.
Histopathological findings: neoplastic:
no effects observed
Details on results:
For the group-based data tables please refer to the attached document in the section "Attached background material".
Dose descriptor:
NOAEL
Effect level:
39.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
not specified

HISTOPATHOLOGY

Treatment-related findings were observed in the liver of male and female animals and in the thyroid gland of males with incidences and grading according to the tables below:

LIVER Male animals Female animals
Test group 0 1 2 3 0 1 2 3
(mg/kg bw/d) 0 50 200 1000 0 50 200 1000
No. of animals 10 10 10 10 10 10 10 10
Hypertrophy, centrilobular 0 0 0 4 0 0 5 10
Grade 1       2     5 5
Grade 2       2       4
Grade 3               1

Centrilobular hepatocellular hypertrophy was noted in 4 out of 10 males of test group 3 and in females of test group 2 (5 out of 10) and 3 (all animals).

THYROID GLANDS Male animals Female animals
Test group 0 1 2 3 0 1 2 3
(mg/kg bw/d) 0 50 200 1000 0 50 200 1000
No. of animals 10 10 10 10 10 10 10 10
Hypertrophy / hyperplasia, follicular 2 1 6 7 0 1 0 2
Grade 1 2 1 5 6   1   2
Grade 2     1 1        
Alteration, colloid 2 2 7 7 0 1 0 0
Grade 1 1 1 6 4   1    
Grade 2 1 1 1 3        

The follicular hypertrophy/ hyperplasia and colloid alteration in males of test groups 2 and 3 was regarded as treatment-related, whereas the two females of test group 3 with only minimal follicular hypertrophy/hyperplasia lacking colloid alteration were not considered to be affected by treatment.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Conclusions:
The administration of gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of toxicity at a dose level of 200 mg/kg bw/d and above. The adverse pathological findings were related to male animals only. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 50 mg/kg bw/d (38.5 mg/kg bw/d; corrected for the recovery values determined during concentration control analyses) for male and 1000 mg/kg bw/d for female Wistar rats.
Executive summary:

The test article was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 50 (test group 1), 200 (test group 2) and 1000 mg/kg body weight/day (mg/kg bw/d;test group 3) over a period of 3 months. Deionized water containing 0.5% sodium carboxymethyl cellulose and 5 mg/100 mL Tween 80 served as vehicle, control animals were dosed daily with the vehicle only. Note: For test group 1 (50 mg/kg bw/d) an average recovery rate of 79% was found during concentration control analyses. Thus, the calculated dose level was 39.5 mg/kg bw/d instead of 50 mg/kg bw/d. Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.Ophthalmological examinations were performed before the beginning and towards the end of the administration period.Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out towards the end of the administration period. Clinico-chemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all animals were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. In the high dose and mid dose animals, minimal to slight follicular hypertrophy/ hyperplasia in the thyroid glands in male animals were reported, accompanied by colloid alteration (minimal to slight). No other findings were noted.  No treatment-related, adverse effects were observed in the mow dose group. In conlcusion, the administration of gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of toxicity in male animals at a dose level of 200 mg/kg bw/d and above. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 50 mg/kg bw/d (39.5 mg/kg bw/d; corrected for the recovery values determined during concentration control analyses) for male and 1000 mg/kg bw/d for female Wistar rats.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
39.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is compliant to OECD guideline 408 and GLP regulations (Klimisch score: 1).

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 article was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 50 (test group 1), 200 (test group 2) and 1000 mg/kg bw/d (test group 3) over a period of 3 months. Note: For test group 1 (50 mg/kg bw/d) an average recovery rate of 79% was found during concentration control analyses. Thus, the calculated dose level was 39.5 mg/kg bw/d instead of 50 mg/kg bw/d. The only treatment-related effects observed were limited to organ weights and histopathology. The significant absolute and relative liver weight increases in both sexes of test group 3(1000 mg/kg bw/d)were regarded as treatment-related. These changes correlated with hepatocellular centrilobular hypertrophy in test group 3 males (4 out of 10, graded minimal to slight) and females (all females, graded minimal to moderate). Since these changes were not associated with changes regarding clinical chemistry parameters related to the liver, the pathology findings were considered treatment-related but not adverse. In males, the thyroid glands showed minimal to slight follicular hypertrophy/ hyperplasia (7 out of 10 males) accompanied by minimal to slight colloid alteration (7 out of 10 males). These changes were regarded as treatment-related and adverse. In test group 2 (200 mg/kg bw/d), the significant increase of relative liver weight in females correlated with minimal hepatocellular centrilobular hypertrophy (5 out of 10 females). These changes were judged as treatment-related but not adverse, due to the lack of hepatic alterations in clinical chemistry. In males, the marginal increase of relative weight had no histopathological correlate and was assumed as possibly treatment-related. In the thyroid glands of males, minimal to slight hypertrophy/hyperplasia of follicular cells and altered colloid were increased compared to the control group and approached the same incidence and grading as in the high-dose group. As mentioned before, these histomorphological changes were considered treatment-related and adverse. In test group 1 (50 mg/kg bw/d),treatment-related findings were not observed in liver and thyroid gland of male and female animals. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. In conclusion, the administration of the test item by gavage to male and female Wistar rats for 3 months caused test substance-related, adverse signs of toxicity at a dose level of 200 mg/kg bw/d and above. The adverse pathological findings were related to male animals only. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 50 mg/kg bw/d (39.5 mg/kg be/d corrected for the recovery values determined during concentration control analyses) for male and 1000 mg/kg bw/d for female Wistar rats.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008. Based on the criteria laid down in Regulation (EC) No. 1272/2008, as amended in Directive EC 286/2011, no classification for STOT RE is warranted.