Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-31 to 2016-09-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted September 21, 1998
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Characteristics: Slightly yellowish liquid
Content: ≥ 99.75%
Batch no.: MMS 15-15.13

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS:
- Species: Rat
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Rat / CD®-1 / Crl:CD(SD)
- Age: Males 54 days, females: 61 days
- body weight: Males: 261.0 g - 294.1 g, females: 209.2 g - 245.0 g
- Diet: ad libitum, Commercial ssniff-R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum
- Acclimatisation period: 6 days
-Housing: kept singly in MAKROLON cages
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
ADMINISTRATION: 
- Frequency: once daily for 90 days
- Dose volume: 2 mL/kg b.w./day
- Dose: 0, 100, 300 or 1000 mg/kg/bw
- Vehicle: Corn oil, Batch no. 15296404, supplier: Caesar & Loretz GmbH, 40721 Hilden, Germany.
- Animals:
Main study animals: 80 animals (40 male and 40 female rats); 10 animals/sex/group
Recovery animals: 20 animals (10 male and 10 female rats); 5 animals/sex for groups 1 and 4.

- DOSAGE PREPARATION:
- The administration formulations were freshly prepared every day.
- The test item was suspended in the vehicle (corn oil) to the appropriate concentrations. The suspensions were constantly stirred with a magnetic stirrer during the administration period.
- The stability, homogeneity and concentration of the administration formulations were monitored
- The test item formulations were administered orally at a constant volume once daily.
- The amount of the test item was adjusted to the animal's current body weight daily up to and including test week 6, thereafter weekly.
- The control animals received the vehicle at the same administration volume daily in the same way
Analytical verification of doses or concentrations:
yes
Remarks:
The analytical method was validated by LPT. The following parameters were determined: - Linearity - Accuracy - Precision - Sensitivity - Specificity - Stability
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 2 mL were taken (divided into 2 aliquots of 2 mL) at the following times and stored at
-20°C or colder until analysis:
On the first administration day:
Analysis of stability and concentration
Immediately after preparation of the formulations as well as after 8 and 24 hours storage of formulations at room temperature.
(3 samples/test item group).
Number of samples: 2 x 3 x 3 = 18
Homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the test item group.
(3 samples/test item group).
Number of samples: 2 x 3 x 3 = 18

On the last administration day:
Analysis of concentration
During treatment always before administration to the last animal of the group (1 sample/test item group).
Number of samples: 2 x 1 x 3 = 6

Sum of all samples: 2 x 21 = 42
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- The dose levels for this study were selected in agreement with the Monitor based on available toxicological data generated during a preliminary dose-range-finding study.
- In this dose-range-finding study, the test item was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks.
- Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 1000 mg test itemkg b.w./day for the male and female animals.
- None of the rats died prematurely and no signs of toxicity were noted.
- An increased liver weight was noted at the high dose level (1000 mg/kg b.w./day) for the male and female rats caused by an increased work load on the liver.
- Based on the obtained data dose levels of 100, 300 and 1000 mg/kg were employed in this repeated dose 90-day toxicity study.

Selection of species:
Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Identification of animals:
Each rat received a continuous number from 1 to 100. Points were set on paws and/or tail by tattoo according to a number scheme. Additionally, the animal cages were labelled with study number, animal number, sex, and treatment group.

Positive control:
not required

Examinations

Observations and examinations performed and frequency:
Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documentation. In addition, observations related to individual animals made throughout the study were recorded.
The following observations were made during the course of the study:

Clinical signs
Cage side observations
- The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness.
- Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
- Special attention was paid to ascertain any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc.
Detailed clinical observations
- Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals. In test week 13 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. The observations were made within 2 hours after dosing. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). 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) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

Mortality
- Further checks were made early in the morning and again in the afternoon of each work-ing day to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately 4.00 p.m.

Body weight
- The weight of each rat was recorded at the time of group allocation, daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, and thereafter weekly throughout the experimental period. Weekly values are stated in the report.

Food and drinking water consumption
- The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week.
- The drinking water consumption was monitored daily by visual appraisal throughout the study.

Neurological screening
In test week 13 (before any blood sampling for laboratory examinations) and at the end of the recovery period in test week 17, screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli, based on GAD ), as well as the assessment of grip strength (according to MEYER ), and motor activity assessment were conducted in all animals outside the home cage as described below. The observations were made 1 to 2 hours after dosing.
Observational screening:
Righting reflex
Body temperature
Salivation
Startle response
Respiration
Mouth breathing
Urination
Convulsions
Pilo-erection
Diarrhoea
Pupil size
Pupil response
Lacrimation
Impaired gait
Stereotypy
Toe pinch
Tail pinch
Wire manoeuvre
Hind-leg splay
Positional passivity
Tremors
Positive geotropism
Limb rotation
Auditory function

Functional tests:
Grip strength
Locomotor activity

Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under light isoflurane anaesthesia from animals fasted overnight. The blood samples collected were divided into tubes as follows:
EDTA anticoagulant (whole blood) for haematological investigations
Citrate anticoagulant (plasma) for coagulation tests
Li-Heparin anticoagulant (plasma) for biochemical tests
No anticoagulant (serum) for bile acid determination

Blood samples were collected at the following times:
- At main study termination (on the 1st day of dissection, test day 91) ): All main study animals
- At the end of the recovery period (on the day of dissection, tedt day 119): All recovery animals

Haematology: ADVIATM 120 Siemens Diagnostics GmbH, 35463 Fernwald, Germany
Haemoglobin content (HGB), mmol/L blood
Erythrocytes (RBC), 10E6/µL blood
Leucocytes (WBC), 10E3/µL blood
Reticulocytes (Reti), %
Platelets (PLT), 10E3/µL blood
Haematocrit value (HCT), %
Differential blood count (relative)#, %
Differential blood count (absolute)#, 10E3/µL blood
Mean corpuscular volume (MCV), fL
Mean corpuscular haemoglobin(MCH), fmol
Mean corpuscular haemoglobin concentration (MCHC), mmol/L blood
#: Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells were simultaneously quantified during measurement of the differential blood count.

Coagulation: Amax Destiny Plus™ Tcoag Deutschland GmbH, 32657 Lemgo, Germany
Thromboplastin time (TPT) sec
Activated partial thromboplastin time (aPTT), sec

Clinical biochemistry: KONELAB 30i Thermo Fisher Scientific, 63303 Dreieich, Germany
Albumin g/L plasma
Globulin g/L plasma (by substraction)
Albumin/globulin ratio (non-dimensional) (by substraction)
Bile acids µmol/L serum
Bilirubin (total) µmol/L plasma
Cholesterol (total) mmol/L plasma
Creatinine µmol/L plasma
Glucose mmol/L plasma
Protein (total) g/L plasma
Triglycerides mmol/L plasma
Urea (in blood) mmol/L plasma
Calcium mmol/L plasma
Chloride mmol/L plasma
Potassium mmol/L plasma
Sodium mmol/L plasma
Alanine amino-transferase (ALAT) U/L plasma
Alkaline phosphatase (aP) U/L plasma
Aspartate aminotransferase (ASAT) U/L plasma
Lactate dehydrogenase (LDH) U/L plasma

Urinalysis:
Urine samples were collected from animals fasted overnight at the following times and the parameters listed below were determined:
- At the end of test week 13 (on test days 90 to 91): All study animals
- At the end of the recovery period (on test days 118 to 119): All recovery animals

The urine was collected for 16 hours in an URIMAX funnel cage. The collection of urine was determined immediately prior to starting the blood withdrawals for the haematological and clinical biochemical examinations at study termination.
The following parameters were measured using the methods given below:
Parameter Units Method
- Volume mL graded measuring cylinder
- pH n/a using a digital pH meter type WTW InoLab pH 720
- Specific gravity g/mL using Atago Refractometer, type Uricon sample compared with water (nominal value of 1.000)
The following tests were also performed using qualitative indicators (Combur 9® Test, Roche Diagnostics GmbH, 68305 Mannheim, Germany) of analyte concentration:
Protein, g/L
Glucose, mmol/L
Bilirubin
Urobilinogen, µmol/L
Ketones
Haemoglobin (Hb, approx. values), ery/µL
Nitrite
Microscopic examinations of urine samples were carried out by centrifuging samples and spreading the resulting deposit on a microscopic slide. The deposits were examined for the presence of the following parameters:
- Epithelial cells (E)
- Leucocytes (L)
- Erythrocytes (R)
- Organisms (B)
- Further constituents (i.e. sperm, casts) (C)
- Crystalluria (A)
The colour and turbidity of the urine were examined visually.

Ophthalmological and auditory examinations
Examinations were performed on all animals before first dosing, at main study (test week 13) and at the end of the recovery period (test week 17).
The eyes were examined with a HEINE ophthalmoscope. After examination of the pupillary reflex, mydriasis was produced after instillation of STULLN® eye drops into the cornea. The following ocular structures were then examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
The auditory acuity was checked with a simple noise test.


Sacrifice and pathology:
Necropsy
- On test day 91, the main study animals were dissected following a randomisation scheme. Animals not dissected on test day 91 were dosed again in test day 91 and dissected on test day 92.
- Necropsy of all animals allocated to the recovery period was performed on test day 119.
- The animals were euthanized under CO2 atmosphere, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist.
- All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
- The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined.
- The lungs were removed and all pleural surfaces examined under suitable illumination.
- The liver and the kidneys were examined.
- Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
adrenal gland (2)
brain
epididymis (2)
heart
kidney (2)
liver
ovary (2)
pancreas
spleen
testicle (2)
thymus
as a whole: prostate, seminal vesicles with coagulating glands
uterus (incl. cervix)
Paired organs were weighed individually and identified as left or right
The relative organ weight [g/kg b.w.] was calculated

Histopathology
- The following organs or parts of organs with the exception of the eyes and testicles of all animals were fixed in 7% buffered formalin.
- The eyes were preserved in Davidson’s solution and the testicles were preserved in modified Davidson’s solution for optimum fixation.
adrenal gland (2)
aorta abdominalis
bone (os femoris with joint)
bone marrow (os femoris)
brain (3 levels: cerebrum, cerebellum, medulla/pons)
epididymis (2)
eye with optic nerve (2)
gross lesions observed
heart (3 levels: right and left ventricle, septum)
intestine, large (colon, rectum)
intestine, small (duodenum, jejunum, ileum, incl. Peyer´s patches), Swiss roll method
kidney and ureter (2)
liver
lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion))
lymph node (1, cervical)
lymph node (1, mesenteric)
mammary gland
muscle (skeletal, leg)
nerve (sciatic)
oesophagus
ovary (2) (and oviducts)
pancreas
pituitary
prostate and seminal vesicles with coagulating glands
salivary glands (mandibular, sublingual and parotid gland)
skin (left flank)
spinal cord (3 levels: cervical, mid-thoracic, lumbar)
spleen
stomach
testicle (2)
thymus
thyroid (2) (incl. parathyroids)
tissue masses or tumours (including regional lymph nodes)
trachea (incl. larynx)
urinary bladder
uterus (incl. cervix)
vagina
- The afore-listed organs of all main study and recovery animals of groups 1 and 4 were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining.
- In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically.
- Parathyroids cannot always be identified macroscopically; they were examined micro-scopically if in the plane of section and in all cases where they are noted as grossly en-larged.

Other examinations:
no other examinations
Statistics:
Toxicology and pathology data were captured, whenever possible, using the departmental computerized systems (Provantis®). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups 2 to 4 were compared with the control group 1.
The following statistical methods were used for the data captured with the Provantis system:

Multiple t-test based on DUNNETT, C. W.
New tables for multiple comparisons with a control Biometrics, 482 – 491 (September 1964)
Body weight / food consumption / haematology / coagulation / clinical biochemistry / urinalysis / relative and absolute organ weights (p
STUDENT's t-test:
All numerical functional tests: Body temperature / hind leg splay / grip strength / spontaneous motility (p The following limits were used:
- p = 0.05 / 0.01 ^ t = 2.0484 / 2.7633 (for 28 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.0687 / 2.8073 (for 23 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.3060 / 3.3554 (for 8 degrees of freedom)

Exact test of R. A. FISHER : Histopathology (p
The following settings were used for the statistical evaluation of the parametrical values captured by Provantis:

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).



Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
None of the male and female rats treated with 100 mg test item/kg b.w./day by oral administration revealed any changes in behaviour, external appearance, or consistency of faeces.
Moderate salivation was observed for all animals treated with the intermediate dose of 300 mg test item/kg b.w./day starting on test day 11 (females) or 37 (males), and for all animals treated with the high dose of 1000 mg test item/kg b.w./day starting on test day 9 (males and females). Salivation started within 5 minutes after the respective administration and lasted up to 60 minutes on 54 and 80 days for the intermediate dosed males and females, respectively, and on 82 days for the high dosed animals.
No premature deaths occurred during the treatment period.
The faeces of all animals were normally formed at all dose levels.

Recovery period (restricted to groups 1 and 4)
Salivation had completely subsided with end of treatment. None of the male and female rats previously treated with 1000 mg test item/kg b.w./day by oral administration revealed any changes in behaviour, external appearance, or consistency of faeces during the 28 day recovery period.
Mortality:
no mortality observed
Description (incidence):
No premature deaths occurred during the treatment period.
No premature deaths occurred during the recovery period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period and recovery period (restricted to groups 1 and 4)
The body weight, body weight gain and body weight at necropsy were not test item-relatedly influenced in the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day, neither during the 90 day treatment period nor during the 28 day treatment-free recovery period.
The decrease of the body weight of the male animals of the previously high dose group noted during the recovery period resulted from the allocation of the animals (4 of 15 males with the lowest body weight of the high dose animals at the end of the treatment period entered the recovery period). Therefore, this finding is a change finding and without any toxicological relevance.
Statistically significant differences in body weight compared to the control are not considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period and recovery period (restricted to groups 1 and 4)
No test item-related influence was noted on the relative food consumption of the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day, neither during the 90 day treatment period nor during the 28 day treatment-free recovery period.
Statistically significant differences in the food consumption compared to the control are not considered to be test item-related.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Treatment period and recovery period (restricted to groups 1 and 4)
No changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, lens, vitreous body and fundus were noted in the male and female rats of the main study treated with 100, 300 or 1000 mg test item/kg b.w./day for 90 days or of the recovery animals previously treated with 1000 mg test item/kg b.w./day.
There was no indication of any impairment to auditory acuity.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was noted on any of the haematological parameters of the female rats after treatment with 100 mg test item/kg b.w./day for 90 days and of the male rats after treatment with 100 or 300 mg test item/kg b.w./day for 90 days compared to the control group at the end of the treatment period on test day 91.

At 300 or 1000 mg test item/kg b.w./day the following changes were noted at the end of the treatment period on test day 91:
Changes in haematological parameters compared to the control on test day 91 [%]
Parameter Group 3 Group 4
males females males females
Haemoglobin content none none -6** -8**
Erythrocytes none -6* -6* -7*
Haematocrit value none none -5** -7**
*: statistically significant at p <= 0.05
**: statistically significant at p <= 0.01

No test item-related influence was observed for the number of leucocytes (WBC) and platelets (PLT), the percentage of reticulocytes (Reti), the relative and absolute count of neutrophilic granulocytes (Neut), lymphocytes (Lym), monocytes (Mono), eosinophilic granulocytes (Eos), large unstained cells (LUC) and basophilic granulocytes (Baso), the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).

Recovery period (restricted to groups 1 and 4)
All changes noted for the male and female animals previously treated with 1000 mg test item/kg b.w./day had completely subsided at the end of the 28 day recovery period.

Statistically significant differences in haematological parameters compared to the control which are not considered to be test item-related are given in the table below:
Parameter Ref. table no. Increase/Decrease Group/Sex Test day Statistical significance Reason
MCV 7-1 Decrease 4 f 119 p ≤ 0.05 A
f: female
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group

No test item-related influence was observed for the haemoglobin content (HGB), the number of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the percentage of reticulocytes (Reti), the haematocrit value (HCT), the relative and absolute count of neutrophilic granulocytes (Neut), lymphocytes (Lym), monocytes (Mono), eosinophilic granulocytes (Eos), large unstained cells (LUC) and basophilic granulocytes (Baso), the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period and recovery period (restricted to groups 1 and 4)
No test item-related influence was noted on any of the biochemical parameters of the male and female rats after treatment with 100, 300 or 1000 mg test item/kg b.w./day for 90 days compared to the control group, neither at the end of the treatment period on test day 91 nor at the end of the 28 day treatment-free recovery period on test day 119.
Statistically significant differences in biochemical parameters compared to the control are not considered to be test item-related.
Parameter Ref. table no. Increase /Decrease Group/Sex Test day Statistical significance Reason
Globulin 8-1 In 4 f 91 p ≤ 0.01 A
Albumin/globulin ratio De 4 f 91 p ≤ 0.05 A
Bile acids De 4 f 91 p ≤ 0.01 A
Bilirubin De 3 - 4 m 91 p ≤ 0.01 A
De 2 - 4 f 91 p ≤ 0.01 A
Protein In 4 f 91 p ≤ 0.05 A
Triglycerides In 4 f 91 p ≤ 0.01 A
Urea In 4 m 91 p ≤ 0.05 A
Calcium In 4 f 91 p ≤ 0.05 A
ASAT De 2 m 91 p ≤ 0.05 A
De 4 m 91 p ≤ 0.05 A
De 4f 91 p ≤ 0.05 A
LDH De 4 m 119 p ≤ 0.05 A
m: male
f: female
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group

No test item-related influence was noted for the plasma levels of albumin and globulin, the albumin/globulin ratio, the plasma level of bile acids, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium. Further, the plasma activity of alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) was not influenced.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period
No test item-related toxicologically relevant changes in the urinary status were noted for the male and female animals treated with 100, 300 or 1000 mg test item/kg b.w./day for 90 days compared to the control group.
However, the following changes were noted:
The pH value of the urine of the male animals treated with 300 or 1000 mg test item/kg b.w./day for 90 days and of the female animals treated with 1000 mg test item/kg b.w./day for 90 days was slightly decreased compared to the control group possibly caused by the acid nature of the test item. Details are given in the following table:
Changes in urinary parameters on test day 91 compared to the control [%]
Parameter Group 3 Group 4
males females males females
pH -9** none -10** -5*
*: statistically significant at p ≤ 0.05
**: statistically significant at p ≤ 0.01

These marginal changes are still within the LPT background data. Details are given in the table below:
Parameter Mean value observed in this study LPT background data mean value ± standard deviation LPT background data range of individual data#
pH Group 1 m: 6.94
Group 2 m: 6.61 7.01 ± 0.42 6.0 – 8.2
Group 3 m: 6.34**
Group 4 m: 6.26**

Group 1 f: 6.29
Group 2 f: 6.11 6.55 ± 0.44 5.5 – 8.1
Group 3 f: 6.18
Group 4 f: 6.01*
*: statistically significant at p <= 0.05
**: statistically significant at p <= 0.01
#: n= 127, data taken from 2013-2016, data not audited by the LPT QAU
The statistical significance of the differences in urinary parameters compared to the control which are not considered to be test item-related is described in the following table:
Parameter Ref. table no. Increase/Decrease Group/Sex Test day Statistical significance Reason
Specific gravity 9-1 In 4 f 91 p ≤ 0.01 A
Urine volume De 3 f 91 p ≤ 0.05 A
De 4 f 91 p ≤ 0.01 A
m: male
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group

No test item-related changes were noted for the specific gravity of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.

Recovery period
No test item-related changes in the urinary status were noted for the male and female animals previously treated with 1000 mg test item/kg b.w./day compared to the control group.
The statistical significance of the differences in urinary parameters compared to the control which are not considered to be test item-related is described in the following table:
Parameter Ref. table no. Increase/Decrease Group/Sex Test day Statistical significance Reason
Urine volume 9-1 In 4 m 119 p ≤ 0.05 C
m: male
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group

No test item-related changes were noted for the specific gravity and the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.









Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Neurological screening
Treatment period and recovery period (restricted to groups 1 and 4)
The neurological screening performed at the end of the treatment period in test week 13 did not reveal any test item-related influence in the male and female rats treated with 100, 300 or 1000 mg test item/kg b.w./day, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility. Furthermore, no test item-related influence was noted in the rats previously treated with 1000 mg test item/kg b.w./day at the end of the recovery period.
Salivation as noted during the detailed clinical observations was not observed during the neurological screening as it had already subsided when the functional observations were started within 2 hours following dosing.
Statistically significant differences in neurological parameters compared to the control which are not considered to be test item-related are given in the table below:
Parameter Ref. table nos. Increase/Decrease Group/sex Testweek Statistical significance Reason
Body
temperature 2-1, 2-2 De 3 m 13 p ≤ 0.05 A
Hind leg splay In 3 m 13 p ≤ 0.05 A
De 4 f 16 p ≤ 0.05 C
m: male
f: female
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group

Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was noted on the relative and absolute organ weights of the male and female animals treated with 100 or 300 mg test item/kg b.w./day for 90 days compared to the control group at the end of the treatment period on test day 91/92.
The following changes were noted in the animals treated with 1000 mg test item/kg b.w./day at the end of the treatment period on test day 91/92:

Changes in organ weights at the end of test week 13 compared to the control [%]
Organ Group 4
males females
Kidney, left: - relative +31** +16**
- absolute +28** +16**
Kidney, right: - relative +31** +16**
- absolute +28** +15**
Liver: - relative +30** +48**
- absolute +28** +47**
*: statistically significant at p  0.05
**: statistically significant at p  0.01
The weight increase of the kidneys and liver is probably a non-specific change caused by the high dose level administered causing a high work load/metabolic stress on the kidneys and liver, in particular, as no histopathological changes were noted for the liver (both sexes) and the kidneys (females).
The even more increased kidney weights of the males can be correlated with the histopathological findings for the kidneys in form of tubular basophilia, tubular dilation, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli for nearly all male animals of the high dose group.
The test item related changes in the kidneys of the male animals are classified as alpha 2u-globulin nephropathy. They are rat-specific and not relevant in man.

Statistically significant differences in organ weights compared to the control which are not considered to be test item-related are given in the table below:
Organ Ref. table nos. Increase/Decrease Group/Sex Test day Statistical significance Reason
Spleen
(relative) 13-1 In 3 f 91/92 p ≤ 0.05 B
Spleen
(absolute) 14-1 In 3 f 91/92 p ≤ 0.05 B
f: female
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group
D: effect is due to the reduced body weight

Recovery period (restricted to groups 1 and 4)
All changes noted for the male and female animals previously treated with 1000 mg test item/kg b.w./day had completely subsided at the end of the 28 day recovery period.

Statistically significant differences in organ weights compared to the control which are not considered to be test item-related are given in the table below:
Organ Ref. table nos. Increase/Decrease Group/Sex Test day Statistical significance Reason
Heart (absolute) 14-1 De 4 m 119 p ≤ 0.05 A
Kidney, left (absolute) 14-1 De 4 m 119 p ≤ 0.05 A
Pancreas (relative) 13-1 In 4 m 119 p ≤ 0.01 C
Pancreas (absolute) 14-1 In 4 m 119 p ≤ 0.05 C
Prostate and seminal vesicle (relative) 13-1 De 4 m 119 p ≤ 0.05 A
Prostate and seminal vesicle (absolute) 14-1 De 4 m 119 p ≤ 0.01 A
m: male
A: the slight alteration in comparison to control animals is without any biological relevance
B: lacking dose dependence
C: effect is due to the relative low or high value observed for the control group
D: effect is due to the reduced body weight
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period
No test item-related influence was noted for the male and female rats treated with 100, 300 or 1000 mg test item/kg b.w./day for 90 days at terminal sacrifice on test day 91/92.
Macroscopic changes were noted in the thyroid (reduced in size) in one male animal of the control group. This change is an incidental finding as it occurred in the vehicle-treated control group.

Recovery period (restricted to groups 1 and 4)
No pathological changes were observed in the organs of the male and female rats previously treated with 1000 mg test item/kg b.w./day for 90 days at the end of the 28 day recovery period.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment period (restricted to group 1 (control) and group 4 (high dose))
Morphological lesions considered to be related to the test item were noted at the high dose of 1000 mg test item/kg b.w./day in the kidneys of the male animals.
A minimal tubular basophilia was also noted in the kidneys of individual male control animals.
At the high dose (group 4), statistical significance (at p ≤ 0.01 or p ≤ 0.05, Fisher's two-tailed exact test) was calculated for the occurring of tubular basophilia, tubular dilation, cystic tubular degeneration, and hyaline droplets in the proximal tubuli in the kidneys. The increased lymphocytic infiltration was statistically not significant.
The test item related changes in the kidneys of the male rats are described as alpha 2u-globulin nephropathy. They are rat-specific and not relevant in man.
The histopathological findings in the kidneys of the male high dose animals correlated with the increased kidney weights noted for these animals.
The coincidental findings in various organs in a few control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.

Recovery period (restricted to group 1 (control) and group 4 (high dose))
Morphological lesions were still noted in the kidneys of the male animals previously treated with 1000 mg test item/kg b.w./day at the end of the 28-day recovery period.
At the high dose (group 4), statistical significance (p ≤ 0.05, Fisher's two-tailed exact test) was still calculated for the occurring of cystic tubular degeneration in both kidneys. An increased lymphocytic infiltration, tubular basophilia and tubular dilation (left kidney only) were still noted in the kidneys without being statistically significant.
The coincidental findings in various organs in a few control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.
see tables below (overall remarks)
Description (incidence and severity):
see above
Other effects:
not specified
Details on results:
None of the animals died or had to be sacrificed prematurely.
No test item-related changes were observed for the neurological screening, the body weight, body weight gain and body weight at autopsy, food and drinking water consumption, or for any of the clinical chemical or urinary parameters of the male and female rats during the course of the study. No changes were noted for the eyes and the auditory.
The macroscopic inspection at necropsy did not reveal any morphological changes, which are considered to be related to the administration of the test item. acuity.

Moderate salivation was observed for all animals treated with 300 or 1000 mg test item/kg b.w./day starting on test day 11 (females) or 37 (males) at the intermediate dose and as of test day 9 (males and females) at the high dose.
Treatment with 300 mg test item/kg b.w./day led to a slight decreased number of erythrocytes.
Treatment with 1000 mg test item/kg b.w./day led to slight changes in the haematological parameters (decreased number of erythrocytes, decrease of the haemoglobin content and haematocrit value) and to increased relative and absolute liver and kidney weights in both sexes.
The weight increase of the kidneys and liver is probably a non-specific change caused by the high dose level administered causing a high work load/metabolic stress on the kidneys and liver, in particular, as no histopathological changes were noted for the liver (both sexes) and the kidneys (females).
The histopathological examination revealed test item-related morphological lesions at the high dose of 1000 mg test item/kg b.w./day in the kidneys of the male animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli. The test item related changes in the kidneys of the male animals are classified as alpha 2u-globulin nephropathy. They are rat specific and not relevant in man.

All changes observed during the main study had completely subsided in all male and female previously high-dosed animals at the end of the recovery period. Histopathology still revealed morphological lesions in the kidneys of the male high dosed animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, and lymphocytic infiltration.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect observed up to a concentration of 1000 mg/kg bw

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Findings

Treatment period

 

Mortality: None of the animals died or had to be sacrificed prematurely.

 

Clinical signs / Detailed clinical observations: Moderate salivation was observed for all animals treated with the intermediate dose of 300 mg test item/kg b.w./day starting on test day 11 (females) or 37 (males), and for animals treated with the high dose of 1000 mg test item/kg b.w./day starting on test day 9 (males and females). Salivation started within 5 minutes after the respective administration and lasted up to 60 minutes on 54 and 80 days for the intermediate dosed males and females, respectively, and on 82 days for the high dosed animals.

 

Neurological screening: The neurological screening did not reveal any test item-related influence in the male and female rats treated with 100, 300 or 1000 mg test item/kg b.w./day, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.

 

Body weight and body weight gain: No test item-related changes were noted.

 

Food and drinking water consumption: No test item-related changes were noted.

Haematology and coagulation on test day 91:

At 1000 mg test item/kg b.w./daythe following changes were noted at the end of the treatment period on test day 91:

Changes in haematological parameters

compared to the control on test day 91 [%]

Parameter

Group 3

300 mg/kg

Group 4

1000 mg/kg

males

females

males

females

Haemoglobin content

none

none

-6**

-8**

Erythrocytes

none

-6*

-6*

-7*

Haematocrit value

none

none

-5**

-7**

  *:       statistically significant at p <= 0.05

**:       statistically significant at p <= 0.01

Clinical chemistry:   No test item-related changes were noted.

 

Urinalysis:  No test item-related toxicologically relevant changes were noted.

 

Ophthalmological and auditory examinations:  No test item-related changes were noted.

 

Macroscopic post mortem findings: No test item-related changes were noted.

Organ weights: The following changes were noted in the animals treated with 1000 mg test item/kg b.w./dayat the end of the treatment period on test day 91/92:

                

Changes in organ weights at the end oftest week 13

compared to the control [%]

Organ

 

Group 4

1000 mg/kg

 

males

females

Kidney, left:

- relative

+31**

+16**

 

- absolute

+28**

+16**

Kidney, right:

- relative

+31**

+16**

 

- absolute

+28**

+15**

Liver:

- relative

+30**

+48**

 

- absolute

+28**

+47**

 *:       statistically significant at p <= 0.05

**:       statistically significant at p <= 0.01

The weight increase of the kidneys and liver is probably a non-specific change caused by the high dose level administered causing a high work load/metabolic stress on the kidneys and liver, in particular, as no histopathological changes were noted for the liver (both sexes) and the kidneys (females).

The even more increased kidney weights of the males can be correlated with the histopathological findings for the kidneys in form of tubular basophilia, tubular dilation, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli for nearly all male animals of the high dose group.

Histopathology (restricted to group 1 (control) and group 4 (high dose)):

Morphological lesions considered to be related to the test item were noted in the kidneys of the male animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli.

The test item related changes in the kidneys of the male animals are classified as alpha 2u-globulin nephropathy. They are rat-specific and not relevant in man.

Test item formulation analysis:

The analysis of the test item concentration in the test item formulations revealed that the suspensions used for the administration in groups 2 to 4 were correctly prepared, homogenous and stable for at least 24 hours after storage at room temperature. The measured concentrations ranged from 101.5% to 103.8% (low dose), from 101.5% to 108.4% (intermediate dose) and from 97.4% to 106.0% (high dose). The results were well within the admissible limits of 90% to 110%.

Recovery period:

All changes noted for the male and female animals previously treated with 1000 mg test item/kg b.w./day had completely subsided at the end of the 28 day recovery period.

No premature deaths occurred and no signs of systemic toxicity were noted. No test item-related changes were noted at the neurological screening, body weight, body weight gain and body weight at autopsy, food and drinking water consumption, haematological, biochemical and urinary parameters, at ophthalmological and auditory examinations, at necropsy as well as for organ weights at the end of the 28‑day recovery period.

Histopathology still revealed morphological lesions in the kidneys of the male high dosed animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, and lymphocytic infiltration

Applicant's summary and conclusion

Conclusions:
In conclusion, the experimental no-observed-adverse-effect level (NOAEL) was 1000  mg test item/kg b.w./day by daily oral administration.

All changes observed during the main study had completely subsided in all male and female previously high-dosed animals at the end of the recovery period. Histopathology still revealed morphological lesions in the kidneys of the male high dosed animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, and lymphocytic infiltration.The test item related changes in the kidneys of the male animals are classified as alpha 2u-globulin nephropathy. They are rat specific and not relevant in man.
Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity of test item administered daily by oral administration to rats for 90 consecutive days and to assess the reversibility of any effects at the end of a 28‑day recovery period. The rats were treated with 100, 300 or 1000 mg test item/kg b.w./day. The control animals received vehicle (corn oil).

None of the animals died or had to be sacrificed prematurely.

Moderate salivation was observed for all animals treated with 300 or 1000 mg test item/kg b.w./day starting on test day 11 (females) or 37 (males) at the intermediate dose and as of test day 9 (males and females) at the high dose.

Treatment with 300 mg test item/kg b.w./day led to a slight decreased number of erythrocytes.

Treatment with 1000 mg test item/kg b.w./day led to slight changes in the haematological parameters (decreased number of erythrocytes, decrease of the haemoglobin content and haematocrit value) and to increased relative and absolute liver and kidney weights in both sexes.

The weight increase of the kidneys and liver is probably a non-specific change caused by the high dose level administered causing a high work load/metabolic stress on the kidneys and liver, in particular, as no histopathological changes were noted for the liver (both sexes) and the kidneys (females).

No test item-related changes were observed for the neurological screening, the body weight, body weight gain and body weight at autopsy, food and drinking water consumption, or for any of the clinical chemical or urinary parameters of the male and female rats during the course of the study. No changes were noted for the eyes and the auditory acuity.

The macroscopic inspection at necropsy did not reveal any morphological changes, which are considered to be related to the administration of the test item.

The histopathological examination revealed test item-related morphological lesions at the high dose of 1000 mg test item/kg b.w./day in the kidneys of the male animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, lymphocytic infiltration, and hyaline droplets in the proximal tubuli. The test item related changes in the kidneys of the male animals are classified as alpha 2u-globulin nephropathy. They are rat specific and not relevant in man.


All changes observed during the main study had completely subsided in all male and female previously high-dosed animals at the end of the recovery period. Histopathology still revealed morphological lesions in the kidneys of the male high dosed animals in form of tubular basophilia, tubular dilation, cystic tubular degeneration, and lymphocytic infiltration.

 

In conclusion, the experimental no-observed-adverse-effect level (NOAEL) was 1000 mg test item/kg b.w./day by daily oral administration.