Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Regulation (EC) No. 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
CD® rats supplied by Charles River Laboratories Germany GmbH were used in this experiment (Test species / Strain / Stock: Rat / CD® / Crl:CD(SD)). Species selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals and environmental conditions:
An initial health check was performed upon delivery of the animals. Only animals free of signs of illness were selected for the study. The animals were allocated to the 4 test groups based on body weight by means of a computerised randomisation program. Animals of the extremes of the weight distribution and/or any animal showing signs of illness during the period between allocation and the start of treatment were replaced with a surplus animal selected from the pool. Animal replacement after the first dosing was not planned nor did it occur.
Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number and sex of animals: 100 animals (50 male and 50 female rats).
Main study: 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/group for groups 1 and 4.
Additionally, 10 spare animals (5 males and 5 females) were reserved for possible replacement during the adaptation period.
Body weight (at 1st dosing): Males: 285.6 g - 320.2 g, females: 226.0 g - 266.4 g
Age of animals (at 1st dosing): Males: 62 days, females: 63 days
Identification of animals: Each rat received an identification number, which was tattooed on the tail. The animal cages were labelled with the study number, animal number, sex, and treatment group.
Adaptation period: 8 (males) or 9 (females) days.
Housing and feeding
Diet: A certified commercial diet (ssniff®-R/M-H V1534, ssniff® Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This diet was offered ad libitum and food residue was removed and weighed.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Institut für Tiergesundheit und Lebensmittelqualität GmbH, 24107 Kiel, Germany). Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data.
Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm at a room temperature of 22 °C ±3 °C (maximum range) and a relative humidity of 55% ±10% (maximum range). Deviations from the maximum range caused for example during cleaning procedures are dealt with in SOPs. The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL.
Environmental enrichment: The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
Drinking water: Drinking water was offered ad libitum. Drinking water is examined according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on Drinking Water 2001] by the Hamburger Wasserwerke, 20539 Hamburg, Germany, at least four times a year.
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001, Anlage 1' [German Regulations on Drinking Water 2001, Addendum 1].

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Vehicle / Control used: 0.5% aqueous hydroxypropyl methylcellulose gel (Batch no. 16H23-B02, Fagron Services B.V., 1911 DB UITGEEST, The Netherlands)
Vehicle:
other: aqueous hydroxypropyl methylcellulose gel
Details on oral exposure:
Administration volume: 10 mL/kg b.w./day
The administration formulations were continuously stirred throughout the entire administration procedure to ensure homogeneity. The formulations were administered by oral administration at a constant volume once daily. The dose of the test item was adjusted to the animal's 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.
Preparation of the test item formulations: The test item formulations were freshly prepared every day. The test item was suspended in the vehicle (0.5% aqueous hydroxypropyl methylcellulose gel) to the appropriate concentrations. Test item formulations with concentrations of 10, 30 and 100 mg Grilbond® IL 6 100%/mL were prepared. In order to avoid bubble formation, the test item was first moistened with a small amount of the vehicle and stirred slowly. Then the suspension was filled up to the final volume and stirred thoroughly for approximately 15 minutes.
The vehicle chosen is a commonly used vehicle for the preparation of homogenous suspensions for water-insoluble test items. The administration formulations were continuously stirred throughout the entire administration procedure to ensure homogeneity. The homogeneity and concentration of the administration formulations were monitored.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the administration formulations, samples of approximately 5 mL were taken at the following times and stored at ≤-20 °C until analyses:
On the first administration day (on test day 1): Analysis of concentration and homogeneity by sample collection from the top, middle and bottom of the container of each dose formulation (3 samples/test item group; number of samples is 3 × 3 = 9).
On the last administration day (on test day 90): Analysis of concentration during treatment always before administration to the last animal of the group (1 sample/test item group; number of samples is 1 x 3 = 3.
Therefore, the total number of all samples is 12 for dose analysis.
The samples were labelled with study number, test species, type of sample, concentration, test day, sampling time and date.
The analytical method was validated and the stability of the formulations at room temperature for 6 hours was confirmed by LPT Study No. 35348. The samples were analysed at LPT and results confirming homogeneity and stability are annexed to the original study report.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Frequency of administration: Once daily for 90 consecutive days by gavage
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control, no test substance applied
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
The animals were allocated to the test groups based on body weight by means of a computer-generated randomisation program.
Group 1: 0 mg/kg b.w./day, p.o. (vehicle control); 10 + 5 m and 10 + 5 f; animals 1 – 15 and 16 – 30 (includes 5 animals per sex for recovery period)
Group 2: 100 mg/kg b.w./day, p.o. (low dose); 10 m and 10 f; animals 31 – 40 and 41 – 50
Group 3: 300 mg/kg b.w./day, p.o. (mid dose); 10 m and 10 f; animals 51 – 60 and 61 – 70
Group 4: 1000 mg/kg b.w./day, p.o. (high dose); 10 + 5 m and 10 +5 f; animals 71 – 85 and 86 – 100 (includes 5 animals per sex for recovery period)
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection: The dose levels were selected in agreement with the sponsor based on the results of a 28-day dose-range finding study (LPT Study No. 35348). In this dose-range-finding study rats were treated with 100, 300 or 1000 mg N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide)/kg b.w./day by oral administration for 28 consecutive days.
None of the animals treated with 100, 300 or 1000 mg N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide)/kg b.w. revealed any change in behaviour or external appearance. None of the animals died or had to be sacrificed prematurely. No test item-related influence was noted on the body weight and the food and drinking water consumption for any group during the course of the study. The macroscopic inspection at necropsy did not reveal any abnormalities. Based on above results the aforementioned dose levels of N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide) were also selected for this main study.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Dated and signed records of all activities relating to the day-to-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: The animals were observed individually before and after each dosing for any signs of behavioural changes, reaction to treatment or illness. Cage-side 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. Animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals. Special attention was paid to ascertain any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity etc. No such effects were noted during the course of this study. Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (always on the first day of the test week), 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 of day, each time. 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.
- Neurological screening: In test weeks 13 and 17 (before any blood sampling for laboratory examinations), 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 (MEYER) and motor activity assessment were conducted in all animals approximately 2 hours after dosing outside the home cage as described below.
● Observational screening:
Righting reflex: The animal was grasped by its tail and flipped in the air (approx. 60 cm) above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet, in this case zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature: An electronic probe thermometer (with a blunt probe) was used to take a rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
Salivation: The animals were observed for discharge of clear fluid from mouth, most frequently seen as beads of moisture on lips in rats. Normal state is to see none, in which case a zero (0) was recorded. If present, a plus sign (+) was recorded.
Startle response: With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, in which case a zero (0) was recorded. If there was no response, a plus sign (+) was recorded.
Respiration: While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing: Rats are normally obligatory nose-breathers. Each animal was observed, whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded. Mouth breathing was documented by a plus sign (+).
Urination: When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyurea) with 3 being normal.
Convulsions: If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.
Pilo-erection: The fur of the animal's back was observed, whether it was raised or elevated. In the normal animal no pilo-erection should be observed and a score of zero (0) was recorded. If pilo-erection was present, a plus sign (+) was recorded.
Diarrhoea: In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. Normal state is for there to be none (0), in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
Pupil size: It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
Pupil response: The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign was recorded.
Lacrimation: The animal was observed for the secretion and discharge of tears. In rats the tears contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded. If a discharge was present, a plus sign was recorded.
Impaired gait: The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).
Stereotypy: Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns, occurring out of context and with an abnormal high frequency). These were graded on a scale of 0 (absent = normal) to 5 (marked).
Toe pinch: The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.
Tail pinch: The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale of 1 to 5 with 3 being the normal response.
Wire maneuver: The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score of from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
Hind leg splay: Using an ink pad, the hind paws were marked with ink. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured.
Positional passivity: The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
Tremors: Periods of continued fine movements, usually starting in the limbs (and perhaps limited to them). The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
Positive geotropism: The animal was placed on the inclined (at an angle of approx. 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face 'up-hill', in which case a score of zero (0) was recorded. If this did not occur, a negative sign was recorded.
Limb rotation: One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly in-creased resistance or rigidity (5), with 3 being normal.
Auditory function: Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+). If there was no response a zero (0) was recorded.

- Functional tests
Grip strength: Prior to testing, the gauge (Chatillon, Model DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus was adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge had been zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. The animal continued to be pulled along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.
Locomotor activity: The motility was measured using the TSE InfraMot system . The infrared sensor was mounted on top of the home cage and any movements were measured for the duration of 12 minutes by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.
- Mortality: Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure was followed except that the final check was carried out at approximately 4:00 p.m. None of the animals died or had to be sacrificed prematurely.
- 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. From these data the food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day) = (Total food given (g) - Total food left (g)) / Number of animal days x Body weight (kg)
Drinking water consumption was monitored by daily visual appraisal throughout the study.
- Laboratory examinations: Blood samples were taken from the retrobulbar venous plexus under 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, and Li-Heparin anticoagulant (plasma) for clinical chemistry tests
Haematology: Blood samples were taken at the following times and the parameters listed below were determined:
• At the end of the treatment period (on the day of dissection, test day 91): All main study animals
• At the end of the recovery period (on the day of dissection, test day 119): All recovery animals
Haematological parameters assessed were: Haemoglobin content (HGB), Erythrocytes (RBC), Leucocytes (WBC), Reticulocytes (Reti), Platelets (PLT), Haematocrit value (HCT), Differential blood count (relative), Differential blood count (absolute), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC).
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.
Coagulation parameters: Blood samples were taken at the following times and the parameters listed below were determined:
• At the end of the treatment period (on the day of dissection, test day 91): All main study animals
• At the end of the recovery period (on the day of dissection, test day 119): All recovery animals
Coagulation parameters assessed are Prothrombin time (PT), and Activated partial thromboplastin time (aPTT).
Clinical chemistry: Blood samples were taken at the following times and the parameters listed below were determined:
• At the end of the treatment period (on the day of dissection, test day 91): All main study animals
• At the end of the recovery period (on the day of dissection, test day 119): All recovery animals
Clinical chemistry parameters assessed were Albumin, Globulin, Albumin/globulin ratio, Bile acids, Bilirubin (total), Cholesterol (total), Creatinine, Glucose, Protein (total), Triglycerides, Urea (in blood), Calcium, Chloride, Potassium, Sodium, Alanine amino-transferase (ALAT) , Alkaline phosphatase (aP), Aspartate aminotransferase (ASAT), Lactate dehydrogenase (LDH).

- Ophthalmological and auditory examinations
Examinations were performed on all animals before first dosing, at the end of the main study (in test week 13) and at the end of the recovery period (in test week 17): The eyes were examined with a HEINE ophthalmoscope. After examination of the pupillary reflex, mydriasis was produced by instillation of STULLN® eye drops onto the cornea. The following ocular structures were then examined: Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva, Cornea, anterior chamber, and 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 allocated to the recovery period were dissected on test day 119. The animals were euthanized by carbon dioxide (CO2) inhalation, 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, Brain, Epididymis, Heart, Kidney, Liver, Ovary, Spleen, Testicle, Thymus, Uterus (including cervix). Paired organs were weighed individually and identified as left or right. Blood smears prepared from all animals for haematological examination are available for possible examination of pathological changes but will be examined and evaluated only depending on necropsy findings and upon agreement of the Sponsor.
Organ preservation and histotechnique: The following organs or parts of organs with the exception of the eyes and testes of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution and the testes in modified Davidson's solution for optimum fixation.
Tissues collected for preservation / Histopathology were: 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 (left and right ventricle, septum), Intestine, large (colon, rectum), Intestine, small (duodenum, jejunum, ileum, including Peyer´s patches), Swiss roll method, Kidney and ureter (2), Liver (2 lobes), 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), Ovary and oviducts (2), Pancreas, Pituitary, Prostate and seminal vesicles with coagulating glands, Salivary glands (mandibular, parotid, sublingual), Skin (left flank), Spinal cord (3 sections: cervical, mid-thoracic, lumbar), Spleen, Stomach, Testicle (2), Thymus, Thyroid (2) (including parathyroids), Tissue masses or tumours (including regional lymph nodes), Trachea (including larynx), Urinary bladder, Uterus (including cervix), Vagina.
The afore-listed organs of all group 1 and 4 main study animals were examined histologically after preparation of haematoxylin-eosin stained paraffin sections. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined as well. The examination of the recovery animals is optional. So far, no evaluation of the recovery animals was performed.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
- Histopathological evaluation
The histotechnique was performed by LPT. The slides (labelled with study number, test species, animal number, block number) were dispatched to Test Site 1 (InSight Pathology BV) on 28 September 2018 via courier. The transport of slides to the histopathology Test Site was arranged by LPT, whereas the return transport to the Test Facility will be arranged by the Test Site. The study phase was recorded under the Test Site reference number ISP18221 according to the relevant ISP SOPs. The Principal Investigator performed the required histopathological evaluation (i.e. the organs and tissues as defined in the Study Plan and any subsequent Study Plan amendments) and entered the microscopic findings into the PathData system (version V6.2e). The Individual Animal Pathology Table and the Histopathology Incidence Table were created automatically by the PathData system. In the pathology Phase Report, the Principal Investigator included the tables mentioned above and reported/interpreted the terminal body weight changes, the organ weight changes (absolute and relative to body weight), the macroscopic findings and the microscopic findings. If an organ weight change or a macroscopic finding could be consistently correlated with a microscopic finding, then this was also included in the narrative. The report of this phase of the study comprised a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comment/review. Comments were addressed and the Draft Final Phase Report was presented to the Test Site Quality Assurance Unit (TSQAU) for auditing. The audited Draft Final Phase Report was sent electronically to the Study Director who will provide documented approval on the contents of the Phase Report by fax or e-mail to the Principal Investigator.
Statistics:
Data for toxicology and pathology were captured using the departmental computerized systems (Provantis® Integrated Preclinical Software, version 9.4.0, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs and test item groups 2 to 4 were compared to the control group 1.
The following statistical methods were used:
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 chemistry/Relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
Exact test of R. A. FISHER (if applicable): Histopathology (p ≤ 0.05)
The following settings were used for the statistical evaluation of the parametrical values captured by Provantis (see flow chart of decision tree on the next page):
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s 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), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
The following statistical methods were used for the data not captured with the Provantis system: STUDENT's t-test - Numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p  0.05 and p  0.01); 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)
These statistical procedures were used for all data. Significantly different data are indicated in the text tables of the results section and in the result tables of the tables section of this report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
All parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination on test day 1. All male and female control animals revealed normal values for each parameter set examined throughout the course of the study. None of the animals treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration for 90 days revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13.
Mortality:
no mortality observed
Description (incidence):
None of the male and female rats died or had to be sacrificed prematurely after repeated oral administration of 100, 300 or 1000 mg test substance/day during the 90 day treatment period or during the 28 day treatment-free recovery period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration compared to the control group, neither during the 90 day treatment period nor during the 28 day treatment-free recovery period. See also tables below and graphical display attached.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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 substance/kg b.w./day by oral administration compared to the control group, neither during the 90 day treatment period nor during the 28 day treatment-free recovery period.
Food efficiency:
no effects observed
Description (incidence and severity):
As food consumption and body weight development were not affected, food efficiency is considered non affected too.
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):
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 substance/kg b.w./day by oral administration for 90 days or of the recovery animals previously treated with 1000 mg test substance/kg b.w./day by oral administration. See also tables below!
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on any of the haematological parameters of the male and female rats treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration 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.
A slight decrease in neutrophilic granulocytes (absolute) observed in females (p ≤ 0.01) at the end of the recovery period compared to controls was not considered of biological relevance.
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 prothrombin time (PT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC). See also tables below!
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted on any of the clinical chemistry parameters of the male and female rats treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration 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.
No test item-related influence was noted for the plasma levels of albumin and globulin, the albumin/globulin ratio, the plasma levels of bile acids, bilirubin (total), cholesterol (total), 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 test item-relatedly influenced. See also tables below!
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
None of the male and female rats treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration revealed any changes of behaviour or external appearance during the 90 day treatment period or during the 28 day treatment-free recovery period. 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 substance/kg b.w./day by oral administration, 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 substance/kg b.w./day at the end of the recovery period in test week 17. An increase in forelimb grip strength observed in males of the low-dose group at the end of the treatment period was considered non-treatment related in lack of any dose dependency. See also tables below!
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the relative and absolute organ weights of the male and female animals treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration for 90 days compared to the control group at the end of the treatment period on test days 91 nor at the end of the 28 day treatment-free recovery period on test day 119.
Statistically significant differences in relative and absolute organ weights compared to the control which are not considered to be test item-related were as follows: Increased relative thymus weight (p ≤ 0.05) in mid dose group females with only slight alteration compared to control, considered of no biological relevance; Increased relative uterus weight (p ≤ 0.05) in low dose group females, lacking dose dependency, considered not treatment-related; Increased absolute uterus weight (p ≤ 0.01) in low dose group females, lacking dose dependency, considered not treatment-related. See also tables below!
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related influence was noted for the male and female rats treated with 100, 300 or 1000 mg test substance/kg b.w./day by oral administration for 90 days at terminal sacrifice on test day 91.
A very few macroscopic changes were noted in the liver (increased lobular pattern - one animal in low dose group / yellowish discoloured - one animal in high dose group), stomach (adhered to liver, tissue enlargement - one animal in low dose group) and/or uterine horns (dilated, filled with clear liquid - one control female and 1 high dose female in recovery group as well as one female in low dose group) in individual animals of the test item-treated groups. These changes were considered to be incidental findings due to the low number of animals affected.
Following the recovery period, no test item-related pathological changes were observed in the organs or tissues of the male and female rats previously treated with 1000 mg test substance/kg b.w./day by oral administration for 90 days at the end of the 28 day treatment-free recovery period. See also tables below!
Macroscopic changes in form of a dilated uterus were noted in individual animals of the previously test item-treated group as well as of the control group and were considered to be an incidental finding.
Neuropathological findings:
no effects observed
Description (incidence and severity):
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 substance/kg b.w./day by oral administration, 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.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Daily repeated oral administration of the test item at dose levels of 0, 100 mg/kg b.w., 300 mg/kg b.w. and 1000 mg/kg b.w. for a period of 90 days did not result in test item-related mortalities, changes in body weight, organ weights, gross findings or in relevant histopathologic findings in a broad range of organs and tissues.
Because of the lack of relevant histopathologic changes and the lack of a dose-response, the increase in relative mean thymic weight in males of the mid dose group is considered to represent an incidental finding.
The apparent test-item related increase in the incidence of perivascular mononuclear inflammatory cell infiltrates in the lungs of high dose animals is believed to be the result of reflux after administration of the relative high dosing volume (10 mL/kg b.w.) and not directly related to the test-item, also because the nature and severity of the findings was similar in affected high dose and control animals.
Based on the pathology, the systemic No-Observed-Adverse-Effect-Level (NOAEL) is considered to be 1000 mg test item/kg b.w./day.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
The faeces of all animals were of normal consistency throughout the experimental period. There was no indication of any impairment to auditory acuity. See also tables below!
Details on results:
The results of the analysis revealed that the test item formulations were correctly prepared and were very homogenous. The measured concentrations ranged from 97.4% to 102.9% (low dose), from 95.6% to 103.4% (mid dose) and from 101.0% to 106.4 (high dose) on test day 1 and/or on test day 90. The results were within the admissible limits of 90% to 110%. Values recorded incl. assessment of significant outliers are presented in tables below.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Detailed Clinical Observations Males [number of affected animals / number animals examined]

Males

Group

Pre-dose

week

Criteria

1

2

3

4

5

6

7

8

9

10

11

12

13

External observations

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Body posture

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Movement/
coordination

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Behaviour

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

 

Detailed Clinical Observations Females [number of affected animals / number animals examined]

Males

Group

Pre-dose

week

Criteria

1

2

3

4

5

6

7

8

9

10

11

12

13

External observations

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Body posture

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Movement/
coordination

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

Behaviour

1

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

2

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

3

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

0/10

4

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

0/15

 

Functional observations males [number affected animals/number assessed animals]

Parameter

Group 1

Group 2

Group 3

Group 4

Salivation

TW 13

TW 17

TW 13

TW 13

TW 13

TW 17

Respiration

0/15

0/5

0/10

0/10

0/15

0/5

Mouth breathing

0/15

0/5

0/10

0/10

0/15

0/5

Convulsion

0/15

0/5

0/10

0/10

0/15

0/5

Pilo-erection

0/15

0/5

0/10

0/10

0/15

0/5

Diarrhoea

0/15

0/5

0/10

0/10

0/15

0/5

Lacrimation

0/15

0/5

0/10

0/10

0/15

0/5

Impaired gait

0/15

0/5

0/10

0/10

0/15

0/5

Stereotypy

0/15

0/5

0/10

0/10

0/15

0/5

Tremors

0/15

0/5

0/10

0/10

0/15

0/5

Startle response

0/15

0/5

0/10

0/10

0/15

0/5

Auditory function

0/15

0/5

0/10

0/10

0/15

0/5

Toe pinch

0/15

0/5

0/10

0/10

0/15

0/5

Tail pinch

0/15

0/5

0/10

0/10

0/15

0/5

Urination

0/15

0/5

0/10

0/10

0/15

0/5

Limb rotation

0/15

0/5

0/10

0/10

0/15

0/5

Wire maneuver

0/15

0/5

0/10

0/10

0/15

0/5

Positional passivity

0/15

0/5

0/10

0/10

0/15

0/5

Positive geotropism

0/15

0/5

0/10

0/10

0/15

0/5

Righting reflex

0/15

0/5

0/10

0/10

0/15

0/5

Body temperature

37.4 °C

37.3 °C

37.3 °C

37.3 °C

37.3 °C

37.3 °C

Hind leg splay

6.8 cm

8.6 cm

6.4 cm

6.3 cm

6.4 cm

8.5 cm

Pupil size

0/15

0/5

0/10

0/10

0/15

0/5

Pupil response

0/15

0/5

0/10

0/10

0/15

0/5

 

Functional observations females [number affected animals/number assessed animals]

Parameter

Group 1

Group 2

Group 3

Group 4

Salivation

TW 13

TW 17

TW 13

TW 13

TW 13

TW 17

Respiration

0/15

0/5

0/10

0/10

0/15

0/5

Mouth breathing

0/15

0/5

0/10

0/10

0/15

0/5

Convulsion

0/15

0/5

0/10

0/10

0/15

0/5

Pilo-erection

0/15

0/5

0/10

0/10

0/15

0/5

Diarrhoea

0/15

0/5

0/10

0/10

0/15

0/5

Lacrimation

0/15

0/5

0/10

0/10

0/15

0/5

Impaired gait

0/15

0/5

0/10

0/10

0/15

0/5

Stereotypy

0/15

0/5

0/10

0/10

0/15

0/5

Tremors

0/15

0/5

0/10

0/10

0/15

0/5

Startle response

0/15

0/5

0/10

0/10

0/15

0/5

Auditory function

0/15

0/5

0/10

0/10

0/15

0/5

Toe pinch

0/15

0/5

0/10

0/10

0/15

0/5

Tail pinch

0/15

0/5

0/10

0/10

0/15

0/5

Urination

0/15

0/5

0/10

0/10

0/15

0/5

Limb rotation

0/15

0/5

0/10

0/10

0/15

0/5

Wire maneuver

0/15

0/5

0/10

0/10

0/15

0/5

Positional passivity

0/15

0/5

0/10

0/10

0/15

0/5

Positive geotropism

0/15

0/5

0/10

0/10

0/15

0/5

Righting reflex

0/15

0/5

0/10

0/10

0/15

0/5

Body temperature

37.4 °C

37.3 °C

37.4 °C

37.4 °C

37.3 °C

37.3 °C

Hind leg splay

5.3 cm

7.3 cm

4.4 cm

5.0 cm

5.0 cm

7.6 cm

Pupil size

0/15

0/5

0/10

0/10

0/15

0/5

Pupil response

0/15

0/5

0/10

0/10

0/15

0/5

 

Grip strength [g] summary (* p ≤ 0.05)

 

 

Group 1

Group 2

Group 3

Group 4

forelimb

Males TW 13

mean

555.5

612.2

534.8

572.4

SD

54.4

45.5

34.8

57.6

t

-

2.717*

not significant

not significant

Males TW 17

mean

778.8

-

-

803.2

SD

21.7

-

-

18.1

t

-

-

-

not significant

Females TW13

Mean

508.7

492.2

469.6

507.2

SD

61.0

61.1

54.7

57.7

t

-

not significant

not significant

not significant

Females TW 17

mean

690.7

-

-

684.9

SD

59.0

-

-

24.6

t

-

-

-

not significant

hindlimb

Males TW 13

Mean

174.3

171.4

168.7

168.2

SD

21.8

18.8

14.4

24.7

t

-

not significant

not significant

not significant

Males TW 17

292.5

-

-

275.7

 

SD

29.8

-

-

22.0

t

-

-

-

not significant

Females TW13

mean

162.1

157.2

157.2

161.9

SD

16.9

21.3

21.9

17.6

t

-

not significant

not significant

not significant

Females TW 17

Mean

246.6

-

-

243.0

SD

25.9

-

-

19.2

t

-

-

-

not significant

 

Spontaneous motility [number of movements/12 min] summary

 

 

Group 1

Group 2

Group 3

Group 4

Males TW 13

mean

56.6

71.6

58.9

56.4

SD

49.3

61.7

52.1

46.8

t

-

not significant

not significant

not significant

Males TW 17

mean

38.6

-

-

37.6

SD

38.7

-

-

38.6

t

-

-

-

not significant

Females TW13

Mean

56.9

63.1

74.1

70.5

SD

47.8

64.4

60.2

56.6

t

-

not significant

not significant

not significant

Females TW 17

mean

51.6

-

-

63.0

SD

41.0

-

-

65.5

t

-

-

-

not significant

 

Statistically significant changes of neurological screening parameters unrelated to the test item

Parameter

Increase/Decrease

Group/sex

Test week

Statistical significance

Reason

Forelimb grip strength

2 m

13

p ≤ 0.05

B

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

 

Body weight [g] males, summary

 

Day(s) Relative to Start Date

-8

1

8

15

22

29

36

43

50

57

64

71

78

85

90

97

104

111

118

Group 1:

mean

207.25

302.55

351.36

382.07

410.85

434.29

460.93

483.17

506.04

522.03

537.67

550.29

562.35

569.25

79.06

570.82

577.36

590.84

604.12

SD

7.47

9.32

15.07

22.68

29.85

30.63

33.30

37.38

36.93

40.15

39.68

41.10

41.83

41.56

43.66

36.29

39.64

37.20

39.88

N

15

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

Group 2:

mean

208.54

303.88

353.42

392.64

426.25

449.12

475.31

495.86

520.08

538.80

557.55

569.26

580.63

591.05

595.46

-

-

-

-

SD

8.20

10.28

9.31

11.03

14.50

14.76

19.91

21.58

22.75

23.08

26.50

29.22

30.62

33.67

35.25

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

%Diff

0.6

0.4

0.6

2.8

3.7

3.4

3.1

2.6

2.8

3.2

3.7

3.4

3.3

3.8

2.8

-

-

-

-

Group 3:

mean

208.55

300.70

340.94

377.24

407.21

423.03

446.36

462.09

489.55

507.73

522.78

536.36

548.71

550.85

559.90

-

-

-

-

SD

9.27

8.01

14.23

15.05

19.18

21.43

26.70

28.51

29.79

34.06

35.68

36.27

39.48

37.36

39.85

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

%Diff

0.6

-0.6

-3.0

-1.3

-0.9

-2.6

-3.2

-4.4

-3.3

-2.7

-2.8

-2.5

-2.4

-3.2

-3.3

-

-

-

-

Group 4:

mean

207.57

301.61

346.75

384.15

410.31

430.84

455.55

475.86

498.11

515.26

530.11

542.57

553.90

563.62

570.39

595.06

605.82

614.74

627.70

SD

7.47

8.89

13.14

20.99

26.86

31.19

34.45

35.23

38.28

40.73

39.36

40.01

42.67

42.95

43.70

57.77

51.18

62.41

62.80

N

15

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

%Diff

0.2

-0.3

-1.3

0.5

-0.1

-0.8

-1.2

-1.5

-1.6

-1.3

-1.4

-1.4

-1.5

-1.0

-1.5

4.2

4.9

4.0

3.9

 

Body weight [g] females, summary

 

Day(s) Relative to Start Date

-8

1

8

15

22

29

36

43

50

57

64

71

78

85

90

97

104

111

118

Group 1:

mean

199.61

246.95

266.71

275.36

287.71

294.76

302.59

304.41

312.15

321.86

323.93

321.61

328.00

331.48

329.42

351.26

352.46

360.34

356.12

SD

8.30

12.11

12.44

11.93

13.90

18.00

17.66

19.32

23.73

23.43

22.52

23.86

25.83

26.93

29.48

26.15

29.58

26.18

23.44

N

15

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

Group 2:

mean

200.20

247.61

261.30

274.47

283.73

294.75

302.67

306.72

315.58

325.98

327.63

328.11

335.48

336.75

336.98

-

-

-

-

SD

8.06

5.83

4.74

7.60

10.92

8.43

9.66

10.55

14.72

16.35

17.12

18.87

17.00

21.89

21.86

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

%Diff

0.3

0.3

-2.0

-0.3

-1.4

0.0

0.0

0.8

1.1

1.3

1.1

2.0

2.3

1.6

2.3

-

-

-

-

Group 3:

mean

198.90

244.28

255.31

269.80

278.77

286.47

293.83

299.65

306.39

312.55

315.43

317.89

323.06

326.76

326.35

-

-

-

-

SD

7.60

9.79

13.63

14.70

15.85

18.48

19.87

16.47

18.00

21.75

20.54

22.57

20.68

21.19

19.91

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

%Diff

-0.4

-1.1

-4.3

-2.0

-3.1

-2.8

-2.9

-1.6

-1.8

-2.9

-2.6

-1.2

-1.5

-1.4

-0.9

-

-

-

-

Group 4:

mean

199.21

244.62

260.45

272.80

282.40

289.45

297.21

303.32

310.23

316.78

320.37

322.45

325.28

327.85

326.97

345.42

348.18

356.12

352.52

SD

7.97

6.97

10.78

11.07

13.39

15.47

15.13

15.71

17.14

20.40

20.27

19.58

19.88

20.57

18.95

15.47

20.07

25.53

23.06

N

15

15

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

%Diff

-0.2

-0.9

-2.3

-0.9

-1.8

-1.8

-1.8

-0.4

-0.6

-1.6

-1.1

0.3

-0.8

-1.1

-0.7

-1.7

-1.2

-1.2

-1.0

 

Body weight gain, males [%]

 

Day(s) Relative to Start Date

1 → 8

1 → 15

1 → 22

1 → 29

1 → 36

1 → 43

1 → 50

1 → 57

1 → 64

1 → 71

1 → 78

1 → 85

1 → 90

90 → 97

90 → 104

90 → 111

90 → 118

Group 1:

Mean

16.12

26.26

35.73

43.49

52.27

59.62

67.16

72.44

77.62

81.78

85.77

88.04

91.29

1.13

2.27

4.68

7.02

SD

2.94

5.95

7.98

8.14

8.52

9.95

9.14

10.34

10.15

10.44

10.75

10.43

11.21

1.60

2.16

2.05

2.01

N

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

Group 2:

Mean

16.34

29.29

40.38

47.92

56.57

63.37

71.37

77.55

83.72

87.60

91.40

94.81

96.24

-

-

-

-

SD

2.14

4.37

5.96

6.43

8.35

9.54

10.48

10.95

11.93

13.11

14.47

14.81

15.00

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

Group 3:

Mean

13.39

25.49

35.48

40.80

48.58

53.83

62.97

69.04

74.04

78.56

82.69

83.37

86.41

-

-

-

-

SD

3.91

4.97

6.86

8.52

10.40

11.30

11.79

13.42

13.84

14.05

15.38

14.29

15.49

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

Group 4:

Mean

14.97

27.35

36.01

42.83

51.01

57.76

65.14

70.80

75.75

79.91

83.67

86.90

89.14

1.07

3.00

4.38

6.60

SD

2.89

5.52

7.32

9.22

10.17

10.57

11.55

12.01

11.90

12.43

13.40

13.66

13.79

1.01

1.51

1.20

1.66

N

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

 

Body weight gain, females [%]

 

Day(s) Relative to Start Date

1 → 8

1 → 15

1 → 22

1 → 29

1 → 36

1 → 43

1 → 50

1 → 57

1 → 64

1 → 71

1 → 78

1 → 85

1 → 90

90 → 97

90 → 104

90 → 111

90 → 118

Group 1:

Mean

8.05

11.66

16.63

19.41

22.65

23.40

26.48

30.36

31.27

30.34

32.89

34.34

33.47

1.38

1.64

3.99

2.82

SD

2.73

5.55

5.45

5.47

6.77

7.87

8.64

7.55

8.48

9.06

9.41

10.39

11.10

2.79

1.43

1.57

1.78

N

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

Group 2:

Mean

5.55

10.87

14.59

19.06

22.27

23.91

27.48

31.70

32.39

32.57

35.55

36.09

36.19

-

-

-

-

SD

1.79

2.63

3.47

3.03

4.13

4.62

6.01

6.92

7.64

8.15

7.55

9.75

9.88

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

Group 3:

Mean

4.53

10.49

14.15

17.28

20.31

22.73

25.49

27.95

29.15

30.13

32.28

33.83

33.65

-

-

-

-

SD

4.05

5.17

5.34

5.90

6.84

6.11

6.78

6.99

6.87

7.21

7.07

7.96

7.01

-

-

-

-

N

10

10

10

10

10

10

10

10

10

10

10

10

10

-

-

-

-

Group 4:

Mean

6.46

11.53

15.42

18.31

21.51

24.01

26.82

29.47

30.96

31.82

32.95

33.99

33.65

2.32

3.10

5.42

4.37

SD

2.83

3.48

3.63

4.97

5.41

5.80

5.98

7.05

7.38

7.21

6.82

6.91

6.58

1.03

1.87

4.09

3.15

N

15

15

15

15

15

15

15

15

15

15

15

15

15

5

5

5

5

 

 

Haematological parameters males, summary, relative to start date

 

 

HGB
(mmol/L)

RBC
(*10⁶/µL)

WBC
(*10³/µL)

Reti
(%)

PLT
(*10³/µL)

HCT
(%)

Neut
(%)

Lym
(%)

Mono
(%)

Eos
(%)

LUC
(%)

Baso
(%)

PT
(s)

aPTT
(s)

MCV
(fL)

MCH
(fmol)

MCHC
(mmol/L)

Group 1, day 91:

mean

9.29

9.340

10.653

2.08

730.1

46.05

17.55

76.48

2.18

2.68

0.76

0.33

9.73

14.25

49.37

0.999

20.205

SD

0.38

0.600

1.619

0.46

134.2

2.13

3.94

4.56

0.53

0.75

0.23

0.11

0.45

0.56

1.80

0.041

0.227

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, day 91:

mean

9.08

8.957

11.420

2.43

877.8

45.31

16.38

78.21

2.43

2.03

0.66

0.29

9.93

14.53

50.60

1.014

20.070

SD

0.22

0.254

2.482

0.34

175.4

1.02

4.68

5.36

0.67

0.88

0.25

0.07

0.40

1.00

0.57

0.017

0.203

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-2.3

-4.1

7.2

16.8

20.2

-1.6

-6.7

2.3

11.5

-24.3

-13.2

-12.1

2.1

2.0

2.5

1.5

-0.7

Group 3, day 91:

mean

9.32

9.112

11.486

2.30

821.4

46.47

20.27

74.00

2.96

1.76

0.71

0.28

9.63

14.49

50.98

1.020

20.044

SD

0.42

0.397

1.298

0.75

106.5

2.24

7.41

8.33

1.10

0.75

0.36

0.04

0.39

1.13

1.00

0.022

0.138

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

0.3

-2.4

7.8

10.6

12.5

0.9

15.5

-3.2

35.8

-34.3

-6.6

-15.2

-1.0

1.7

3.3

2.1

-0.8

Group 4, day 91:

mean

9.25

9.080

10.523

1.98

866.9

45.64

17.84

76.94

2.31

1.79

0.76

0.30

9.76

14.68

50.30

1.018

20.243

SD

0.35

0.333

2.604

0.42

99.4

1.87

5.25

5.77

0.71

0.47

0.30

0.09

0.25

0.36

1.80

0.036

0.183

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-0.4

-2.8

-1.2

-4.8

18.7

-0.9

1.7

0.6

6.0

-33.2

0.0

-9.1

0.3

3.0

1.9

1.9

0.2

Group 1, day 119:

mean

8.96

9.104

11.364

2.46

782.4

44.68

22.14

72.12

3.02

2.00

0.46

0.30

9.80

14.44

49.06

0.982

20.020

SD

0.30

0.222

2.809

0.30

139.0

1.34

4.19

5.19

0.99

0.48

0.09

0.07

0.46

0.52

1.01

0.022

0.211

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, day 119:

mean

8.92

8.850

11.998

2.70

858.8

44.16

20.36

74.50

2.72

1.56

0.54

0.32

10.08

14.12

49.92

1.006

20.166

SD

0.24

0.203

1.674

0.49

179.0

0.96

6.54

6.44

1.02

0.44

0.30

0.04

0.39

0.59

2.13

0.045

0.191

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-0.4

-2.8

5.6

9.8

9.8

-1.2

-8.0

3.3

-9.9

-22.0

17.4

6.7

2.9

-2.2

1.8

2.4

0.7

 

Haematological parameters females, summary, relative to start date

 

 

HGB
(mmol/L)

RBC
(*10⁶/µL)

WBC
(*10³/µL)

Reti
(%)

PLT
(*10³/µL)

HCT
(%)

Neut
(%)

Lym
(%)

Mono
(%)

Eos
(%)

LUC
(%)

Baso
(%)

PT
(s)

aPTT
(s)

MCV
(fL)

MCH
(fmol)

MCHC
(mmol/L)

Group 1, day 91:

mean

8.83

8.381

7.050

1.54

936.7

42.71

14.29

80.73

2.60

1.46

0.68

0.22

9.02

14.96

51.01

1.054

20.672

SD

0.31

0.338

1.248

0.40

153.1

1.55

3.39

3.79

0.57

0.42

0.15

0.08

0.30

0.50

1.67

0.039

0.248

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, day 91:

mean

8.71

8.234

6.459

1.81

892.1

42.48

14.39

80.49

2.66

1.60

0.65

0.24

9.25

15.25

51.59

1.055

20.459

SD

0.45

0.304

1.520

0.48

156.1

1.84

5.02

5.52

0.74

0.58

0.19

0.07

0.16

0.76

1.33

0.034

0.286

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-1.4

-1.8

-8.4

17.5

-4.8

-0.5

0.7

-0.3

2.3

9.6

-4.4

9.1

2.5

1.9

1.1

0.1

-1.0

Group 3, day 91:

mean

8.89

8.414

6.693

1.75

980.3

43.03

12.36

82.79

2.25

1.69

0.65

0.27

9.04

14.88

51.17

1.058

20.655

SD

0.31

0.377

2.295

0.36

154.6

1.63

3.01

3.37

0.66

0.66

0.26

0.12

0.23

0.59

1.51

0.032

0.218

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

0.7

0.4

-5.1

13.6

4.7

0.7

-13.5

2.6

-13.5

15.8

-4.4

22.7

0.2

-0.5

0.3

0.4

-0.1

Group 4, day 91:

mean

9.01

8.487

5.436

1.74

890.7

43.89

15.31

78.32

2.47

3.00

0.67

0.23

8.77

14.84

51.74

1.063

20.534

SD

0.31

0.298

1.725

0.31

199.5

1.53

4.80

6.30

0.97

1.73

0.21

0.07

0.21

0.59

1.63

0.035

0.213

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

2.0

1.3

-22.9

13.0

-4.9

2.8

7.1

-3.0

-5.0

105.5

-1.5

4.5

-2.8

-0.8

1.4

0.9

-0.7

Group 1, day 119:

mean

8.84

8.360

8.416

1.64

854.2

42.42

16.30

76.44

3.52

2.88

0.54

0.30

8.88

15.40

50.76

1.058

20.792

SD

0.34

0.395

1.122

0.18

135.1

1.69

4.60

4.85

1.08

1.39

0.18

0.14

0.28

1.11

0.73

0.016

0.157

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, day 119:

mean

8.66

8.086

7.218

1.60

924.6

41.98

11.00

82.80

2.74

2.42

0.72

0.32

8.92

15.62

51.92

1.072

20.672

SD

0.25

0.227

1.033

0.29

172.6

1.15

1.27

1.61

0.71

1.04

0.37

0.08

0.18

0.65

1.13

0.029

0.108

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-2.0

-3.3

-14.2

-2.4

8.2

-1.0

-32.5

8.3

-22.2

-16.0

33.3

6.7

0.5

1.4

2.3

1.3

-0.6

 

Statistically significant changes in haematological parameters unrelated to the test item

Parameter

Increase/Decrease

Group/sex

Test day

Statistical significance

Reason

Neutrophilic granulocytes (absolute)

4 f

119

p ≤ 0.01

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

 

Biochemical parameters, males, summary

 

 

Albumin (g/L)

Globulin (g/L)

Alb./Glob. Ratio

Bile Acids (µmol/L)

Bilirubin (total) (µmol/L)

Cholesterol (total) (mmol/L)

Creatinine (µmol/L)

Glucose (mmol/L)

Protein (total)(g/L)

Triglycerides (mmol/L)

Urea (in blood) (mmol/L)

Calcium (mmol/L)

Chloride (mmol/L)

Potassium (mmol/L)

Sodium (mmol/L)

ALAT (U/L)

aP (U/L)

ASAT (U/L)

LDH (U/L)

Group 1, day 91:

mean

30.39

31.71

0.960

9.44

3.21

1.668

38.1

7.164

62.1

0.668

4.081

2.742

99.8

3.774

138.3

37.4

90.6

80.3

71.2

SD

0.66

1.37

0.036

6.75

0.50

0.262

1.3

0.813

1.8

0.272

0.621

0.043

1.3

0.229

0.7

6.7

13.1

16.1

45.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, day 91:

mean

30.26

32.04

0.946

8.82

3.42

1.891

39.1

6.815

62.3

0.705

4.227

2.744

99.4

3.747

138.1

38.6

88.8

80.0

51.8

SD

0.53

1.36

0.034

5.46

0.50

0.238

2.0

0.516

1.7

0.361

0.498

0.072

1.2

0.155

0.9

8.3

16.1

11.7

17.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-0.4

1.0

-1.5

-6.6

6.5

13.4

2.6

-4.9

0.3

5.5

3.6

0.1

-0.4

-0.7

-0.1

3.2

-2.0

-0.4

-27.2

Group 3, day 91:

mean

30.01

30.79

0.976

11.43

3.64

1.700

38.9

7.612

60.8

0.599

4.264

2.720

99.7

3.766

137.8

40.9

82.2

77.9

63.0

SD

0.32

1.12

0.035

6.20

0.49

0.270

1.6

0.930

1.2

0.232

0.475

0.042

0.8

0.249

0.9

5.6

8.0

14.2

9.4

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-1.3

-2.9

1.7

21.1

13.4

1.9

2.1

6.3

-2.1

-10.3

4.5

-0.8

-0.1

-0.2

-0.4

9.4

-9.3

-3.0

-11.5

Group 4, day 91:

mean

30.37

30.83

0.990

12.32

3.22

1.798

39.6

7.350

61.2

0.696

4.342

2.714

100.2

3.756

138.5

41.2

85.1

84.4

61.0

SD

0.75

2.45

0.077

16.85

0.36

0.510

2.5

0.949

2.7

0.405

0.654

0.105

1.0

0.257

1.0

12.4

12.5

17.0

22.0

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-0.1

-2.8

3.2

30.5

0.3

7.8

3.9

2.6

-1.4

4.2

6.4

-1.0

0.4

-0.5

0.1

10.2

-6.1

5.1

-14.3

Group 1, day 119:

mean

30.62

32.58

0.944

8.80

3.46

2.022

42.6

6.880

63.2

0.712

4.018

2.740

101.2

3.774

138.2

40.8

88.0

80.4

62.6

SD

0.78

2.71

0.065

4.74

0.32

0.542

3.0

0.499

3.3

0.372

0.640

0.038

0.8

0.181

0.8

11.9

20.8

11.3

13.0

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, day 119:

mean

30.38

32.62

0.935

7.06

3.80

2.034

43.8

7.710

63.0

0.792

3.980

2.750

100.8

3.750

138.2

35.2

76.8

76.4

60.4

SD

1.24

2.77

0.050

3.88

0.61

0.423

1.1

0.892

3.9

0.351

0.204

0.076

0.8

0.239

0.4

3.1

4.3

10.4

11.3

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-0.8

0.1

-1.0

-19.8

9.8

0.6

2.8

12.1

-0.3

11.2

-0.9

0.4

-0.4

-0.6

0.0

-13.7

-12.7

-5.0

-3.5

 

Biochemical parameters, females, summary

 

 

Albumin (g/L)

Globulin (g/L)

Alb./Glob. Ratio

Bile Acids (µmol/L)

Bilirubin (total) (µmol/L)

Cholesterol (total) (mmol/L)

Creatinine (µmol/L)

Glucose (mmol/L)

Protein (total)(g/L)

Triglycerides (mmol/L)

Urea (in blood) (mmol/L)

Calcium (mmol/L)

Chloride (mmol/L)

Potassium (mmol/L)

Sodium (mmol/L)

ALAT (U/L)

aP (U/L)

ASAT (U/L)

LDH (U/L)

Group 1, day 91:

mean

35.31

34.19

1.035

26.50

3.97

2.400

43.9

6.923

69.5

0.366

4.364

2.870

100.9

3.394

138.5

81.4

40.6

175.4

82.2

SD

2.36

2.73

0.051

39.34

0.55

0.476

3.1

0.626

4.8

0.167

0.736

0.107

1.0

0.172

0.7

73.8

19.3

161.7

77.7

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, day 91:

mean

36.31

34.09

1.067

9.36

3.99

2.481

44.2

7.592

70.4

0.342

4.009

2.910

101.0

3.406

138.7

60.1

35.5

114.5

75.2

SD

2.40

2.62

0.040

4.73

0.87

0.334

2.8

0.778

4.9

0.088

0.364

0.107

1.1

0.175

1.3

33.2

13.1

64.0

45.1

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

2.8

-0.3

3.1

-64.7

0.5

3.4

0.7

9.7

1.3

-6.6

-8.1

1.4

0.1

0.4

0.1

-26.2

-12.6

-34.7

-8.5

Group 3, day 91:

mean

35.49

33.81

1.051

12.45

4.15

2.441

45.0

6.680

69.3

0.366

4.554

2.868

101.3

3.415

138.7

45.2

41.3

97.0

66.6

SD

2.55

2.19

0.065

7.39

0.73

0.311

3.2

0.504

4.2

0.080

0.631

0.091

0.9

0.276

0.8

10.8

10.1

12.4

18.5

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

0.5

-1.1

1.6

-53.0

4.5

1.7

2.5

-3.5

-0.3

0.0

4.4

-0.1

0.4

0.6

0.1

-44.5

1.7

-44.7

-19.0

Group 4, day 91:

mean

35.75

32.95

1.086

9.02

4.07

2.437

44.6

6.833

68.7

0.324

4.591

2.844

100.7

3.215

138.9

46.0

42.8

96.5

60.3

SD

1.73

1.93

0.045

4.23

0.63

0.593

1.5

0.578

3.4

0.041

0.388

0.103

0.9

0.251

0.6

14.3

7.5

23.7

21.3

N

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

1.2

-3.6

5.0

-66.0

2.5

1.5

1.6

-1.3

-1.2

-11.5

5.2

-0.9

-0.2

-5.3

0.3

-43.5

5.4

-45.0

-26.6

Group 1, day 119:

mean

39.10

36.50

1.073

13.66

4.46

2.894

46.2

7.390

75.6

0.644

4.288

2.986

100.2

3.408

137.2

43.6

28.2

96.8

79.0

SD

1.06

1.75

0.049

6.22

0.25

0.310

1.1

0.641

2.4

0.190

0.555

0.029

0.8

0.195

0.8

9.4

5.7

27.5

30.9

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, day 119:

mean

38.22

35.98

1.064

16.10

4.68

2.678

47.0

8.140

74.2

0.558

4.670

2.954

100.8

3.486

137.4

48.0

28.0

87.4

61.8

SD

1.56

2.48

0.046

13.11

0.99

0.408

3.2

0.804

3.8

0.332

0.267

0.105

1.6

0.223

0.9

29.2

3.9

31.1

18.4

N

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-2.3

-1.4

-0.8

17.9

4.9

-7.5

1.7

10.1

-1.9

-13.4

8.9

-1.1

0.6

2.3

0.1

10.1

-0.7

-9.7

-21.8

 

Ophthalmological Examination [number animals with findings/number animals examined]

 

TW 0

TW 13

TW 17

 

Left eye

right eye

Left eye

right eye

Left eye

right eye

Male Group 1

0/15

0/15

0/15

0/15

0/5

0/5

Male Group 2

0/10

0/10

0/10

0/10

-

-

Male Group 3

0/10

0/10

0/10

0/10

-

-

Male Group 4

0/15

0/15

0/15

0/15

0/5

0/5

Female Group 1

0/15

0/15

0/15

0/15

0/5

0/5

Female Group 2

0/10

0/10

0/10

0/10

-

-

Female Group 3

0/10

0/10

0/10

0/10

-

-

Female Group 4

0/15

0/15

0/15

0/15

0/5

0/5

 

Auditory Examination [number animals with findings/number animals examined]

 

TW 0

TW 13

TW 17

Male Group 1

0/15

0/15

0/5

Male Group 2

0/10

0/10

-

Male Group 3

0/10

0/10

-

Male Group 4

0/15

0/15

0/5

Female Group 1

0/15

0/15

0/5

Female Group 2

0/10

0/10

-

Female Group 3

0/10

0/10

-

Female Group 4

0/15

0/15

0/5

 

Macroscopic Post Mortem Findings

Animal no.

Sacrifice / Dead

Affected organs

Findings

Group 1 males

1 - 10

TS / TD 91

-

no pathological findings

11 – 15

RS / TD 119

-

no pathological findings

Group 2 males

31 – 35 & 37 - 40

TS / TD 91

-

no pathological findings

36

TS / TD 91

Liver

increased lobular pattern

Stomach

adhered to liver, tissue enlargement (yellow brown coloured, solid, diameter 8 mm)

Group 3 males

51 - 60

TS / TD 91

-

no pathological findings

Group 4 males

71 - 74 & 76 - 80

TS / TD 91

-

no pathological findings

75

TS / TD 91

Liver

yellowish discoloured

81 - 85

RS / TD 119

-

no pathological findings

Group 1 females

16 - 25

TS / TD 91

-

no pathological findings

26

RS / TD 119

Uterus

dilated

27 – 30

RS / TD 119

-

no pathological findings

Group 2 females

41

TS / TD 91

Uterine horns

dilated, filled with clear liquid

42 - 50

TS / TD 91

-

no pathological findings

Group 3 females

61 - 70

TS / TD 91

-

no pathological findings

Group 4 females

86 - 95

TS / TD 91

-

no pathological findings

96 & 98 - 100

RS / TD 119

-

no pathological findings

97

RS / TD 119

Uterus

dilated

 

TD = test day

TS = terminal sacrifice

RS = recovery sacrifice

 

Relative Organ Weights, males [g/kg bw]

 

Adren. gland (left)

Adren. gland (right)

Brain

Epididymis (left)

Epididymis (right)

Testis (left)

Testis (right)

Heart

Kidney (left)

Kidney (right)

Liver

Spleen

Thymus

Group 1, D91:

Mean

0.0594

0.0598

4.090

1.505

1.513

3.412

3.253

3.105

3.542

3.596

30.10

1.759

0.836

SD

0.0097

0.0075

0.230

0.197

0.156

0.346

0.442

0.271

0.278

0.237

1.67

0.217

0.154

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, D91:

Mean

0.0638

0.0613

3.868

1.437

1.469

3.119

3.107

3.068

3.526

3.590

30.61

1.754

0.829

SD

0.0114

0.0067

0.141

0.178

0.112

0.188

0.213

0.144

0.296

0.322

1.52

0.343

0.155

N

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

7.4

2.5

-5.4

-4.5

-2.9

-8.6

-4.5

-1.2

-0.5

-0.2

1.7

-0.3

-0.9

Group 3, D91:

Mean

0.0604

0.0607

4.194

1.418

1.542

3.341

3.347

3.192

3.738

3.790

29.62

1.865

0.800

SD

0.0141

0.0150

0.256

0.164

0.183

0.490

0.453

0.305

0.360

0.331

3.69

0.364

0.176

N

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

1.7

1.5

2.6

-5.7

1.9

-2.1

2.9

2.8

5.5

5.4

-1.6

6.0

-4.3

Group 4, D91:

Mean

0.0562

0.0635

4.068

1.443

1.490

3.365

3.405

3.100

3.460

3.508

30.21

1.844

0.860

SD

0.0106

0.0080

0.303

0.178

0.147

0.402

0.407

0.134

0.303

0.359

1.96

0.223

0.175

N

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-5.4

6.1

-0.5

-4.1

-1.5

-1.4

4.7

-0.2

-2.3

-2.4

0.4

4.8

2.8

Group 1, D119:

Mean

0.0605

0.0624

3.879

1.467

1.523

3.267

3.219

3.157

3.640

3.632

29.99

1.898

0.772

SD

0.0164

0.0124

0.295

0.188

0.179

0.094

0.136

0.294

0.299

0.308

2.43

0.319

0.158

N

5

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, D119:

Mean

0.0538

0.0513

3.735

1.357

1.445

3.073

3.045

3.135

3.350

3.442

28.51

1.687

0.701

SD

0.0104

0.0224

0.303

0.180

0.253

0.247

0.297

0.347

0.100

0.288

2.79

0.257

0.220

N

5

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-11.0

-17.8

-3.7

-7.5

-5.1

-5.9

-5.4

-0.7

-8.0

-5.2

-4.9

-11.1

-9.2

 

Relative Organ Weights, females [g/kg bw] (* = p ≤ 0.05)

 

Adren. gland (left)

Adren. gland (right)

Brain

Ovary (left)

Ovary (right)

Heart

Kidney (left)

Kidney (right)

Liver

Spleen

Thymus

Uterus incl. cervic

Group 1, D91:

Mean

0.1307

0.1282

6.579

0.1457

0.1502

3.627

3.566

3.658

29.92

1.779

0.984

2.458

SD

0.0120

0.0197

0.524

0.0389

0.0269

0.280

0.290

0.332

2.26

0.073

0.231

0.508

N

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, D91:

Mean

0.1217

0.1253

6.374

0.1175

0.1350

3.543

3.516

3.656

30.13

1.798

1.076

3.610*

SD

0.0177

0.0142

0.525

0.0425

0.0493

0.334

0.273

0.375

2.79

0.234

0.118

1.355

N

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-6.9

-2.3

-3.1

-19.4

-10.1

-2.3

-1.4

-0.1

0.7

1.0

9.3

46.8

Group 3, D91:

Mean

0.1193

0.1266

6.535

0.1412

0.1549

3.683

3.555

3.646

29.46

1.879

1.220*

2.433

SD

0.0258

0.0286

0.283

0.0341

0.0344

0.310

0.223

0.257

2.47

0.268

0.206

0.402

N

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-8.7

-1.3

-0.7

-3.1

3.2

1.6

-0.3

-0.3

-1.6

5.6

24.0

-1.0

Group 4, D91:

Mean

0.1480

0.1353

6.569

0.1510

0.1706

3.544

3.747

3.852

31.01

1.799

1.021

2.651

SD

0.0462

0.0254

0.392

0.0333

0.0392

0.269

0.230

0.222

2.99

0.138

0.192

0.688

N

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

13.3

5.5

-0.2

3.6

13.6

-2.3

5.1

5.3

3.6

1.1

3.8

7.8

Group 1, D119:

Mean

0.1289

0.1259

6.183

0.1323

0.1506

3.892

3.766

3.942

31.74

1.749

0.954

2.589

SD

0.0185

0.0181

0.433

0.0330

0.0351

0.359

0.106

0.226

2.31

0.138

0.158

0.898

N

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, D119:

Mean

0.1274

0.1237

5.966

0.1361

0.1687

3.662

3.595

3.743

31.35

1.912

0.982

2.247

SD

0.0141

0.0158

0.467

0.0344

0.0506

0.181

0.146

0.231

2.33

0.360

0.148

0.761

N

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-1.1

-1.8

-3.5

2.9

12.1

-5.9

-4.5

-5.0

-1.2

9.3

2.9

-13.2

 

Absolute Organ Weights, males [g]

 

Adren. gland (left)

Adren. gland (right)

Brain

Epididymis (left)

Epididymis (right)

Testis (left)

Testis (right)

Heart

Kidney (left)

Kidney (right)

Liver

Spleen

Thymus

Group 1, D91:

Mean

0.0317

0.0322

2.194

0.807

0.813

1.828

1.742

1.672

1.907

1.935

16.20

0.944

0.451

SD

0.0036

0.0046

0.075

0.095

0.089

0.124

0.193

0.205

0.215

0.189

1.44

0.106

0.091

N

10

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, D91:

Mean

0.0351

0.0339

2.139

0.795

0.814

1.726

1.719

1.699

1.956

1.993

16.93

0.966

0.456

SD

0.0054

0.0038

0.107

0.106

0.087

0.137

0.137

0.142

0.240

0.259

1.07

0.172

0.075

N

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

10.7

5.3

-2.5

-1.5

0.1

-5.6

-1.3

1.6

2.6

3.0

4.5

2.3

1.1

Group 3, D91:

Mean

0.0311

0.0311

2.157

0.728

0.792

1.717

1.720

1.644

1.923

1.950

15.26

0.959

0.413

SD

0.0074

0.0065

0.069

0.059

0.073

0.237

0.212

0.166

0.172

0.150

2.03

0.187

0.097

N

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-1.9

-3.4

-1.7

-9.8

-2.6

-6.1

-1.3

-1.7

0.8

0.8

-5.8

1.6

-8.4

Group 4, D91:

Mean

0.0293

0.0332

2.121

0.751

0.777

1.752

1.773

1.622

1.804

1.830

15.80

0.965

0.451

SD

0.0051

0.0044

0.069

0.062

0.054

0.153

0.156

0.115

0.108

0.157

1.34

0.134

0.101

N

10

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-7.6

3.1

-3.3

-6.9

-4.4

-4.2

1.8

-3.0

-5.4

-5.4

-2.5

2.2

0.0

Group 1, D119:

Mean

0.0336

0.0346

2.154

0.818

0.850

1.820

1.794

1.754

2.026

2.020

16.78

1.060

0.430

SD

0.0088

0.0062

0.054

0.120

0.121

0.111

0.132

0.119

0.172

0.151

2.42

0.196

0.091

N

5

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, D119:

Mean

0.0310

0.0294

2.156

0.784

0.832

1.778

1.762

1.808

1.944

1.992

16.66

0.970

0.412

SD

0.0046

0.0111

0.070

0.091

0.119

0.139

0.168

0.118

0.173

0.177

3.13

0.082

0.154

N

5

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-7.7

-15.0

0.1

-4.2

-2.1

-2.3

-1.8

3.1

-4.0

-1.4

-0.7

-8.5

-4.2

 

Absolute Organ Weights, females [g] (** = p ≤ 0.01)

 

Adren. gland (left)

Adren. gland (right)

Brain

Ovary (left)

Ovary (right)

Heart

Kidney (left)

Kidney (right)

Liver

Spleen

Thymus

Uterus incl. cervic

Group 1, D91:

Mean

0.0394

0.0386

1.975

0.0440

0.0452

1.094

1.074

1.100

9.03

0.537

0.299

0.741

SD

0.0045

0.0061

0.069

0.0123

0.0080

0.115

0.100

0.091

1.03

0.049

0.081

0.164

N

10

10

10

10

10

10

10

10

10

10

10

10

Group 2, D91:

Mean

0.0376

0.0388

1.967

0.0366

0.0416

1.095

1.087

1.130

9.30

0.555

0.334

1.113**

SD

0.0049

0.0046

0.056

0.0139

0.0143

0.090

0.074

0.107

0.60

0.059

0.044

0.413

N

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-4.6

0.5

-0.4

-16.8

-8.0

0.1

1.2

2.7

3.0

3.4

11.7

50.2

Group 3, D91:

Mean

0.0362

0.0384

1.984

0.0433

0.0474

1.116

1.081

1.110

8.95

0.571

0.372

0.735

SD

0.0073

0.0083

0.077

0.0121

0.0120

0.054

0.092

0.116

0.79

0.083

0.071

0.097

N

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

-8.1

-0.5

0.5

-1.6

4.9

2.0

0.7

0.9

-0.9

6.3

24.4

-0.8

Group 4, D91:

Mean

0.0443

0.0406

1.972

0.0456

0.0516

1.066

1.128

1.160

9.33

0.541

0.308

0.797

SD

0.0128

0.0072

0.071

0.0107

0.0135

0.091

0.100

0.101

1.00

0.046

0.063

0.210

N

10

10

10

10

10

10

10

10

10

10

10

10

%Diff

12.4

5.2

-0.2

3.6

14.2

-2.6

5.0

5.5

3.3

0.7

3.0

7.6

Group 1, D119:

Mean

0.0424

0.0414

2.034

0.0432

0.0492

1.280

1.242

1.304

10.48

0.576

0.314

0.844

SD

0.0053

0.0053

0.027

0.0089

0.0094

0.072

0.065

0.147

1.03

0.038

0.051

0.242

N

5

5

5

5

5

5

5

5

5

5

5

5

Group 4, D119:

Mean

0.0418

0.0406

1.962

0.0444

0.0548

1.210

1.186

1.234

10.34

0.628

0.326

0.746

SD

0.0024

0.0036

0.115

0.0090

0.0137

0.132

0.095

0.105

1.03

0.114

0.069

0.276

N

5

5

5

5

5

5

5

5

5

5

5

5

%Diff

-1.4

-1.9

-3.5

2.8

11.4

-5.5

-4.5

-5.4

-1.3

9.0

3.8

-11.6

 

Statistically significant changes in organ weights unrelated to the test item

Organ

Increase ­Decrease¯

Group/Sex

Test day

Statistical significance

Reason

Thymus (relative)

3 f

91

p ≤ 0.05

A

Uterus (including cervix, relative)

2 f

91

p ≤ 0.05

B

Uterus (including cervix, absolute)

2 f

91

p ≤ 0.01

B

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

Applicant's summary and conclusion

Conclusions:
In this subchronic oral toxicity study to rats according to OECD 408, dosed up to 1000 mg/kg bw/d no adverse effects were observed on any of the parameters assessed. Consequently, the No Observed Effect Level (NOEL) was set to 1000 mg/kg bw/d (high dose in this study).
Executive summary:

The aim of this repeated dose toxicity study was to obtain information on the toxicity of N,N'-(methylenedi-4,1-phenylene)bis(2-oxoazepane-1-carboxamide) (CAS no. 54112-23-1, EC no. 258-981-8, Batch no. 9116213/13) 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 treatment-free recovery period. The animals were treated with 100, 300 or 1000 mg of the test substance/kg b.w./day. The control animals received the vehicle (0.5% aqueous hydroxypropyl methylcellulose gel).

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

No test item-related changes were observed for the behaviour or external appearance of the animals, the detailed clinical observations, the neurological screening, the body weight, body weight gain and body weight at autopsy, the food and drinking water consumption, for any of the haematological and clinical chemical parameters, the eyes or optic region, the auditory acuity, at macroscopic inspection at necropsy, and for the relative and absolute organ weights at any dose level.

The histopathological examination of the main study animals treated with 1000 mg test substance/kg b.w./day did not reveal any test item-related morphological changes at the end of the 90 day treatment period.

No test item-related changes were noted during or at the end of the 28 day treatment-free recovery period.

The experimental no-observed-effect level (NOEL) was found above 1000 mg test substance/kg b.w./day by daily oral administration via gavage.