Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-24 - 2017-11-30
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Name: Ukanol FR 70
Batch no.: 4251318
Appearance: white solid
Composition: 9,10-Dihydro-9-oxa~10-[2,3-Di-(2-hydroxyethoxy) carbonylpropyl]-10-phosphaphenanthren-10-oxid; Bis(2-hydroxyethyl)-(6H-dibenz[c,e][1,2]oxaphos-phorin-6-yl-methyl)succinat-P-oxide
CAS No.: 63562-34-5
EINECS-No.: 264-313-6
Molecular formula: C21H23O8P
Molecular weight: 434 g/mo
Purity: app. 90% (HPLC)
Homogeneity: homogeneous
Specific details on test material used for the study:
CAS no. 63562-34-5

EC no. 264-313-6

Batch no. 7505294

Receipt no. 61985

Date of receipt December 22, 2016

Characteristics Solid, yellow to yellowish

Stability / Expiry date August 08, 2018

Storage conditions At room temperature in a tightly closed container.

Purity ≥95%

Test animals

Species:
rat
Strain:
other: CD/ Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld , Germany

Body weight (at 1st administration, TD15):
Males:406.3 g - 491.3 g
Females: 237.0 g - 317.6 g
Age (at 1st administration) : Males + Females: 77 days

Selection of species: The rat is a commonly used rodent species for such studies.
Number and sex of animals Pre-exposure period (TD 1 to TD 14): 60 female animals
A sufficient number of animals in order to grant at least 40 females with a normal oestrus cycle for the main study.
Main study (start on TD 15): 80 animals (40 males and 40 females)
A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.
Adaptation period: 5 days

Diet:
A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany; served as food. This food was offered ad libitum. Food residue was removed and weighed.
Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
Drinking water:
Tap water was offered ad libitum.
Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001 ].
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.
No contaminants above the limitations were noted.
Housing:
With the exception of the mating period, the males and females (F0-Generation) 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. The room temperature was 22°C ± 3°C (maximum range) and the relative humidity was 55% ± 15% (maximum range). The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dose levels were selected by the Sponsor based on available toxicological data and a 14-day dose range finding study (LPT Study No. 34269).
In the 14-day dose range finding study, Ukanol FR 70 was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks.
Salivation was noted in all dose groups with an increasing incidence and severity from the low to the high dose level. At 1000 mg/kg b.w./day all 5 male and all 5 female animals showed a slight to severe salivation on 10 to 13 test days that started immediately to 5 min after dosing and lasted between 5 to 20 min after dosing.
No further test item-related changes were noted in behaviour, the external appearance and the faeces. No influence was noted on body weight or food consumption. The macroscopic inspection at necropsy revealed no test item-related changes.
Based on the data obtained in this dose range finding study, dose levels of 30, 100 and 300 mg Ukanol FR 70/kg b.w./day were selected for the main study
Vehicle:
polyethylene glycol
Details on oral exposure:
The test item was dissolved in the vehicle to provide dose concentrations of 7.5, 25 or 75 mg/mL
The test item formulations were freshly prepared every day and were adjusted to the animal's actual body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 2 x 2 mL were taken at the following times and stored at ≤-20°C until analysis:
At start of the treatment period (first dosing day):
Analysis of stability and concentration
Immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature
(3 samples (each consisting of 2 x 2 mL)/dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

Towards the end of the treatment period (when the majority of animals was dosed):

Analysis of concentration
During treatment always before administration to the last animal/dose level group
(1 samples (each consisting of 2 x 2 mL)/dose level group; groups 2 - 4).
Number of samples: 3 x 1 = 3

Sum of all samples: 12

The samples were labelled with the study number, species, type of sample, concentration, test day, sampling time and date.
The analytical method was validated by LPT, whereby the following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability
The investigation of the above mentioned validation parameters confirmed that the method employed was suitable for the determination and quantification of the test item Ukanol FR 70.
Duration of treatment / exposure:
Males: 33 days
Females: 53 days
Frequency of treatment:
once dailay
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Clinical signs
Daily observations:
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
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.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
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 of the parental generation. These examinations were performed at least 2 hours after dosing.
These observations were made outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Neurological screening
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 as described on the following pages in five males and five females randomly selected from each study group.
The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group (randomly selected): On test days 43 and 45.

5 female F0 animals per group (randomly selected): On test days 63 and 64 (between lactation days 10 to 12).

Observational screening
Righting reflex
The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; that means 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 the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.

Salivation
Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.

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, whereby a zero (0) was recorded in the blank space of the scoring sheet. 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. The 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 in the blank space.

Lacrimation
The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. 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 abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).

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 from 1 (absent) to 5 (exaggerated), with 3 being the normal response.

Wire manoeuvre
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 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
The hind paws were marked with ink using an ink pad. 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 (in cm).

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 (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.
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 increased 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, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was 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. It was continued to pull the animal 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 placed on the cage and any movements were measured for a duration of 12 min 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
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m.
Premortal symptoms were recorded in detail; as soon as possible after exitus, a post mortem examination was performed. for the pups and the parental animals.
Body weight
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 1 post-partum) and on day 4 and 13 post-partum. Body weights were recorded individually for each adult animal.

ood and drinking water consumption
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 (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined.
Drinking water consumption was monitored daily by visual appraisal throughout the study.

Reproductive performance
The following parameters and indices were evaluated:
Reproductive parameters
Pre-coital time and gestation length
- number of pregnant females
- duration of pre-coital time
- gestation length
(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore)).

Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

Number of pups absolute
- at birth (alive and dead)
- after 4 and 13 days of life

Number of pups per dam
- at birth (alive and dead)
- after 4 and 13 days of life

Number of male and female pups
- at birth
- after 4 and 13 days of life

Number of stillbirths
- absolute
- per dam

Number of pups with malformations
- absolute
- per dam


Reproductive indices
The following indices were calculated for each group:

Female Fertility Index [%] = Number of pregnant rats x 100
Number of rats used

The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.

Gestation Index [%] = Number of dams with live pups x 100
Number of pregnant rats

For each litter and group the following indices were determined:
Birth Index [%] = Total number of pups born
(alive + dead) x 100
Number of implantation scars


Live Birth Index [%] = Number of pups alive on day 0/1 of
lactation x 100
Total number of pups (alive + dead)


Viability Index [%] pre-cull = Number of pups alive on day 4 (pre cull) x 100
Number of pups alive on day 0/1


Viability Index [%] post cull = Number of pups alive on day 13 x 100
Number of pups alive on day 4 (post cull)

Post-implantation loss [%] = Implantations - living fetuses x 100
Implantations

Examination of the pups
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups.
The following examinations/observations were done for the offspring.
Counting
Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.
Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale.
Litter adjustment on PND 4
After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups following a randomization scheme.
Blood sampling for thyroid hormone (T4) determination
On PND4, blood samples for T4 hormone level determination were taken from 2 of the culled surplus pups (see section 4.12 'Thyroid hormone (T4) determination'). If possible, sampling was performed on one male and one female pup.
On PND13, blood samples for T4 hormone level determination were taken from 2 pups (one male and one female pup if possible) of each litter.
Nipples counting
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).

Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnightat the following times:
At the end of the premating period on test day 29 (male and female animals):
5 male and 5 female F0 animals randomly selected from each group.

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 clinical chemistry tests

Haematology
The parameters listed below were determined:
Haemoglobin content
(HGB)
Erythrocytes
(RBC)
Leucocytes
(WBC)
Differential
blood count
(relative and absolute)
Reticulocytes
(Reti)
Platelets
(PCT or PLT)
Haematocrit value
(PCV or HCT)
Mean corpuscular
volume
(MCV)
Mean corpuscular
haemoglobin
(MCH)
Mean corpuscular
haemoglobin concentration
(MCHC)

Clinical biochemistry
The parameters listed below were determined:
Albumin
Globulin
Albumin/Globulin ratio
Bile acids
Bilirubin (total)
Cholesterol (total)
Creatinine
Glucose
Protein (total)
Blood urea (BUN)

Calcium
Chloride
Potassium
Sodium
Sodium/Potassium ratio
BUN/Creatinine ratio
Lactate dehydrogenase (LDH)
Alanine
aminotransferase
(ALAT)
Alkaline
phosphatase
(aP)
Aspartate
aminotransferase
(ASAT/GOT)

Thyroid hormone (T4) determination
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (approx.. between 7.00 a.m. and 10 a.m. for the adult animals).
Blood withdrawal was performed at the day of necropsy in a randomized way for the adult male and female animals and the pups on PND 13. In case of the PND 4 pups the first male and the first female pup, if possible, from the randomly created pup culling list was used.

Sacrifice and pathology:
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.

The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males
On test day 49 (after a dosing period of at least 4 weeks)
Pups
On PND 13
Dams
On lactation day 14, 15 or 16


Examination of the pups
Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
The thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin. Thyroid weight was determined after fixation.

Dissection of adult animals
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes.
Special attention was paid to the organs of the reproductive system and the number of implantation sites was recorded.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI
All superficial tissues were examined visually and by palpation and the cranial roof 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 to 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 the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

Organs to be weighed
The weight of the following organs of all adult male and female animals was determined before fixation (where applicable):
Adrenal gland (2) Spleen
Brain Testicle (2)
Epididymis (2) Thyroid
Heart Thymus
Kidney (2) As a whole: prostate, seminal vesicles
Liver with coagulating glands
Ovary (2) Uterus including cervix

Thyroid weight was determined after fixation; paired organs were weighed individually and identified as left or right.

Organs to be fixed for preservation
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin:
Fixative: modified Davidson'solution
Epididymis (2) Testicle (2)
Fixative: 7 % buffered formalin
Gross lesions observed Thyroid (including parathyroids)
Ovary and oviduct (2) Uterus (including cervix)
Prostate Vagina
Seminal vesicles with coagulating glands

The following organs from the selected animals and from every deceased or prematurely sacrificed animal were fixed for histopathology examination.

Fixed organs from the 5 randomly selected male and female animals per group:
Fixative: Davidson'solution
Eye with optic nerve (2)
Fixative: modified Davidson'solution
Epididymis (2) Testicle (2)
Fixative: 7 % buffered formalin
Adrenal gland (2) Muscle (skeletal)
Bone Nerve (sciatic)
Bone marrow (os femoris) Oesophagus
Brain (cerebrum, cerebellum, brain stem (pons)) Ovary and oviduct
Gross lesions observed Pituitary
Heart (3 levels: right and left ventricle, Prostate and seminal vesicles
septum) with coagulating glands
Intestine, small (duodenum, jejunum, ileum, Spinal cord (3 sections)
incl. Peyer's patches, Swiss roll method) Spleen
Intestine, large (colon, rectum) Stomach
Kidney and ureter (2) Thyroid (including parathyroids)
Liver Thymus
Lungs (with mainstem bronchi and bronchioles), Tissue masses or tumors
preserved by inflation with fixative and then including regional lymph nodes)
immersion Tongue (including base)
Lymph node (1, cervical) Trachea (including larynx)
Lymph node (1, mesenteric) Urinary bladder
Mammary gland Uterus (including cervix)
Vagina
Any other organs displaying macroscopic changes were also preserved.

Histopathology
Animals and organs used for histopathology
Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and 4, and the thyroid of the selected pups.
The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.
The blood smears prepared from all animals during the haematological examination were available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor. So far, no examination was performed.


Statistics:
Parametrical data
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 8.2.0.8, Instem LSS Ltd), using the following settings:
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), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data
The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the following software:
- Using Provantis:
Statistical evaluation of histopathology findings ; see Appendix 4 'Histopathology Phase Report')
- Using StatXact 4.0.1:
Statistical evaluation of the fertility index, the gestation index, the birth and live birth index, the viability indices and the post-implantation loss.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No changes in behaviour, the external appearance or the appearance of the faeces were noted in the control group.
At the low dose level (30 mg test item/kg b.w./day) salivation was noted for a short period after dosing for 2 male and 2 female animals on 1 or 2 test days. Due to the low incidence, these observations were considered as spontaneous and not test item-related.
A dose related increased incidence of post-dosing salivation was noted at the intermediate and the high dose level (100 or 300 mg test item/kg b.w./day) for the male and the female animals. The effect observed maybe a reaction to the taste or irritation of the test article rather than an indication of adverse toxicity .
With the exception of the above mentioned salivation (that was not considered as a sign of adverse toxicity), no changes in behaviour, the external appearance or the faeces that were considered as test item-related were noted at the intermediate and the high dose group (100 or 300 mg test item/kg b.w./day).
Observations that were considered as not test item-related but spontaneous were noted for the male animals for the consistency of the faeces (pultaceous faeces; noted at the intermediate dose level) or the external appearance (haemorrhagic nose/snout; noted at the high dose level). Due to their low incidence (only noted for one animal on one or 2 test days), both observations were considered as incidental.
The detailed clinical observations were performed once weekly, at earliest 2 h after dosing.
Males and females:
No external observations and no changes in body posture, movement and coordination and in behaviour were noted for the animals of the control group and those of the treatment groups (30, 100 or 300 mg test item/kg b.w./day) during this weekly examination that was carried out at least 2 hours after dosing.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the haematological parameters of the male and female animals in any of the treatment groups (30, 100 or 300 mg test item/kg b.w./day) for the haemoglobin content, the number of erythrocytes, the number of leucocytes, the number of reticulocytes and platelets, the haematocrit value, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC) and the parameters of coagulation (TPT, aPTT). No test item-related changes were noted in the relative and absolute differential blood counts.

A slight but statistically significantly decreased haemoglobin content was noted at the low and the intermediate dose level. However, as no dose response relationship was noted and no significant changes were noted for the female animals this was considered as spontaneous and not as test item-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry
Males and females
No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups (30, 100 or 300 mg test item/kg b.w./day), i.e. levels of albumin, globulin, the albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium, sodium/potassium ratio, urea/creatinine, the activity of the alanine aminotransferase (ALAT), of the alkaline phosphatase (aP), of the aspartate aminotransferase (ASAT) and the lactate dehydrogenase (LDH).
However, a slight but statistically significant increase was noted for the male animals of the low dose group for the activity of ASAT. As no dose response relationship was noted and no significant changes were noted for the female animals, this finding was considered as spontaneous and not as test item-related. Furthermore, all individual male and female ASAT values of the control group, the intermediate and the high dose group were in the range of background data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Observational screening
Males and Females:
No test item-related observations of abnormal behaviour or test item-related differences in body temperature or the hind-leg splay in comparison to the control group were noted for the male animals of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
A slight but statistically significantly increased body temperature was noted at the high dose level for the female animals. However, as all individual values were in the range of LPT background data, the slightly increased body temperature that was noted at the high dose level was considered to be spontaneous and not test-item related.
Functional tests
Grip strength
Males and females
No test item-related influence on the fore- and hindlimb grip strength was noted for the male and female animals in any of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
Spontaneous motility
Males
No test item-related differences were noted between the control group and the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
Females
No test item-related differences were noted between the control group and the low and the intermediate dose group (30 or 100 mg test item/kg b.w./day).
A statistically significantly increased spontaneous motility (67,3 % above the value of the control group, p ≤ 0.05) was noted at the high dose level. Though the observed value was distinctly above the LPT background data (see table on the following page), it was considered as spontaneous and not as test item-related, as no signs of an increased motility were noted during the daily observations or the weekly detailed observations for behavioural changes

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Male and female animals
No test item-related influence was noted for the relative and the absolute organ weights of the male and female animals in any of the test item-treated groups (30, 100 or 300 mg test item/kg b.w./day).
However, slight reductions in the absolute and relative weights of the left and the right kidney were noted for the male rats of the low and the high dose group (at maximum 10.1 % below the value of the control group, noted for the relative organ weight of the left kidney; p ≤ 0.01). As no dose response relationship was noted, these changes were considered as spontaneous and not test item-related. Furthermore, no reductions for the relative and absolute organ weights of the female rats were noted (although the female animals were sacrificed around 20 days later as the male animals).
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females
Necropsy revealed no test item-related pathological changes during the macroscopic examination of the internal organs and tissues in the male and female rats of the control group and the male and female rats of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).
The following changes were considered as spontaneous and not test item-related, as in both cases only one animal was affected.
Males (group 2; 30 mg test item/kg b.w./day)
- The left testis and the left epididymis of animal no. 27 were reduced in size and of soft consistency. This was in compliance with reduced absolute organ weights of the left testis and epididymis of animal no. 27.

Females (group 2; 30 mg test item/kg b.w./day)
- Both lobes of the lungs of animal no. 38 were darkly spotted and the right lobe was additionally partly dark discoloured.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Determination
Male animals
No test item-related differences were noted in the T4 levels between the male animals of the control group and the male animals of the treatment groups (30, 100 or 300 mg test item/kg b.w./day).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed up to the highest tested dose

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
No premature deaths were noted during the course of the study.
A transient salivation was noted at the intermediate and the high dose level in a dose related manner. The effect maybe related to the taste or irritant properties of the test item, rather than an indication of toxicity.
No further test item-related changes were noted in behaviour or the external appearance, the neurological screening, body weight, food consumption, the haematological and biochemical parameters, the T4 levels and the organ weights.
The macroscopic inspection at autopsy and the microscopic inspection of selected high dosed animals also revealed no test item-related changes.
The NOAEL was determined as above 300 mg/kg b.w..
Executive summary:

No premature deaths were noted during the course of the study.

A transient salivation was noted at the intermediate and the high dose level in a dose related manner. The effect maybe related to the taste or irritant properties of the test item, rather than an indication of toxicity.

No further test item-related changes were noted in behaviour or the external appearance, the neurological screening, body weight, food consumption, the haematological and biochemical parameters, the T4 levels and the organ weights.

The macroscopic inspection at autopsy and the microscopic inspection of selected high dosed animals also revealed no test item-related changes.

The NOAEL was determined as above 300 mg/kg b.w..