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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2015

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source : Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 7-8 weeks old, females: 7-8 weeks old.
Body weight at the allocation of the animals to the experimental groups:
males: 144 – 173g (mean: 159.52 g, ± 20% = 127.6 – 191.4 g)
females: 125 – 161 g (mean: 141.42 g, ± 20% = 113.1 – 169.7 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the
German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1512, 1526)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological
controls at regular intervals)
- animals of the same sex were housed in groups of up to five in type IV polysulphone cages on Altromin saw fibre bedding (lot no. 200114)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. There were no significant clinical
signs in animals before the study initiation. Before the first administration all animals used for the study were weighed and assigned to the
experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Preparation of the Test Item Formulations
The test item was weighed into a tared plastic vial on a precision balance.
The test item was dissolved in aqua ad iniectabilia. The vehicle was selected as suggested by the sponsor based on the test item’s characteristics and testing guideline. The test item formulation was prepared freshly on each administration day before the administration procedure.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The vehicle was also used as control item.

Experimental Groups and Doses
In consultation with the sponsor the following doses were selected for the 3 dose groups and 1 control group:

Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

Administration of Doses
The test item formulation or vehicle were administered at a single dose to the animals by oral gavage.
The application volume for all groups was 5 mL/kg body weight.
For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured (measured weekly).
In the 2nd week of the study all male animals of control and dose groups (main and recovery) and all females of control and HD groups
(only recovery) received the dose volume calculated based on week-1 body weight instead of most recent body weight.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of the concentration of test item in dosing formulations, samples were retained from all groups once in the first week of
treatment, at the beginning of month 2 and month 3, respectively, and at the end of the treatment period and stored between -15 and -35 °C.
Concentration was tested in all samples.
Stability of the dosing formulations was tested once at the beginning of the treatment period. From the low and high dose groups samples of
dosing formulations were frozen at 0 hours and 6 hours after the preparation and stored at -15 and -35 °C.
In the first week of treatment, at the beginning of the second month of treatment and at the end of the treatment period, samples for the testing
of homogeneity were taken from the top, middle and bottom of the freshly prepared high, medium and low-dose formulations and stored
between -15 and -35 °C. Homogeneity was tested in samples of dosing formulations of the low dose, medium dose and high dose groups.
At the end of the treatment period all samples of dosing formulations were shipped on dry ice and protected from light to:
CIP
Chemisches Institut Pforzheim GmbH
Schulberg 17
75175 Pforzheim
Germany
The determination of test item concentration in the dosing formulations was performed by CIP GmbH, in accordance with GLP. The procedures
followed were described in a detailed analytical phase plan that was attached to the study plan per amendment. CIP GmbH issued an analytical
phase report which was integrated in the report of this study.
Duration of treatment / exposure:
Period of 90 days
Frequency of treatment:
Once a day; 7 days per week
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg body weight; 300 mg/kg body weight; 1000 mg/kg body weight
Basis:
actual ingested
No. of animals per sex per dose:
80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group). In addition 20 animals
(10 males and 10 females) were included in the control and HD recovery groups (5 male and 5 female animals per group).
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 90 days. 10 animals per gender and group
were subjected to necropsy one day after the last administration (end of treatment period). 5 animals per gender and group were subjected to
necropsy 28 days after the last administration (end of recovery period).

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. The selected high dose also happens
to be a limit dose. A descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received aqua ad iniectabilia (sterile water)
using the same volume as used for the high dose group.

Examinations

Observations and examinations performed and frequency:
Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during
the treatment and recovery period. Food consumption was measured weekly during the treatment and recovery period.

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days.
The recovery animals were observed for an additional period of 28 days following the last administration.
General clinical observations were made once a day, approximately at the same time each day after dosing. The health condition of the animals
was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were
made once daily. Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea,
asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were
made outside the home cage in a standard arena once before the first administration and weekly thereafter.
Ophthalmological examination, using an ophthalmoscope was made on all animals before the first administration and in the last week of the
treatment period as well as at the end of the recovery period in the recovery animals.

Functional Observations
Once before the first exposure and once in the last week of exposure as well as in the last week of the recovery period multiple detailed behavioural
observations were made outside the home cage using a functional observational battery of tests [10]. These tests were conducted in all animals.

Haematology
Haematological parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of deeply anaesthetized animals was collected in EDTA-coated tubes.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC),
mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC),
reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos),
basophils (Baso), large unstained cells (Luc).

Blood Coagulation
Coagulation parameters were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of deeply anaesthetized animals was collected in citrate tubes.
The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical Biochemistry
Parameters of clinical biochemistry were examined at the end of the treatment and recovery period prior to or as part of the sacrifice of the
animals. After overnight fasting, blood from the abdominal aorta of of deeply anaesthetized animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT),
alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bilirubin (TBIL), total bile acids (TBA), total cholesterol (Chol),
glucose (Gluc), sodium (Na), potassium (K).

Urinalysis
A urinalysis was performed with samples collected from all animals at necropsy.
The following parameters (specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (Ket), urobilinogen (UBG), bilirubin (BIL),
erythroctes (Ery), leukocytes (Leu)) were measured using qualitative indicators (Heiland Urine Stripes URI 10SL).
Additionally, urine colour / appearance were recorded.

Serum Phosphorus Analysis
Blood sampling for serum phosphorus analysis was made in 5 male and 5 female animals per group. The sampling was made (4 times) 24 hours
after the 1st dose administration, in week 4, 24 hours after the last administration (at necropsy) and in recovery animals at necropsy.
Approximately 250 µL blood per animal was collected and approximately 100 µL was separated. The serum samples were stored at -20°C
until the analysis.
Blood sampling was made on rest animals at terminal sacrifice. The serum was separated and the samples were stored at -20°C.
These additional serum samples were sent to Clariant GmbH for analysis. The data generated is not part of this study report.
The determination of phosphorus in rat serum (Serum Analysis) was performed by CIP GmbH, in accordance with GLP. The procedures
followed were described in a detailed analytical phase plan that was attached to the study plan per amendment. CIP GmbH issued an analytical
phase report which was integrated in the report of this study.






Sacrifice and pathology:
Pathology
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period and 4 weeks after the last administration
all surviving animals of the recovery period (study day 119) were sacrificed using anesthesia (ketamine, Pharmanovo, lot no: 24863,
expiry date: 10/2015 and xylazin, Serumwerk, lot no. 01213, expiry date: 10/2015) and were subjected to a detailed gross necropsy which
includes careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Animal (no. 85 of HD group) intercurrently euthanized (study day 89) for animal welfare reasons was also subjected to a gross necropsy and
the organs preserved for a histopathological examination. Bones of animals from recovery control and HD groups were collected at necropsy,
preserved at 70% ethanol and sent to Clariant GmbH for analysis. The data generated is not part of this study report.

Organ Weight
The wet weight of the following organs (liver, uterus, kidneys, thymus, adrenal glands, thyroid/parathyroid glands, testes, spleen, epididymides,
brain, prostate gland, pituitary gland, seminal vesicles and coagulating glands after ligation, heart, ovaries) of all sacrificed animals was
recorded as soon as possible. Paired organs were weighed individually. Organ weights of animal euthanised for animal welfare reasons were
not recorded.
The following tissues (brain (cerebrum, cerebellum and pons), spinal cord, eye, liver, kidneys, adrenal glands, stomach,
small and large intestines (including Peyer´s patches), thymus, thyroid / parathyroid glands, spleen, lung and trachea, mammary glands,
skin, pancreas, heart, ovaries (females), uterus with cervix (females), vagina (females), testes (males), epididymides (males),
prostate and seminal vesicles with coagulating glands as a whole (males), urinary bladder, lymphnodes (mesentric and axillary),
peripheral nerve (e.g. sciatic nerve) with skeletal muscle, sternum with bone marrow, pituitary gland, oesophagus, aorta, salivary gland)
from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were
fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

Histopathology
A full histopathological evaluation was performed on organs and tissues (see above) from all animals of non-recovery (main study)
Control and HD groups, as well as all gross lesions from all groups, were examined by light microscopy. Since microscopic changes that
could be attributed to treatment with the test item were not observed in animals of the non-recovery (main study) HD group, further
histoprocessing and histopathology evaluation were not performed.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services,
Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified
contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test
site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1]
(Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to
the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement
of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a
pathology phase report to the study director upon the completion of the study.
Statistics:
A statistical assessment of the results of the body weight, parameters of haematology, blood coagulation and clinical biochemistry and absolute
and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way
ANOVA and a post-hoc Dunnett Test. Statistical comparisons of data acquired during the recovery period were performed with a Student’s t-Test.
These statistics were performed with E-Workbook software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
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):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality
There was no mortality caused due to test item treatment during the study period. However one female no. 85 of the HD group was euthanized
for ethical reasons during the course of the study on treatment day 89. The cause of animal’s morbidity was an accidental aspiration
(aspiration pneumonia) of the dosing formulation. The remainder of animals survived the scheduled study period.

Clinical Observations
There were no clinical signs of toxicological relevance in test item treated animals during the treatment and recovery period. However, there were
incidences of nasal discharge (males= 2/10 control, 2/10 MD; Female 1/10 HD group), alopecia (males= 2/10 control, 1/10 HD; females= 5/10 control, 1/10 LD and 3/10 MD), crust (males= 1/5 HD recovery; female= 1/10 control) and scratch (female= 1/10 LD). These clinical signs were apparent in
few isolated animals and were not attributed to treatment. Clinical signs of dehydration, eyes half closed and tachypnea were observed in
female no. 85 before necropsy. The cause of animal’s morbidity was an accidental aspiration.
During the weekly detailed clinical observation, no significant changes or differences between the groups were found.
There were no ophthalmoscopic findings in any of the animals of this study.

Body Weight Development
IIn both males and females, there was no test item related effect on body weight in dose groups during the study period.
There were no statistically significant differences between the dose groups and the control group.
The mean body weight gain of both males and females in the dose groups showed transient changes (higher or lower) compared to corresponding
controls during the entire study period without statistical significance except the lower weight gain (days 1-90) of females HD group compared
to corresponding control. However, this statistical significant change in female HD group was not evident during the treatment period in female
recovery HD group compared to control. Hence, the statistical significance was not considered to have biological significance.

Food Consumption
In both males and females, no considerable effect of test item on food consumption was observed throughout the study period.
The food consumption in both males and females were within the normal range of variations.

Functional Observation Battery
No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period.
There were no biologically relevant differences in body temperature between the groups.

Haematology and Blood Coagulation
In males and females, at the end of treatment and recovery period there were no effects on haematology and blood coagulation parameters.
However, there was statistically significantly higher basophil count in male LD group. This change was not dose dependent and was within the
historical control data range. There was also slightly, but not statistically significantly lower, platelet count in male MD and HD groups
compared to controls. The mean and the individual values were within the historical control data range and the finding was not considered to
be related to test item treatment. At the end of the recovery period, there was slightly higher neutrophil (Neu) count and prothrombin time (PT)
in females of the recovery HD group, when compared to controls. However, the mean and most individual values were within the historical
control data range. Besides, all haematological and blood coagulation parameters were within the normal range of variation.

Clinical Biochemistry
In males, at the end of treatment and recovery period there was no effect on clinical biochemistry parameters. There were no statistically
significant differences between the dose and control groups. However, there was higher mean TBA in MD and HD groups, which was due to
substantially higher TBA value of isolated males of MD (No. 30; TBA valus 77.17 µmol/L) and HD (no. 35 and 40; TBA values 50 µmol/L and
80 µmol/L, respectively) groups. The mean and most individual values of TBA were within the historical control range and there were no
associated microscopic changes in liver except for lower intensity of inflammatory cell focus/foci of animal no. 40.
The lower mean TBA value in male recovery HD group compared to control was within the range of historical control data.
At the end of the treatment period there were statistically significantly lower serum ASAT levels in females of LD group and significantly lower
serum ALAT levels in females of LD and HD groups. The changes were only slight and not dose dependent. Moreover, slightly reduced ALAT
and ASAT values are generally not associated with toxicity.
At the end of the recovery period there was slightly higher mean AP and TBA values in females of the HD group compared to controls.
However, as there were no statistically significant differences, no effect at the end of the treatment and no associated histopathological changes,
the finding was not considered to be an adverse effect of test item treatment.
Besides, all parameters of clinical biochemistry were within the normal range of variation for this strain and statistically significant differences
between the dose and control groups were not assumed to be biologically relevant.

Urinalysis
All urinary parameters were in the normal range of variation and no conspicuous differences between the dose and control groups were observed.

Pathology
There were no gross lesions that could be attributed to treatment with the test item. In the lung of female no. 85 (HD group) which was
euthanized for ethical reasons, “Irregular surface” and “Not collapsed”, which correlated microscopically with aspiration pneumonia, were recorded.
The remainder of gross lesions recorded was within the range of normal background alterations which may be recorded in animals of this strain
and age.

Organ Weight
In males, there were no statistically significant differences in absolute and relative (to brain and terminal body weight) organ weights between
dose and control groups. However, there were slightly lower absolute or relative weights of pituitary gland and prostate (including seminal vesicle
and coagulating gland) by 12% to 15% in recovery HD group compared to control. There was also slightly higher relative thymus weight to brain
weight (15% higher than controls) in recovery HD group and relative thymus weight to body weight in MD group (23 % higher than controls) and
recovery HD group (25% higher than controls), respectively compared to corresponding control.
In females, there was a statistically significantly higher absolute and relative (relative to brain and terminal body weight) pituitary gland weight
(23% to 28% higher than controls) in HD group; statistically significantly higher absolute and relative (to brain weight) spleen weight
(Approx. 23% higher than controls) in recovery HD group and statistically significantly lower relative brain weight to body weight
(approx. 7% lower than controls) in recovery HD group.
Furthermore, there was higher relative thyroid weight to body weight in MD group (18% higher than control) and pituitary weight in LD group
(17% higher than control); slightly lower relative (to body weight) uterus and thyroid weight (12-15% lower than controls) and higher spleen weight
(15% above controls) in recovery HD group. These changes were not statistically significantly different from the corresponding controls.
In the absence of relevant macroscopic and microscopic findings the above organ weight changes of both males and females were not considered
to be of toxicological relevance. The statistically significant changes were considered to have no biological relevance.

Histopathology
There were no microscopic lesions that could be attributed to treatment with the test item.
In female no. 85 (HD group) which was euthanized for ethical reasons, aspiration pneumonia which was characterized by inflammation with
intra-bronchial and alveolar fluid contents as well as with aggregation of substance-laden macrophages and reactive hypertrophy of bronchial/
bronchiolar epithelium was observed in the lung. This was caused by an accidental aspiration of the dosing solution and considered to be cause
of animal’s morbidity. Marked thymic atrophy (ie. accelerated atrophy) was recorded in this animal. This was deemed to be a secondary change
as a response to a stressful condition related to aspiration pneumonia. The remainder of microscopic findings recorded in this study was
within the range of normal background lesions which may be recorded in
animals of this strain and age.

Dose Formulation Analysis
The analytical method for the determination of test item in dose formulation samples was successfully validated according to
SANCO/3029/99 rev. 4 (11/07/2000). The determinations were performed by ICP-OES. The requirements of SANCO/3029/99 rev.4 (11/07/2000)
regarding linearity, precision (repeatability), accuracy (recovery) and specificity were fulfilled.
Test substance concentration, stability and homogeneity in dosing formulations were obtained by ICP-OES. The analytical results obtained for the
individual dose groups were consistent with analysis of the % of the nominal of the test item for the concentration, stability and homogeneity analysis.
The concentration analysis of formulation samples was determined in study weeks 1, 5, 9 and 13 for all dose groups. The recoveries observed in
the low dose (LD), medium dose (MD) and high dose (HD) groups were in the range of 94.6% to 103.6% of the nominal dose of test item.
Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD groups. After 6 hours storage at room
temperature recovery compared to starting value was 102.9% and 95.7% of % nominal dose of test item in LD and HD groups, respectively.
All samples were stable as concentration after storage did not differ from start value by more than 20%.
Homogeneity of formulation samples was determined in study weeks 1, 5 and 13 for the LD, MD and HD groups. The recovery observed for the LD, MD and
HD groups were in the range of 90% to 110.4 % of the % nominal dose of tes item.

Effect levels

Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects of the test item were found at dose levels of 1000 mg/kg body weight/ day. The NOAEL of the test item in this study is considered to be 1000 mg/kg body weight/ day.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with the test item in male and female Wistar rats, with dose
levels of 100, 300, and 1000 mg/kg body weight/ day the following conclusions can be made:
No adverse effects of the test item were found at dose levels of 1000 mg/kg body weight/ day. The NOAEL of the test item in this study is
considered to be 1000 mg/kg body weight/ day.
Executive summary:

This study was performed in order to be compliant with Chinese regulations for notification.

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test item via

oral administration to rats over a period of 90 days.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia(sterile water), the vehicle

used in this study. The 4 groups comprised of 10 male and 10 femaleWistarrats. In order to allow a detection of possible delayed occurrence

or persistence of or recovery from toxic effects, the animals in the recovery groups (5 males and 5 females for control and high dose group)

were observed for a period of 28 days following the last administration. All animals were housed in groups of 5 animals/sex/cage.

The following doses were evaluated:

Control:                             0      mg/kg body weight/ day

Low Dose:                    100     mg/kg body weight/ day

Medium Dose:              300    mg/kg body weight/ day

High Dose:                   1000  mg/kg body weight/ day

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in aqua ad iniectabilia(sterile water)

and administered daily during a 90-day treatment period to male and female animals. Dose volumes were adjusted individually based on

weekly body weight measurements.

During the period of administration, the animals were observed precisely each day for signs of toxicity. Female (no. 85 of HD group)

killed intercurrently due to ethical reason was examined macroscopically and at the conclusion of the test, surviving animals were

sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly. At the conclusion of the treatment period, all animals of the non recovery groups

were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken and a set of organs/tissues was preserved.

Blood samplesfrom the abdominal aortawere collected afterovernight fastingat the end of the treatment and recovery period as part of the

sacrifice of the animals to examine the haematology, clinical biochemistry and blood coagulation parameters.

A urinalysis was performed with samples collected from urinary bladder of all animals at necropsy.

A full histopathological evaluation of the tissues was performed on organs and tissues from all animals of non-recovery (main study) Control

and HD groups, as well as all gross lesions from all groups, were examined by light microscopy. Since microscopic changes that could

be attributed to treatment with the test item were not observed in animals of the non-recovery (main study) HD group, further histoprocessing

and histopathology evaluation were not performed.


Summary Results

There was no mortality caused due to test item treatment during the study period, except one female no. 85 of HD group (the non-recovery

high dose group), which was euthanized for ethical reasons during the course of the study on treatment day 89. The cause of animal’s

morbidity was an accidental aspiration (aspiration pneumonia) of the dosing solution.

There were no clinical signs of toxicological relevance observed in the test item treated animals during the study period.

During the weekly detailed clinical observation, no significant changes or differences between the groups were found.

There were no ophthalmoscopic findings in any of the animals of this study.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment or

recovery period. There were no biologically relevant differences in body temperature between the groups.

There were no effects on body weight, body weight gain and food consumption in both male and female dose groups during the study period.

In males and females, at the end of treatment and recovery period there were no effects on haematology and blood coagulation parameters.

There were no adverse effect of test item treatment on clinical biochemistry parameters and noconspicuous differences in urinary parameters between the dose and control group observed.

There were no gross lesions that could be attributed to treatment with the test item. In female no. 85 (HD group) which was euthanized for

ethical reasons, “Irregular surface” and “Not collapsed”, which correlated microscopically with aspiration pneumonia, were recorded in the lung.

In both males and females, there were no effects of test item treatment on absolute and relative (to brain and terminal body weight) organ weights.

There were no microscopic lesions that could be attributed to treatment with the test item. In female no. 85 (HD group) which was euthanized for ethical reasons, aspiration pneumonia which was characterized by inflammation with intra-bronchial and alveolar fluid contents as well as with aggregation of substance-laden macrophages and reactive hypertrophy of bronchial/ bronchiolar epithelium was observed in the lung. This was caused by an accidental aspiration of the dosing solution and considered to be cause of animal’s morbidity. Marked thymic atrophy

(ie. accelerated atrophy) was recorded in this animal. This was deemed to be a secondary change as a response to a stressful condition

related to aspiration pneumonia.

The remainder of microscopic findings recorded in this study was within the range of normal background lesions which may be recorded in animals of this strain and age.

The determinations of serum phosphorus were performed by ICP-OES using two independent wavelengths highly specific for phosphorus, one wavelength for quantification and one for confirmation.The level of phosphorus was determined in serum sampled at24 hours after the 1stdose administration, in week 4, 24 hours after the last administration (at necropsy) and in recovery animals at necropsy.The level of serum phosphorus in the test item treated groups and corresponding controls were comparable indicating no accumulation of test item in the body.

Test substance concentration, stability and homogeneity in dosing formulations were obtained by ICP-OES. The analytical results obtained for the individual dose groups were consistent with analysis of the % of the nominal of the test item for the concentration, stability and homogeneity analysis.

The concentration analysis of formulation samples was determined in study weeks 1, 5, 9 and 13 for all dose groups. The recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were in the range of 94.6% to 103.6% of the nominal dose of test item.

Stability of formulation samples was investigated in study week 1 based on concentration in the LD and HD groups. After 6 hours storage at room temperature recovery compared to starting value was 102.9% and 95.7% of % nominal dose of test item in LD and HD groups, respectively. All samples were stable as concentration after storage did not differ from start value by more than 20%.

Homogeneity of formulation samples was determined in study weeks 1, 5 and 13 for the LD, MD and HD groups. The recovery observed for the LD, MD and HD groups were in the range of 90% to 110.4% of the % nominal dose of tes item.

Conclusion

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with the test item in male and female Wistar rats, with dose levels of 100, 300, and 1000 mg/kg body weight/ day the following conclusions can be made:

No adverse effects of the test item were found at dose levels of 1000 mg/kg body weight/ day. The NOAEL of the test item in this study is considered to be 1000 mg/kg body weight/ day.