Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The objective of this study was to assess the systemic toxic potential of CP-163-625-BV
when administered orally by gavage to rats for 7 days. The study was not designed to meet any specific regulatory guidelines:

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The objective of this study was to assess the systemic toxic potential of CP-163-625-BV
when administered orally by gavage to rats for 7 days. The rat was chosen as the test species because it is accepted by regulatory agencies. The CD
rat was used because of the historical control data available in this laboratory. The oral route of administration was chosen to simulate the conditions of potential human
exposure.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: CP-163-625-BV

Action: Chemical Intermediate in Pharmaceutical/animal health
synthesis

Description: White to off-white crystalline powder

Storage conditions: Room temperature in the dark

Supplier: Sponsor

Batch number: 05C5D0007

Date of receipt: 19 October 2006
Quantity received: 300g
Expiry date: 22 November 2006
Purity: 100%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because it is accepted by regulatory agencies. The CD
rat was used because of the historical control data available in this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
Animal supply, acclimatisation and allocation
A total of 15 male and 15 female CD rats were received from Charles River (UK) Ltd. The
rats were ordered at 49-56 days of age and were within a weight range of 230-257 g for males
and 173-187 g for females on arrival.
On arrival, the animals were removed from the transit boxes and allocated to study cages.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with
the procedure being repeated until each cage held the appropriate number of animals. Each
sex was allocated separately.
The cages constituting each group were blocked by sex and the groups dispersed in a battery
so that possible environmental influences arising from their spatial distribution were
equilibrated, as far as was practicable. Additionally, the battery of cages was rotated around
the room at weekly intervals (between pretreatment and treatment phase) to further minimise
possible spatial variations.
Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
Each cage label was colour-coded according to group and was numbered uniquely with cage
and study number, as well as the identity of the occupants.
The animals were allowed to acclimatise to the conditions described below for 5 days before
treatment commenced. For those animals selected for this study, their age at the start of
treatment was 54 to 61 days and their bodyweights were in the range of 279 to 315g for males
and 201 to 211 g for females.
The spare animals were removed from the study room after treatment commenced.

Animal housing, diet and water supply
Animals were housed inside a barriered rodent facility (Building F21, Room 6C). The
facility was designed and operated to minimise the entry of external biological and chemical
agents and to minimise the transference of such agents between rooms. Before the study the
room was cleaned and disinfected with a bactericide.
The animal room was kept at positive pressure with respect to the outside by its own supply
of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature
and relative humidity controls were maintained within the range of 19 to 23°C and 40 to 70%
respectively. Artificial lighting was controlled to give a cycle of 12 hours continuous light
and 12 hours continuous dark per 24 hours.
Periodic checks were made on the number of air changes in the animal rooms. Temperature
and humidity were monitored continuously. Since these data show that there were no
significant deviations from target values they are not presented.
Alarms were activated if there was any failure of the ventilation system, or temperature limits
were exceeded. A stand-by electricity supply was available to be automatically brought into
operation should the public supply fail.
The animals were housed three of one sex per cage. The cages were made of a stainless steel
body with a stainless steel mesh lid and floor, and were suspended above absorbent paper
which was changed at appropriate intervals. Cages, cage-trays, food hoppers and water
bottles were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet), except overnight before routine blood sampling on Day 8. This diet
contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles
fitted with sipper tubes.
Each cage of animals was supplied with an Aspen chew block for environmental enrichment.
Chew blocks were provided throughout the study, except overnight before the preterminal
blood sampling.
Each batch of diet was analysed routinely by the supplier for various nutritional components
and chemical and microbiological contaminants. Supplier’s analytical certificates were
scrutinised and approved before any batch of diet was released for use. The quality of the
water supply is governed by regulations published by the Department for Environment, Food
and Rural Affairs. Certificates of analysis were received routinely from the water supplier.
Certificates of analysis were received routinely from the supplier of the Aspen chew blocks.
Since the results of these various analyses did not provide evidence of contamination that
might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were
analysed, as none that may have interfered with or prejudiced the outcome of the study was
known.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was chosen to simulate the conditions of potential human
exposure.
Vehicle:
methylcellulose
Details on oral exposure:
The test substance, CP-163-625-BV, was prepared for administration as a series of graded
concentrations in the vehicle. The required amount of test substance was ground in a mortar
using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were
gradually added and mixed to produce a smooth, pourable suspension. The suspension was
quantitatively transferred and diluted to volume and finally mixed using a high-shear
homogeniser. A series of suspensions at the required concentrations were prepared by
dilution of individual weighings of the test substance.

The test substance was used as supplied. All formulations were prepared freshly each day
and dosed within three hours of preparation.
Detailed records of compound usage were maintained. The amount of test substance
necessary to prepare the formulations and the amount actually used were determined on each
occasion. The difference between these amounts was checked before the formulations were
dispensed.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
7 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 per sex per dose
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
Clinical observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the
occupant(s). Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
Daily during the treatment period, detailed observations were recorded at the following times
in relation to dose administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day.
In addition, a more detailed weekly physical examination was performed on each animal to
monitor general health.
During the acclimatisation period, observations of the animals and their cages were recorded
at least once per day.
Mortality
Animals were observed carefully for debilitated animals.
Bodyweight
The weight of each rat was recorded five days before treatment commenced (Day -5), on the
day that treatment commenced (Day 1), once during mid-week (Day 4), on Day 7 and before
necropsy on Day 8.
Food consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for five days before treatment started (Day -5 to -1), and twice during the treatment
period. From these records the mean daily consumption per animal (g/rat/week) was
calculated for each cage.


Sacrifice and pathology:
Method of kill
Animals surviving until the end of the scheduled treatment period were killed by carbon
dioxide asphyxiation. The sequence in which the animals were killed after completion of
treatment was selected to allow satisfactory inter-group comparison.
Macroscopic pathology
All animals were subject to a detailed necropsy.
After a review of the history of each animal, a full macroscopic examination of the tissues
was performed. All external features and orifices were examined visually. The cranial roof
was removed to allow observation of the brain, pituitary gland and cranial nerves. After
ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and
pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position,
morphology or interaction was recorded.
The requisite organs were weighed and external and cut surfaces of the organs and tissues
were examined as appropriate. Any abnormality in the appearance or size of any organ and
tissue was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Organ weights
The following organs, taken from each animal killed after 7 days of treatment, were dissected
free of adjacent fat and other contiguous tissue and the weights recorded:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus

Fixation
Testes and epididymides were fixed in Bouin’s solution prior to transfer to 70% industrial
methylated spirit. Samples (or the whole) of the other tissues listed below from all animals
were preserved in 10% neutral buffered formalin:
Abnormalities Oesophagus
Adrenals Ovaries
Brain Prostate
Caecum Rectum
Colon Sciatic nerves
Duodenum Spinal cord
Epididymides Spleen
Head Stomach
Heart Testes
Ileum (including Peyer’s patches) Thymus
Jejunum Thyroid with parathyroids
Kidneys Trachea
Liver Urinary bladder
Lungs and bronchi Uterus and cervix
Lymph nodes - mandibular
- mesenteric

Samples of any abnormal tissues were also retained and processed for examination. In those
cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly
affected region and sectioned as appropriate.
Samples of the head (including nasal cavity, paranasal sinuses and nasopharynx) were not
examined histologically, but are retained against any future requirement for microscopic
examination.

PATHOLOGY
Light microscopy
Microscopic examination was performed as follows:
All tissues preserved for examination (as specified above) were examined for all
animals of Groups 1 (Control) and 4 (300 mg/kg/day) sacrificed on completion of the
scheduled treatment period. Trackdown was performed in animals of Groups 2 and 3
(75 and 150 mg/kg/day) on the liver only.
Tissues reported at macroscopic examination as being grossly abnormal were examined
for all animals.
The following tissues which were considered to exhibit a reaction to treatment at the
high dosage were examined for all animals.
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus

Findings were either reported as "present" or assigned a severity grade. In the latter case one
of the following five grades was used - minimal, slight, moderate, marked or severe. A
reviewing pathologist undertook a peer review of the microscopic findings.
Other examinations:
Haematology, peripheral blood
On Day 8 (prior to necropsy), blood samples were obtained from all animals after overnight
withdrawal of food. Animals were held under light general anaesthesia induced by isoflurane
and blood samples were withdrawn from the sublingual vein.
Blood samples (nominally 0.5 mL) were collected into tubes containing EDTA as
anticoagulant and examined for the following characteristics:
The following were measured using a Bayer Advia 120 haematology analyser:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Red blood cell distribution width (RDW)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Morphology flags were generated by the Advia 120 analyser. The most common
morphological changes, anisocytosis, micro/macrocytosis and hypo/hyperchromasia
were recorded as follows:
- = no abnormalities detected
Reticulocyte count (Retic) - using a a Bayer Advia 120 haematology analyser.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate
anticoagulant and examined in respect of:
Prothrombin time (PT) - using an ACL 3000 Plus analyser and IL PT-Fibrinogen
reagent.
Activated partial thromboplastin time (APTT) - using an ACL 3000 Plus Analyser
and IL APTT reagent.

Blood chemistry
At the same time and using the same animals as for peripheral haematology, further blood
samples (nominally 0.7 mL) were collected into tubes containing lithium heparin as
anticoagulant. All tubes were mechanically agitated for at least two minutes and the sample
subsequently centrifuged at 6000 rpm for 10 minutes at ambient temperature in order to
separate the plasma. After separation, the plasma was examined in respect of:
Using a Hitachi 917 Clinical Chemistry Analyser:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and
analysed albumin concentration.
Statistics:
Summary statistics (e.g. means and standard deviations) presented in this report were
calculated from computer-stored individual raw data. The summary statistics and the
individual data were stored in the computer to a certain number of decimal places, different
for each parameter. For presentation purposes, however, they were usually rounded to fewer
places. It is, therefore, not generally possible to reproduce the presented means and standard
deviations exactly using the presented individual data.
Throughout the report the following abbreviations are used:
N [or n] Number of animals examined
SD [or sd] Standard deviation

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Post-dose salivation was recorded for animals from all treatment levels. The onset of the
salivation was generally from the end of dosing the group and had generally resolved by 1 to
2 hours post-dose, although on occasions salivation was seen at the last check of the day
(approximately 5 hours post dose) Animals receiving 300 mg/kg/day tended to show a
slightly higher overall incidence than those at lower levels. This sign was not seen in the
control animals.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
All females receiving 300 mg/kg/day showed weight loss (between 1 and 12g) during the
study, with 2/3 females showing lower absolute weights on Day 7 compared with Day 1. The
remaining female at 300 mg/kg/day did show an overall gain but this was lower then the
lowest concurrent control individual gain. A female receiving 75 mg/kg/day also showed an
overall small weight loss (2g), however, overall gains were apparent for the other two
females in this group and also for females receiving 150 mg/kg/day. Therefore there was
considered to be no clear effect of treatment on weight gain for females receiving 75 or
150 mg/kg/day. Bodyweight gain up to Day 7 was similar between controls and all male
treated groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption in females receiving 300 mg/kg/day was slightly lower than control
animals. Food consumption was generally lower in the latter 4 days of the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
One male receiving 300 mg/kg/day (Animal 12 - 4M) exhibited a moderate prolongation of
the Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT), in
comparison with other males, including controls. The remaining two males, however, showed
PT and APTT values which were similar to concurrent controls, thus there was no conclusive
evidence that clotting time was affected by treatment.
Females receiving 150 or 300 mg/kg/day showed higher than control mean haematocrit,
haemoglobin and red blood cell values, which were dosage related in degree. An association
with treatment could not be discounted, however, as the majority of values for the
aforementioned parameters were within or very close to the background ranges (expected
background 5 and 95 percentiles: Hct 3.87-4.54, Hb 13.8-15.9, RBC 6.77-8.04), these
differences were not considered to be toxicologically important. Similarly, although females
receiving 300 mg/kg/day also tended to exhibit elevated mean white blood cell counts
compared with controls, as individual values for these females were within the expected
background ranges, if treatment-related they were considered not to be of toxicological
importance.
There were no other differences from controls in the haematology parameters which were
considered possibly related to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean potassium levels for all treated groups of both sexes were clearly higher than
concurrent controls, this was dosage related in degree. Calcium concentrations were elevated
above control for both sexes receiving 300 mg/kg/day and also females receiving 150
mg/kg/day, with the magnitude of difference in females appearing dosage related. Individual
calcium values for the aforementioned animals were, however, within the background range
and thus considered not to be toxicologically important. Both sexes receiving 300 mg/kg/day
also showed lower than control A/G ratios. In males this was mainly due to slightly lower
albumin values, while in females the plasma globulin levels were elevated. Females receiving
300 mg/kg/day also had higher than control total protein values, although these remained
within the normal background range.
There were no other differences from control which were considered to be possibly related to
treatment. The mean ALP values for females receiving 150 or 300 mg/kg/day were lower
than controls. There was, however, a lack of dose relationship and individual values were
within the expected range.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Individual and group mean absolute liver weights for males receiving 300 mg/kg/day were
higher than controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic changes recorded at termination were considered not to be related to
treatment, all findings being within the background range for animals of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Liver
Two males receiving 300 mg/kg/day showed minimal centrilobular hepatocyte hypertrophy,
compared with none in the controls or other treated groups. These same two males, as well
as two males receiving 150 mg/kg/day and a single female receiving 300 mg/kg/day showed
minimal condensed cytoplasmic centrilobular material. Minimal generalised hepatocyte
vacuolation, often correlated with positive ORO staining was noted for animals from all
treated groups, the group incidence in males being dose-related.
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Critical effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver

Any other information on results incl. tables

Oral gavage administration of CP-163-625-BV to rats for 7 days at dosages of 75 or 150 mg/kg/day did not elicit any adverse treatment-related findings. There was however evidence of toxicity at 300 mg/kg/day, with the females being more obviously affected. The absolute bodyweights at the end of the treatment period for 2/3 females receiving 300 mg/kg/day were below the weights recorded on Day 1. For both of these females there was evidence of progression over-time that is the weight losses were more apparent during the period Day 4 to 7, than Day 1 to 4. The remaining female in this group did exhibit an overall weight gain but it was lower than the lowest gain recorded for concurrent controls and this female did show an initial minor weight loss. These weight losses/lower gains were considered to be indicative of toxicity, despite a lack of similar effects in males. Microscopic examination did reveal the liver as a target organ for toxicity, although the extent of toxicity seen was considered to be minor and likely to be reversible. Animals of both sexes from all treated groups showed generalised hepatocyte vacuolation which showed a good correlation with ORO staining. This indicates that the vacuolation is associated with fat accumulation. Fat accumulation in the liver is a classic sign of toxicity which can show reversibility. The severity of the vacuolation was only minimal and considering that control animals of both sexes, although not exhibiting generalised vacuolation, did show a similar degree of ORO staining, these findings in the treated groups are considered not to represent overt toxicity. In addition, the degree of the findings indicate that they would be reversed if treatment was ceased. The aetiology of the condensed cytoplasmic centrilobular material is not known; there is no clear consistent correlation with the other liver findings. The two males at 300 mg/kg/day which showed condensed material were the same two males which showed hepatocyte hypertrophy, but this was not true for the two males at 150 mg/kg/day and the females at 300 mg/kg/day in which condensed material was present. The centrilobular hepatocyte hypertrophy is considered to be indicative of an adaptive change in the liver and thus not of toxicological importance on this study. The higher liver weights recorded for males at 300 mg/kg/day were considered attributable to the microscopic changes in the liver. There were some haematology and blood chemistry disturbances apparent for both sexes receiving 150 or 300 mg/kg/day. These can not be clearly linked with the treatment-related microscopic changes in the liver and the majority of values were within the expected background ranges. Therefore within the context of this study, these differences were considered not to be of toxicological importance. Transient post-dose salivation, which was seen at all treatment levels, is a common finding on oral gavage studies and in the absence of any correlating findings is considered not to be indicative of toxicity. The lower food intake recorded for both sexes receiving 75 or 300 mg/kg/day is of uncertain relationship to treatment. There is an absence of a similar effect for both sexes at 150 mg/kg/day and no corresponding effects on bodyweight gain in either sex receiving 75 mg/kg/day or males receiving 300 mg/kg/day

Applicant's summary and conclusion

Conclusions:
The effects seen on bodyweight gain for females at 300 mg/kg/day were considered to
indicate overt toxicity. There is evidence of fat accumulation in the liver but the extent of
this finding is considered to be indicative of reversible toxicity and thus within the context of
this study 150 mg/kg/day can be classed as the highest NOAEL.
Executive summary:

The systemic toxic potential of CP-163-625-BV to CD rats by oral administration was assessed over a period of 7 days. Three groups, each comprising three male and three female rats received CP-163-625-BV at dosages of 75, 150 or 300 mg/kg/day. A similarly constituted Control group received the vehicle, aqueous methylcellulose 1% (w/v), at the same volume dosage. During the study clinical condition, bodyweight, food consumption, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. Salivation after dosing was recorded for all treated groups. Females receiving 300 mg/kg/day showed an overall group mean loss in bodyweight. Food consumption in males and females receiving 75 or 300 mg/kg/day, but not those at 150 mg/kg/day, was lower than control animals. Females receiving 150 or 300 mg/kg/day showed higher mean haematocrit, haemoglobin concentration and red and white blood cell counts. All treated groups showed elevated potassium values with females at 150 or 300 mg/kg/day also showing higher calcium values. Lower A/G ratios were evident for both sexes at 300 mg/kg/day. Higher liver weights were recorded in all males at 300 mg/kg/day. There were no macroscopic changes observed for any organs and tissues at post mortem examination. There was increased incidence of hepatocyte hypertrophy in males following treatment at 300 mg/kg/day and an increase in condensed cytoplasmic material in centrilobular hepatocytes in males treated at 150 mg/kg/day and males and females receiving 300 mg/kg/day. An increase in generalised vacuolation of hepatocytes was evident in all treatment groups, which did show a degree of correlation with an increase in Oil Red O stained vacuoles. Conclusion The effects seen on bodyweight gain for females at 300 mg/kg/day were considered to indicate overt toxicity. There is evidence of fat accumulation in the liver but the extent of this finding is considered to be indicative of reversible toxicity and thus within the context of this study 150 mg/kg/day can be classed as the highest NOAEL.