Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Designed to establish the effects of repeated administration of the test material to rats over a period of seven consecutive days
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12th February to 04 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other:
Principles of method if other than guideline:
The study was designed to establish the effects of repeated administration of the test material to rats over a period of seven consecutive days.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
White to yellow tan colour

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD
Details on species / strain selection:
Animals: Rat, Sprague Dawley (SPF)
Rationale: Conducted to GLP guidelines
Breeder: Charles River Deutschland GmbH, Stolzenseeweg 32-36, D-88353 Kisslegg / Germany
Number of Animals per Group: 12 females/12 males
Total Number of Animals: 24 females
Age when treated: 9 weeks
Identification: Unique cage number and corresponding color-coded spots on the tail. The animals were marked at acclimatization start.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.
Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with ranges for room
temperature 22 ± 3 °C and for relative humidity between 30-70 % (values above 70 % during cleaning process possible), automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.
Accommodation: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Duration of treatment / exposure:
seven consecutive days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 males and 3 female
Control animals:
yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:
Clinical signs, bodyweight development and food and water consumption were monitored during the study. Haematology, blood chemistry and organ weight data were evaluated at the end of the study and all animals were subjected to a gross necropsy examination. Histopathological examination of selected tissues was also performed.
Sacrifice and pathology:
On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination. All tissues were despatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford) for processing (Principal Investigator: T Hilling). Tissues from all control and 1000 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 pm and stained with haematoxylin and eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed together with the liver and spleen from all 150 and 500 mg/kg/day dose group animals. Since there were indications of treatment-related changes in the kidneys, brain, stomach and thymus, examination was subsequently extended to include similarly prepared sections of these tissues from all animals in the other treatment groups
Other examinations:
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 7). Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Increased salivation was evident soon after dosing for males treated with 1000 mg/kg/day from Day 1 and for females treated with 1000 mg/kg/day from Day 3. Associated incidents of staining of the external body surface were also evident following dosing on Day 4 and Day 7. One female treated with 1000 mg/kg/day displayed tiptoe gait and hunched posture from Day 4 and these signs were prevalent for all females from this dose group by Day 6. On Day 7, the females displayed further clinical signs, including ataxia, diuresis, lethargy, diarrhoea, dehydration and decreased respiratory rate. Similar signs were also evident for the males, although less severe than the females in appearance. Blood sampling procedures were scheduled for Day 7. Due to concerns for the survival of the high dose females following the removal of blood, it was considered necessary on welfare grounds to terminate the 1000 mg/kg/day females on Day 7 and withdraw the blood at termination for blood analysis. Similar effects were also evident at 500 mg/kg/day. Increased salivation was detected soon after
dosing from Day 2 for males and Day 4 for females. Incidents of diarrhoea were also observed for two males from Day 5 and hunched posture was evident for one female on Day 6. By Day 7, two males displayed signs including splayed gait, hunched posture, lethargy, and diarrhoea, decreased and laboured respiration.The remaining male displayed hunched posture and ataxia. Tiptoe gait, hunched posture, ataxia and staining of the snout and mouth were also evident for one
female on Day 7 and another female showed hunched posture. The remaining female treated at this dose level displayed staining around the mouth on Day 7.
Clinical signs at 150 mg/kg/day were confined to isolated incidents of increased salivation following dosing for one male on Day 5 and one male on Day 6.
Mortality:
mortality observed, treatment-related
Description (incidence):
Females treated 1000 mg/kg/day were terminated on Day 7 due to the poor physical condition and concerns for the survival of these animals
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced bodyweight gains were evident for animals of either sex treated at all dose levels when compared to control.
Substantial bodyweight losses were evident for animals of either sex treated with 1000 mg/kg/day (p<0.05 — p<0.01). All males treated with 500 mg/kg/day also displayed actual bodyweight losses resulting in a statistically significant reduction in bodyweight change when compared to controls
(p<0.01). Two females treated with 500 mg/kg/day also showed actual bodyweight losses, however statistical significance was not achieved.
Bodyweight losses were evident for two females treated with 150 mg/kg/day. Slight reductions in bodyweight gain were evident for males treated with 150 mg/kg/day when compared to controls; however, these animals did not show any bodyweight losses.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reductions in dietary intake were evident for all treated animals in comparison to controls throughout the treatment period. Animals of either sex treated with 1000 mg/kg/day showed a 26-28 % reduction between Days 1- 4 when compared to controls. Males treated with 1000 mg/kg/day showed a 59 % reduction in
dietary intake when compared to controls between Days 4-8. Substantial reductions in dietary intake were also evident for animals of either sex treated with
500 mg/kg/day during the treatment period. Animals of either sex treated with 150 mg/kg/day also displayed slight reductions in dietary intake (4.9 — 6.5%) during the treatment period
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency (the ratio of bodyweight to dietary intake) was similarly reduced for animals of either sex treated with 1000 and 500 mg/kg/day. Females treated with 150 mg/kg/day also showed a reduction in food efficiency when compared to controls
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No obvious differences in water intake were evident for treated animals when compared to controls.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 1000 and 500 mg/kg/day showed statistically significant reductions in leucocyte counts, specifically in the lymphocyte fraction (p<0.05 — p<0.01), when compared to controls.
Males treated with 1000 and 500 mg/kg/day displayed statistically significant increases in clotting times (p<0.01) when compared to controls. Furthermore, increases in erythrocyte and haematocrit counts were also evident for these animals when compared to control, however statistical significance was not achieved. Statistically significant increases in mean cell haemoglobin concentration (p<0.01) and haemoglobin (p<0.05) were however evident for males treated with
1000 and 500 mg/kg/day when compared to controls. No treatment-related effects were detected for animals of either sex treated with 150 mg/kg/day.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 1000 mg/kg/day showed statistically significant reductions in total protein (p<0.05 — p<0.01) and albumin levels (p<0.05 — p<0.01) in comparison to controls. Furthermore, increases in plasma glucose levels were also evident for animals of either sex treated with 1000 mg/kg/day, although statistical significance was only achieved for males (p<0.05). Reductions in plasma calcium levels were also evident for 1000 mg/kg/day males (p<0.01) and
females treated with this dose level showed a statistically significant increase in albumin/globulin ratio (p<0.05) when compared to controls. Increases in plasma urea levels were evident at 1000 mg/kg/day, however statistical significance was only achieved for females (p<0.01).
Animals of either sex treated with 1000 mg/kg/day also showed statistically significant increases in cholesterol (p<0.01) in comparison to controls. Reductions in triglyceride levels were also evident at 1000 mg/kg/day, although statistical significance was only achieved for males (p<0.05).
Females treated with this dose level displayed reductions in aspartate aminotransferase, alkaline phosphatase and creatinine when compared to controls. Increases in alanine aminotransferase levels were also observed, however statistical significance was not achieved for any of these parameters. Females showed an increase in inorganic phosphate when compared to controls and animals of either sex treated with 1000 mg/kg/day displayed statistically significant reductions in potassium (p<0.05 —p<0.01). Similar effects were evident at 500 mg/kg/day. Increases in plasma urea were evident for animals
of either sex, together with reductions in total protein and albumin levels, however statistical significance was only achieved for males (p<0.05 — p<0.01). Males treated with 500 mg/kg/day also showed a statistically significant reduction in calcium when compared to controls (p<0.01).
Statistically significant increases in cholesterol levels were evident for animals of either sex treated with 500 mg/kg/day when compared to controls (p<0.01) and 500 mg/kg/day males showed reductions in triglyceride levels (p<0.05). Reductions in potassium ions was also evident for animals of either sex treated with 500 mg/kg/day, although statistical significance was only achieved for males (p<0.01).
Effects at 150 mg/kg/day were confined to reductions in potassium levels which were evident for animals of either sex when compared to controls, although statistical significance was only achieved for males (p<0.05). There were no further blood chemical changes considered attributed to treatment with the test
material. Slight increases in plasma bilirubin levels were also evident for animals from all treatment group when compared to controls, however, statistical significance was not achieved and a convincing dose-related response was not evident for the female treatment groups. The toxicological significance of this finding is therefore unclear. Statistically significant reductions in sodium ions were also evident for all male treatment groups when compared to controls. The significance achieved in each case was minimal (p<0.05) however, a dose-related response was not evident, therefore these reductions may have arisen incidentally and may be unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Females treated with 1000 mg/kg/day were killed one day before scheduled kill. Organ weights were taken from these females at termination and the inclusion of this data for comparison with control data was considered necessary for the interpretation of the results obtained. A difference of one day was considered not to be sufficient to invalidate comparison between groups. Females treated with 1000, 500 and 150 mg/kg/day showed statistically significant increasein absolute and relative liver weights when compared to controls (p<0.01). Reduced absolute and relative thymus weights were evident for animals of either sex from all treatment groups when compared to controls. Statistical analysis of these data did not however reveal any significant
differences. Animals of either sex treated with 1000 mg/kg/day displayed elevated relative adrenal and kidney weights when compared to controls. Elevated relative adrenal weights were also evident for animals of either sex treated with 500 mg/kg/day when compared to controls. Statistical significance was however not achieved. Reductions in relative spleen weights were also evident for animals of either sex treated with 1000 and 500 mg/kg/day when compared to controls, although statistical significance was only achieved for females (p<0.01). Furthermore, reductions in absolute heart and brain weights, with increases in relative heart and brain weights were evident for animals of either sex treated with 1000 mg/kg/day. The changes in brain weights were
also evident for animals of either sex treated with 500 mg/kg/day. Organ weight changes in the reproductive organs were also noted, although statistical significance was never achieved. Females from all treatment groups showed a reduction in uterus weights, both absolute and relative to terminal bodyweights when compared to controls. Males treated with 1000 mg/kg/day showed a reduction in absolute testis and epididymis weights when compared to controls, and an increase in relative testis weights. Reduced absolute epididymis weights were also observed for males treated with 500 mg/kg/day when compared to controls. There were no further treatment-related changes. Females treated with 500 mg/kg/day displayed statistically significant increases in absolute and
relative kidney and ovary weights. The significance in each case was minimal (p<0.05) and in the
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Two females treated with 1000 mg/kg/day displayed small spleens at termination on Day 7. One
female treated at this dose level also showed coloured contents in the small intestines and
reddened small and large intestines. Dark foci were also evident on the gastric mucosa of the
stomach and a small thymus was evident for one high dose female.
Two males treated with 1000 mg/kg/day showed congestion of the stomach and two males also
displayed small seminal vesicles. All males treated with 500 mg/kg/day displayed small seminal
vesicles and one male also displayed a flaccid left testis. A small thymus were also evident for
one female treated with 500 mg/kg/day. The remaining two females treated at this dose level
revealed a small spleen at terminal kill.
Macroscopic abnormalities at 150 mg/kg/day were confined to one male displaying a cavity in the
left kidney and an enlarged liver and one female treated with 150 mg/kg/day displayed a fluid
filled sac on the median lobe of the liver.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were three unscheduled deaths during the course of the study. Females treated with 1000 mg/kg/day were all killed in extremis on Day 7 of the study. Each of these animals had
treatment-related changes the symptoms of which contributed significantly to the need for early termination.
LIVER: Generalised hepatocyte enlargement frequently with associated fine cytoplasmic vacuolation/clumping of cytoplasm was observed in relation to treatmentfor animals of either sex treated with 1000 or 500 mg/kg/day. One male and two females treated with 150 mg/kg/day also demonstrated generalised hepatocyte enlargement. Lymphoid atrophy and atrophy of the red pulp were seen for females treated with 1000 mg/kg/day but not at any other treatment level.
Hypertrophy of kidney proximal tubular epithelial cells was observed for males treated with 1000 mg/kg/day but not at any other treatment level. Females were not similarly affected.Necrosis of cells in the granular layer of the brain cerebellum and vacuolation of the Purkinji cell layer were seen as a consequence of treatment for all animals of either sex treated with 1000 mg/kg/day, and for all except one female treated with 500 mg/kg/day. Similar effects were not seen among animals of either sex treated with 150 mg/kg/day. Hyperkeratosis of the forestomach epithelium, occasionally with associated acanthosis, was seen for animals of either sex treated with 1000 or 500 mg/kg/day but not at 150 mg/kg/day. Lymphoid atrophy was observed for all animals of either sex treated with
1000 mg/kg/day and for two males and one female treated with 500 mg/kg/day
Histopathological findings: neoplastic:
not specified

Effect levels

Dose descriptor:
NOEL
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable

Target system / organ toxicity

Critical effects observed:
not specified
Organ:
other: Oral (gavage) administration to rats for a period of seven days at dose levels of 1000, 500 and 150 mg/kg/day resulted in treatment-related effects at all dose levels. A 'No Observed Effect Level' (NOEL) could therefore not be established

Applicant's summary and conclusion

Conclusions:
Oral (gavage) administration of CP-902684 to rats for a period of seven days at dose levels of 1000, 500 and 150 mg/kg/day resulted in treatment-related effects at all dose levels. A 'No Observed Effect Level' (NOEL) could therefore not be established