Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-10-1988 to 19-10-1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
GLP compliance:
yes
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:
MK-0217
(L-670,452-005Y),
batch number
008,
was
identified by thin layer chromatography and infrared
spectroscopy.
Purity greater than 99%. was assessed by potentiometry.
Stability was deduced from purity analyses.

Test animals

Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals and environmental conditions:
Age at Initiation of the Study: Approximately 8 months.

Body Weight at Initiation of the Study:
Females: 6.4 to 8.9 kg.
Males 7.4 to 9.3 kg.

Source: Marshall Farms, North Rose, NY, U.S.A.

Identification Method: Printed on a collar.

Assignment to Dosage Groups:
Random allocation scheme.

b. Environmental Conditions:
(l) Housing:
Individual stainless steel cages in an air-conditioned
room with a temperature of approximately 19"C, relative
humidity between 30 and 70%. and a light cycle of
12 hours dark, 12 hours light.

(2) Diet:
Certified UAR 125C Lab Chow (approximately 300 g daily).
Because of decreased food consumption, the male 88-0172
from the mid-dose group was given UAR soft canned food
with the regular food, during 5 consecutive days before
being sacrificed in Drug Week 48.
Diet was given about 2 hours after treatment.
Dogs were fasted overnight prior to bleeding for
hematology, clinical chemistry, necropsy, and during
urine collection.
Tap water ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral, by gavage, via a pliable catheter.
Vehicle:
water
Details on oral exposure:
Preparation of drug solutions in distilled water was
done daily.
A factor of 1.33 was used in preparing drug doses.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Drug solutions were assayed for stability
concentration. Results were within acceptable limits.
Duration of treatment / exposure:
Recovery group: 91 doses (both sexes).
Remaining groups:
Females: 365, 366, or 367 doses.
Males: 364 or 365 doses.
Frequency of treatment:
once daily , seven days a week
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 mg/kg bw/day (actual dose received)
Dose / conc.:
2 mg/kg bw/day (actual dose received)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
recovery group- dosing drug week 1 through 13
No. of animals per sex per dose:
4 (four)
Control animals:
yes, concurrent vehicle
Details on study design:
recovery group treated for 13 weeks and sacrificed at Week 53.
Positive control:
n/a

Examinations

Observations and examinations performed and frequency:
Physical Examination:
All animals daily, with less detailed examination on weekends and holidays.

Body Weights:
All animals pretest and once a week from Drug Week 2 to Drug Week 52..

Food Consumption:
All animals, measured weekly, based on a 4-day consumption period.

Ophthalmologic Examination:
All animals, pretest and in Drug Weeks 4, 12, 2.5, 36, and 52,
using an indirect ophthalmoscope and a slit lamp if
necessary, to examine the different portions of the eye. A
0.5%. tropicamide solution (Mydriaticum, Chibret) was used to
dilate the pupils and facilitate visualization of the fundus
and the lens.

Electrocardiography:
All animals, pretest and in Drug Weeks 4, 12., 25, 36, and 52.
Leads I, II, III, aVL, aVR, aVF, cv recorded.
Heart rate, and PR, QRS, and QT intervals were measured from
the electrocardiogram of each dog.

Hematologic Examination:
All animals, pretest and in Drug Weeks 5, 8, 13, 26, 39, and
51.
Blood collected via the jugular vein.
Parameters determined:
Erythrocyte count
Hemoglobin concentration
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Prothrombin time
Activated partial thromboplastin time
Platelet count
Total leukocyte count
Differential leukocyte count.

Serum Biochemistry Examination (See Appendix II):
All animals, pretest and in Drug Weeks 5, 8, 13, 26, 39, and
51.
Blood collected at the same time and under the same
conditions as for hematologic examinations.
Parameters determined:
Glucose
Urea nitrogen
Creatinine
Total protein
Albumin
A/G ratio
Total cholesterol
Triglycerides
Aspartate aminotransferase actIvity
Alanine aminotransferase activity
Alkaline phosphatase activity
Sodium
Potassium
Chloride
Ionized calcium
Total calcium
Phosphorus.
In all dogs, additional serum (minimum 0.5 ml/dog) and plasma
(about 100 mel/dog) samples were taken at each of the above
time points and were frozen (about -20"C) to be sent to
MSDRL, West-Point, Pa, USA, for possible further analyses.

Urinalysis
(1) Special Urinalyses :
All animals pretest, in Drug Weeks 8, 13, 26, 39, and 51.
Urine was collected with the dogs in their own cages
overnight for approximately 16 hours.
Determinations:
Total calcium
Phosphorus
Creatinine
Total calcium/creatinine ratio
Phosphorus/creatinine ratio.

(2) Routine Urinalyses
All animals pretest and in Drug Weeks 8, 13, 26, 39, and
51.
Urine was collected at the same time and under the same
conditions as in special urinalyses.
Quantitative determinations:
Urinary volume
Specific gravity
Semi guantitative determinations:
pH
Protein
Glucose
Bilirubin
Occult blood
Ketones
Urobilinogen.
Microscopic examinations of sediment for erythrocytes,
leukocytes, epithelial and tubular cells, and casts.
In all dogs, additional urine samples (about 10 ml/dog)
were taken at each of the above time points and were
frozen (about -20"C) to be sent to MSDRL, West-Point,
Pa, USA, for possible further analyses.

Metabolism:
Plasma samples for assaying blood levels of MK-0217 were
taken in two dogs/ sex/ group in Drug Weeks 6, 15, and 27,
before dosing and 30 min., 1, 2, 4, 6, and 24 hours
post-dosing.
After spinning, the samples were frozen (about -80"C) to be
sent to MSDRL, West-Point, Pa. USA for possible analysis.
Sacrifice and pathology:
All dogs, including those sacrificed early, were killed by
exsanguination during intravenous barbiturate anesthesia and
subjected to complete necropsy.
Terminal body weights and weights of adrenals, brains,
hearts, kidneys, livers, lungs, ovaries, pituitaries,
prostates, spleens, testes with epididymides, thyroids, and
uteri of all animals were recorded at the time of necropsy.
From all animals, representative samples of most tissues and
all gross changes were fixed in 10% neutral buffered
formalin, except testes and epididymides in Bouin's fixative,
and eyes in Zenker's acetic fixative. Bone marrow smears
were taken from the rib of each animal and air-dried.
In addition, the following samples were taken: one rib, the
dorsa-medial portion of the ilium, one vertebra (Ll), and the
proximal third of the right femur, and all were fixed in 701.
ethanol. Also the entire left femur, entire left humerus and
radius were taken and frozen at -20°C. All these samples
were sent to MSDRL, West-Point, Pa, USA, for possible
analyses (see Special Toxicity Report).

d. Histology:
Sections of selected sampled tissues were prepared by routine
methods and stained with hematoxylin and eosin (except marrow
smears stained with May-Grlinwald-Giemsa).
Tissues microscopically examined from all
control, 8 mg/kg/day, 8 mg/kg/day recovery group animals and sacrificed
dog 88-0172 of the 2 mg/kg/day group included:
salivary gland, stomach, small intestine, large intestine, liver,
gallbladder,pancreas, adrenals,pituitary, thyroids,
para thyroids (if present in section of thyroids), kidneys,
urinary bladder, ovaries,uterus, testes, epididymides,
prostate, skin, mammary gland (if present in section of skin
from mammary region), lung, heart, spleen, lymph nodes,
thymus, bone marrow (rib), marrow smear, bone (ribs and
femurs), skeletal muscle (thigh), brain, spinal cord, sciatic
nerve, eyes and optic nerve.
Sections of ribs, femurs, and kidneys from dogs of the middle
and the low dose groups were also examined microscopically,
as well as sections of all grossly reported lesions.
Histological sections of the kidneys showing microscopic or
suspected microscopic changes were also stained according to
Perl's, and Nile-Blue methods for hemosiderin and lipofuscin,
respectively.
Statistics:
Data were analyzed using the average, linear, and quadratic
coefficient method with significance established at P ~ 0.05
based on an analysis of variance or covariance using trend
test. If criteria for homogeneity and/or normality were not
met, data were examined after Ranki t transformation followed
by analysis of variance or covariance.
Parameters analyzed:
Body Weight (females)
Food consumption (females).
Data were analyzed for homogeneity of variance by Levene
test, normality by Wilk and Shapiro W. statistics, and
statistical significance at P ~ 0.05 based on an analysis of
variance or covariance using trend test. If criteria for
homogeneity and/or nonmality were not met, data were examined
after Rankit transformation followed by analysis of variance
or covariance.
Parameters analyzed:
Serum total calcium
Serum ionized calcium
Serum phosphorus.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The main clinical signs observed before sacrifice in the
mid-dose male 88-0172 and in the high-dose female 88-0227
were body weight loss, anorexia, coughing (88-0l72M), dyspnea
(88-0227F) and vomiting (both dogs). These clinical signs
suggested a possible faulty intubation;
this was subsequently confirmed by the gross and histopathological
findings
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Two dogs, one mid-dose male (88-0172) and one high-dose female(88-0227).
showing severe signs of respiratory distress, were sacrificed in Drug Weeks 48 and 5O, respective­
ly. Both animals presented gross and microscopical evidence of severe inhalation bronchopneumonia (faulty intubation).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
throughout the study, a lower body weight gain was observed
in the high-dose female group (8 mg/kg/day) when compared to
the control female group (see Figure 1). The time-response
analyses for body weight in female groups during the 0-52 week
period was statistically significant (P ~ 0.05) for quadratic
effect only through the high-dose female group.
At the end of the study, the decrease in mean body weight
gain observed in the high-dose female group was about 33 per­
cent when compared to control females while in the female
group in recovery, treated for 13 weeks at the high dosage
(8 mg/kg/day), the mean body weight gain was roughly similar
(-7%) to that of the female control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no drug-related changes:
the time-response analyses for food consumption in female groups during the
0-52 week period was not statistically significant (P > 0.05)
for average, linear, and quadratic effect
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Before sacrifice, the mid-dose male 88-0172 and the high-dose
female 88-0227 had increased leukocyte counts (19.5 x lo3;mm3
and 30.1 x 103/mm3, respectively) due to increased segmented
neutrophil counts (17 ,062/mm3 and 25,585/mm3, respectively).
These increases were thought to be the reflection of the
inflammatory process observed histologically in the lungs and
consequence of a bronchopneumonia.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
All the transient (from Drug Week 5 to Drug Week 13) drugr elated
changes observed reflected the pharmacological activity of the compound.
In Drug Weeks 5, 8, and 13, a statistically significant
(P ,{ 0.05) decrease in serum total calcium mean values was
observed in both groups treated at the dosage level of 8 mg/kg/day.
'From Drug Week 26 onwards, these mean values returned to control mean values.
In Drug Weeks 5 and 8, a statistically significant (P ~ 0.05)
decrease in serum phosphorus mean values was observed in all
MK-0217-treated groups. In Drug Week 13,all the group mean values (sexes
combined) were lower (10.9 -21. 7%) when compared to
concurrent control; From Drug Week 26 onwards, these
values returned to control values.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no obvious drug-related changes in urine
phosphorus and phosphorus/creatinine ratio.
In Drug Weeks 8 and 13, in the urine from all MK-0217- treated groups,
there were transient increases in total calcium (upto 121% and 109.5% respectively) and in the ratio
total calcium/creatinine (upto 150% and 100% respectively), when compared to the control
values. From Drug Week 26 onwards, these values returned to
control values. There wee no drug related effects on routine urinalyses.
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Bone: Histologically, retention of primary spongiosa was observed
in the ribs of the majority of treated dogs. These changes
were more prominent in the 8 mg/kg/ day group. The severity
of the change ranged from very slight to severe.
Retention of primary spongiosa was also present in the
femurs of the majority of treated dogs (except three dogs of
the 0.5 mg/kg/day group). The extent of the change ranged
from very slight to marked, and the femurs appeared less
affected than the ribs with a larger number of animals within
the range of slight to moderate severity.
Mineralization of the renal papilla accompanied by chronic
focal nephritis was present in one dog of the 8 mg/kg/day
group (88-0129F) and in a second dog of the recovery group
(88-0188M).
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Electrocardiography

Effect levels

Key result
Dose descriptor:
LOAEL
Effect level:
0.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.5 mg/kg bw/day (actual dose received)
System:
musculoskeletal system
Organ:
bone
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
8 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The oral administration of MK-0217 for 53 weeks at doses
ranging from 0.5-8 mg/kg/day induced retention in the primary
spongiosa of the bones examined (ribs and femurs) at all
doses. A trend towards restoration of bone remodelling was
observed in the group kept in recovery for 40 weeks after
receiving the high dose level for 13 weeks.
In addition, the administration of 8 mg/kg/day induced
chronic focal nephritis and mineralization of the papilla. A LOAEL of 0.5 mg/kg bw/day has been derived from the effects noted above.