Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 1999 to 25 Jnauary 2000
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC
(Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27 July 1995).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Details on test material:
brown crystalline solid
Specific details on test material used for the study:
UK 209,947; brown crystalline solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD ®BR strain of rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Sprague-Dawley Crl:CD ®BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. On receipt the
animals were examined for signs of ill-health or injury. The animals were acclimatised for nine days during which time their health status was assessed. A
total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 136 to 179g, and the females
weighed 123 to 156g, and were approximately five to six weeks old.

The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food and water. A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was used.

I. Mains water was
supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. The animals were housed in a single air-conditioned room within the Safepharm Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air
changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly mean temperatures and humidities were included in the study records. The temperature and relative humidity were controlled to remain within target ranges of 21 + 2°C and 55 + 15% respectively. Occasional deviations from these targets were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using random letter tables, and the group mean bodyweights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test material was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg/day of Polyethylene glycol 400.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.
Vehicle:
propylene glycol
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of UK-209,947 in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Standard solutions of test material were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.

HPLC system was a Hewlett-Packard 1050, incorporating autosampler and workstation

The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking in between sampling. Sampling was performed in triplicate.
The test material formulations were sampled and analysed initially and then after storage at approximately +4°C in the dark for fourteen days. The test material formulations were sampled and analysed within two days of preparation.
Duration of treatment / exposure:
28 consecutive days
Frequency of treatment:
Test materia wasl administered daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
incorporating a correction factor for 74.772% purity
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
incorporating a correction factor for 74.772% purity
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
incorporating a correction factor for 74.772% purity
No. of animals per sex per dose:
5 male and 5 female per dose
Control animals:
no
Details on study design:
The dose levels were chosen based on the results of the range-finding study.

Examinations

Observations and examinations performed and frequency:
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the
working week. Animals were observed immediately before dosing and one hour after dosing at weekends and public holidays. Prior to the start of treatment and on Days 6, 13, 21 and 26 all animals were observed for signs of functional/behavioural toxicity. On Day 26 functional performance tests were also performed on all surviving animals together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
Sacrifice and pathology:
On completion of the dosing period all animals (excluding animal number 1 which was killed by intraperitoneal injection) were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Other examinations:
Functional Observation
Behavioural Assessment
Functional Performance Tests: Motor Activity and Forelimb/Hind Limb Grip Strength,
Sensory Reactivity
Bodyweight
Food and Water Consumption
Laboratory Investigations: Haematology, Blood Chemistry, Pathology - organ weights, histopathology
Statistics:
Haematological, blood chemical, organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain and quantitative functional performance and sensory reactivity data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA)
incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg/day showed increased salivation up to ten minutes after dosing from Day 2 onwards. Extensive fur staining and generalised fur loss were prevalent in the males and by Day 10 incidents of hunched posture, pilo-erection, tiptoe gait and respiratory pattern changes were evident in either sex.
One female developed clinical signs during the second week of the study which included laboured and gasping respiration, lethargy, ptosis and abdominal distension. This animal was subsequently killed in extremis at the start of Day 10. Respiratory pattern changes continued throughout the study at this dose level but receded during the final week of treatment to leave observations of hunched posture, noisy respiration, increased salivation, fur staining and fur loss.
Animals of either sex treated with 150 mg/kg/day showed increased salivation up to ten minutes after dosing from Day 9 onwards together with an isolated incident of wet fur. Such observations are often seen when a test material formulation is unpalatable or slightly irritant and, in the absence of any other evidence to suggest an adverse effect at this dose level, were considered not to be indicative of toxicity.
Red/brown fur staining was recorded for one male treated with 50 mg/kg/day and two control females over the first week of the study but this was considered to be entirely incidental.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated with 500 mg/kg/day was killed in extremis at the start of Day 10.
There were no other deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A substantial reduction in bodyweight gain was detected for males and females treated with 500 mg/kg/day during the first two weeks of the study, achieving statistical significance (p <0.01-0.001) for either sex during Week 1. Statistical significance was achieved for the males during Week 2 and one animal showed a bodyweight loss at Day 14. The effect appeared to regress over the final two weeks of the study and bodyweight development was similar to that of controls by the end of Week 4.
Males treated with 150 mg/kg/day showed a statistically significant reduction in bodyweight gain during the first week of study only.
Bodyweight development in 150 mg/kg/day females and animals of either sex treated with 50 mg/kg/day was similar to that of controls over the study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A reduction in food consumption was detected for animals of either sex treated with 500 mg/kg/day during Weeks 1 and 2 of the study. Food efficiency (the ratio of bodyweight gain to dietary intake) was also reduced. Dietary intake was reduced over the first two weeks of the study for 150 mg/kg/day males while a reduction in food efficiency was apparent in this sex during Week 1. Females treated with 150 mg/kg/day and animals of either sex from the 50 mg/kg/day dose group showed a dietary intake and food efficiency similar to that of controls throughout the study period
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Daily visual inspection of water bottles revealed intergroup differences and quantitative measurement was subsequently initiated from Day 15 onwards. Animals of either sex treated with 500 mg/kg/day and females treated with 150 mg/kg/day showed an increased water consumption compared with that of controls from Day 15 onwards. Males treated with 150 mg/kg/day and animals of either sex dosed at 50 mg/kg/day showed no such increase over the measurement period.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant reductions in haemoglobin, haematocrit, erythrocyte count and mean corpuscular volume were detected for males treated with 500 mg/kg/day when compared with that of controls. In addition mean corpuscular haemoglobin concentration (MCHC) was statistically significantly increased for 500mg/kg/day males and neutrophil count was elevated in the females. The neutrophil ia was also evident for 500 mg/kg/day males but, although many of the individual values were outside the normal ranges for either sex, statistical significance was not attained for the males. The anaemic condition was also evident in males treatedwith 150 mg/kg/day with reductions in haemoglobin, haematocrit and erythrocyte count and an increase in MCHC. No such changes were detected for 150 mg/kg/day females or for animals of either sex treated with 50 mg/kg/day.
The remaining statistically significant intergroup differences detected involved a reduction in group mean clotting (prothrombin) time for all female treatment groups compared with that of controls. All values were entirely within the expected normal range and, in isolation, these minimal (p<0.05) intergroup differences were considered to be fortuitous.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg/day showed increases in plasma aspartate aminotransferase and alanine ami notransferase compared with controls. Many of the individual values were outside of the respective normal ranges. A statistically significant increase in plasma cholesterol was also detected at the high dose but this was confined to the males only. Animals treated with 150 or 50 mg/kg/day showed no treatment-related changes in the parameters measured.
The remaining statistically significant intergroup differences detected were incidental changes which, in the absence of any evidence to suggest an effect on a
particular biological system, were considered to be of no toxicological importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Detailed behavioural assessments supported the signs seen clinically in 500 mg/kg/day animals during Weeks 2, 3 and 4. Tiptoe gait was recorded for one male during Week 2 followed by incidents of hunched posture and noisy respiration in animals of either sex during Week 3 and 4 investigations. No such observations were detected in 150 or 50 mg/kg/day animals. All remaining inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used and were, therefore, of no toxicological importance. There were no treatment-related changes in the functional performance parameters measured. There were no treatment-related changes in sensory reactivity
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg/day showed an increased adrenal weight, relative to bodyweight, compared with controls. Absolute adrenal weight was also elevated in the females and the effect extended to 150 mg/kg/day animals of this sex only. Relative heart weight was increased in both sexes treated with 500 mg/kg/day and 150 mg/kg/day females.
An increase in relative kidney weight was detected in either sex at 500 and 150 mg/kg/day (although statistical significance was not achieved for 150 mg/kg/day females) and for 50mg/kg/day males.
Relative liver weight was substantially elevated throughout all treatment groups with the majority of individual values outside the respective normal ranges.
The remaining intergroup difference was confined to a slight but statistically significant (p<0.05) increase in relative brain weight for 500mg/kg/day males.
This, however, was considered to result from the reduced terminal bodyweight seen in these animals.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The decedent from the 500 mg/kg/day dose group showed pale and enlarged lungs at necropsy together with a small spleen, gaseous distension of the intestines and accentuated lobular pattern of the liver. No macroscopic abnormalities were detected in the other 500 mg/kg/day animals at terminal kill. The remaining findings reported for a male and female treated with 150 mg/kg/day were a consequence of damage at removal during the necropsy procedure and were unrelated to test material toxicity
Neuropathological findings:
not examined
Description (incidence and severity):
Treatment-related thyroid gland changes were observed in animals of either sex treated with 500 mg/kg/day identified as an increased incidence and severity of follicular cell hypertrophy. No such changes were identified at the other dose levels. All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

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

Applicant's summary and conclusion

Conclusions:
Oral administration of the test material, UK-209,947, to rats for a period of twentyeight consecutive days at dose levels of up to 500 mg/kg/day resulted in treatmentrelated effects at all dose levels. A "No Observed Effect Level" (NOEL) has, therefore, not been achieved. The liver and kidney weight increases detected at 150 and 50 mg/kg/day were not associated with any histopathological changes and, in isolation, were considered not to represent an adverse health effect. The "No Observed Adverse Effect Level" (NOAEL) was considered to be 50 mg/kg/day for this study.
Executive summary:

The administration of UK-209,947 by oral gavage for a period of up to twenty-eight consecutive days resulted in treatment-related effects at dose levels of 500, 150 and 50 mg/kg/day. Adverse clinical signs developed in 500 mg/kg/day animals during the first two weeks of treatment and included respiratory pattern changes, hunched posture and tiptoe gait. The severity of observations in one 500 mg/kg/day female was such that the animal was killed in extremis at the start of Day 10. Pathology failed to identify any major target organs, although splenic atrophy was observed microscopically. Bodyweight development and dietary intake were adversely affected at this dose level during Weeks 1 and 2 but recovered, as did the clinical signs, over the final two weeks of treatment. Bodyvveight and food consumption were also adversely affected in 1 50 mg/kg/day males. Haematological investigations revealed evidence of a microcytic anaemia in males treated with 500 or 150 mg/kg/day and neutrophilia in animals of either sex treated with 500mg/kg/day. Water intake was affected and, as a consequence, quantitative measurement was initiated on Day 15. This revealed an increased water intake at the high dose and for 150 mg/kg/day females. Kidney weight was elevated for all male treatment groups and for 500 mg/kg/day females. Organ weight changes can often indicate early target organ toxicity, but, in the absence of any microscopic or blood chemical changes to support an adverse kidney effect, the increased weights at 150 or 50 mg/kg/day probably do not to represent an adverse health effect. Other organ weight changes were identified at 500 mg/kg/day in the adrenals and heart. These may have simply been a reflection of the reduced bodyweight gain seen in these animals. The adrenal weight increase may possibly have been a stress response associated with expansion of the adrenal cortex, but, in the absence of pathological changes, is probably of no consequence. Liver weights were substantially elevated throughout the treatment groups but, again, there were no concomitant histopathological changes and it is possible that this liver weight increase could be attributed solely to the bodyweight effect, specifically at the high dose, rather than a "real" effect. Plasma cholesterol and/or alanine aminotransferase and aspartate aminotransferase were increased, however, in both males and females treated with 500 mg/kg/day which may suggest a change in hepatocellular integrity at the highest dose level only. Microscopic examination of tissue sections revealed changes confined to the thyroids. An increased incidence of follicular cell hypertrophy was observed in animals of either sex treated with 500 mg/kg/day. Thyroxine is ultimately excreted via the bile, having first been conjugated in the liver. lt is conceivable that conjugating hepatic -39- SPL PROJECT NUMBER: 1284/003 enzymes may have been induced therefore increasing thyroxine excretion and stimulating compensatory TSH and thyroxine production resulting in the microscopic changes identified. Although no hepatic hypertrophy was identified at 500 mg/kg/day, the syndrome of the increased liver weight, serum chemical and thyroid changes is typical of microsonnal induction, which is a normally expected, physiological response of the liver when challenged with a variety of chemically distinct xenobiotic agents. The possibility of an adaptive response, cannot, therefore, be ruled out.