Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
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:
Identity: CP-163,625-BV
Chemical name: (2-Benzhydry1-1-aza-bicyclo[2.2.2]oct-3-y1)-benzylamine
Intended use: Pharmaceutical intermediate
Appearance: White powder
Storage conditions: Room temperature in the dark
Lot No: 38654-187-3
Purity: 99%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Animals chosen for this study were selected from a stock supply of healthy male and female CD rats of
Sprague - Dawley origin (Hsd:Sprague-Dawley(CD)) obtained from Harlan UK Ltd, Bicester, Oxon,
England.
Animals in the main study were in the weight range of 84 to 144 g and approximately five to seven weeks
of age prior to dosing (Day 1). All the rats were acclimatised to the experimental environment for a
minimum period of five days prior to the start of the study.
The rats were allocated without conscious bias to cages within the treatment groups. They were housed in
groups of up to five rats of the same sex in metal cages (RS Biotech Sub-Dividable Rodent Cages -
polished stainless steel (20cm high x 39cm wide x 39cm long)). The cages were fitted with grid floors to
ensure rapid removal of waste material to undertrays. The cages were suspended in mobile stainless
steel racks in Room 6 of Building R14.
A standard laboratory rodent diet (Special Diet Services RM1(E) SQC expanded pellet) and drinking water
were provided ad libitum. Access to food only was prevented overnight prior to and for approximately 4
hours after dosing. The batch of diet used for the study was analysed for certain nutrients, possible
contaminants and micro-organisms. Results of routine physical and chemical examination of drinking
water, as conducted by the supplier, are made available to Huntingdon Life Sciences Ltd. as quarterly
summaries.
Thermostatic controls were set to maintain a temperature of 22 + 3 °C. Relative humidity was not
controlled but was expected to be in the range 30 - 70%. Temperature and humidity were recorded
continuously using a seven day recorder. Actual measurement of these parameters revealed that animal
room temperature was in the range 20.5 to 25°C and relative humidity was in the range 40 - 62%.
Permanent daily recordings of these parameters were made and these are archived with other Departmental
raw data. Air exchange was maintained at a minimum of 10 air changes per hour and lighting was
controlled by means of a time switch to provide 12 hours of artificial light (0700 - 1900 hours) in each 24-
hour period.
Each animal was identified by cage number and ear punching. Each cage was identified by a coloured label
displaying the dose level, study schedule number, animal mark and the initials of the Study Director and
Home Office licensee.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
methylcellulose
Details on oral exposure:
CP-163,625-BV was formulated at various w/v concentrations in 1% w/v aqueous methylcellulose and
administered at a fixed dose volume of 10 ml/kg bodyweight.
The test substance was prepared on the day of dosing.
The concentration, homogeneity and stability of the test substance in the vehicle was not evaluated.
Doses:
50, 200, 500 and 2000 mg/kg bw

Preliminary study
In the absence of precise toxicological information a preliminary study was conducted to help define the
toxic potential of the test substance and aid in selection of a suitable dosage for the main study. Initially,
one female rat was treated at 500 mg,/kg and for further clarification one further female rat was dosed at
2000 mg/kg bodyweight.
The animal treated at 500 mg/kg survived treatment and this dose level was a considered suitable for
commencement of the main study. However, as a result of mortalities in the main study at 500 mg-/kg
further preliminary investigations were undertaken to further define the toxic potential of the test substance.
In these further investigations, one female was treated at 50 mg/kg and a further female treated at
200 mg/kg.
Main study
A group of ten rats (five males and five females) received a single oral gavage dose of the test substance at a
dose level of 500 mg/kg bodyweight. This dose level was chosen after review of findings in the first phase
of preliminary investigations. However, as a result of mortalities at 500 mg/kg a further group of ten rats
was dosed at 50 mg/kg bodyweight. This latter dosage was selected after consideration of preliminary study
results and in compliance with the test guidelines.
No. of animals per sex per dose:
Stage 1 Preliminary: 1 female @ 500 and 2000 mg/kg ea
Stage 1 Main test: 5 males/5 females @ 500mg/kg bw
Stage 2 Preliminary : 1 female @ 50 and 200 mg/kg bw ea
Stage 2 Main test; 5 males /5 females @ 50 mg/kg bw
Control animals:
no
Details on study design:
ADMINIS1RATION OF THE TEST SUBSTANCE
The appropriate dose volume of the test substance was administered to each rat by oral gavage using a
syringe and plastic catheter (8 choke). The day of dosing was designated Day 1.

OBSERVATIONS
Mortality
Cages of rats were checked at least twice daily for mortalities.

Clinical signs
Animals were observed soon after dosing and at frequent intervals for the remainder of Day 1. On
subsequent days, surviving animals were observed once in the morning and again at the end of the
experimental day (with the exception of the last day of observation - morning only). The nature and
severity of the clinical signs and time were recorded at each observation.
Animals in the preliminary and main studies were observed for up to 7 or 14 days respectively after dosing.

Bodyweight
The bodyweight of each rat in the preliminary study were recorded on Days 1 (prior to dosing) and 8 (or at
death) and in the main study on Days 1 (prior to dosing), 2, 3, 4, 8 and 15 (or at death).

TERMINAL STUDIES
Termination
The animal surviving treatment in the preliminary study was killed on Day 8 and all animals in the main
studies were killed on Day 15 by carbon dioxide asphyxiation.

Macroscopic pathology
All animals were subjected to a macroscopic examination which consisted of opening the cranial,
abdominal and thoracic cavities. The macroscopic appearance of all tissues was recorded.
Statistics:
N/A

Results and discussion

Preliminary study:
Treatment groups each comprising one female rat were treated in the following order; 500, 2000, 50 and
200 mg/kg bodyweight. Study fmdings were as follows:
Clinical signs, characterised by piloerection, were seen in all rats. These signs were accompanied in rats at
one or more dose levels by hunched posture, waddling/unsteady gait, lethargy, abnormal respiration,
partially closed eyelids, pallid extremities, walking on toes, increased salivation, abnormal faeces, increased
lacrimation, increased sensitivity to touch, thin appearance, ungroomed appearance, protruding eyes, body
tremors, blue/cold extremities, dull colouring to eyes and swollen abdomen. Recovery was complete in the
rats dosed at 50 mg/kg and 200 mg,/kg on Day 6. The rat dosed at 500 mg/kg still had minor clinical signs
at study termination on Day 8, and the animal dosed at 2000 mg/kg was killed on humane grounds on
Day 5.
Bodyweight gain was considered satisfactory for studies of this nature and duration.
Macroscopic examination of the decedent at 2000 mg/kg revealed congestion (characterised by darkened
tissues/organs and injected blood vessels) in the brain, liver and kidneys with pallor of the brain and
atrophied spleen. Gaseous distension was observed along the alimentary tract. Macroscopic examination of
animals killed at study termination revealed pallor of the brain in the rat dosed at 500 mg/kg bodyweight.
No abnormalities were evident in the other animals killed at termination.
The results from these investigations indicated 500 mg,/kg phase of the study showed the non-lethal dose to
be less than 2000 mg/kg and that a systemic response to treatment was evident at 50 mg/kg bodyweight.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
discriminating dose
Effect level:
ca. 50 mg/kg bw
Based on:
test mat.
Mortality:
One male dosed at 500 mg/kg died during the study. All remaining animals treated at 500 mg/kg and all
animals subsequently dosed at 50 mg/kg were killed as scheduled at study termination (Day 15).
Clinical signs:
Piloerection was observed in all rats within six minutes of dosing. This sign persisted and was accompanied
in rats at later intervals on Day 1 or thereafter during the study by:
hunched posture, waddling/unsteady gait, lethargy, abnormal respiration, pallid extremities, body
tremors, blue/cold extremities and dull colouring to eyes (notable in all rats at 500 mg/kg). Also
observed in one or more animals were partially closed eyelids, walking on toes, increased
lacrimation, increased sensitivity to touch, protruding eyes and prostration.
Recovery in all surviving rats was complete on Day 3
Body weight:
A notably low bodyvveight gain was observed in one female (No. 16, 500 mg,/kg) on Day 15. All remaining
rats were considered to have achieved satisfactory bodyweight gains during the study.
Gross pathology:
Macroscopic examination of the one decedent in the main study revealed congestive changes to the majority
of tissues and organs with fluid contents in the stomach and along the alimentary tract. No abnormalities
were detected in any of the remaining animals killed at study termination (Day 15).

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
In the main phase of this study, the non-lethal (discriminating) oral dose to rats of CP-163,625-BV was
demonstrated to be 50 mg/kg bodyweight.
As a result of mortality among rats dosed at 500 mg/kg bodyweight, in accord with EU hazard classification
CP-163,625-BV will require labelling with the Hazard Statement H302 "Harmful if swallowed", in accordance with
Commission Regulation 1272/2008/EC
Executive summary:

This study was performed to assess the acute oral toxicity of CP-163,625-BV to the rat. The method followed to be compliance with that described in: EEC Methods for the determination of toxicity, Annex to Directive 92/69/EEC (OJ No. L3 83A, 29.12.92), Part B, Method B.1 bis. Acute toxicity (oral) - fixed dose method. OECD Guideline for Testing of Chemicals No.420 'Acute Oral Toxicity - Fixed Dose Method' Adopted 17 July 1992. A group of ten fasted rats (five males and five females) received a single oral gavage dose of the test substance, formulated in 1% w/v aqueous methylcellulose and administered at a dose level of 500 mg/kg bodyweight. This dose level was chosen after review of results from the preliminary study and in compliance with the test guideline. As a result of a mortality at 500 mg,/kg and after further preliminary investigations a further group of ten rats (five males and five females) was dosed at 50 mg/kg bodyweight. One male dosed at 500 mg/kg died during the study. All remaining animals treated at 500 mg/kg and all animals subsequently dosed at 50 mg/kg were killed as scheduled at study termination (Day 15) and subjected to a macroscopic examination. Clinical signs of reaction to treatment characterised by piloerection (first evident in all rats within six minutes of dosing) was seen in all rats, and accompanied at later intervals on Day 1 or thereafter by hunched posture, waddling/unsteady gait, lethargy, abnormal respiration, pallid extremities, body tremors, blue/cold extremities and dull colouring to eyes (notable in all rats at 500 mg/kg). Also observed in one or more animals were partially closed eyelids, walking on toes, increased lacrimation, increased sensitivity to touch, protruding eyes and prostration. Recovery in all surviving rats was complete on Day 3. A notably low bodyvveight gain was observed in one female (No. 16, 500 mg/kg) on Day 15. All remaining rats were considered to have achieved satisfactory bodyweight gains during the study. Macroscopic examination of the one decedent in the main study revealed congestive changes to the majority of tissues and organs with fluid contents in the stomach and along the alimentary tract. No abnormalities were detected in any of the remaining animals killed at study termination (Day 15). In the main phase of this study, the non-lethal (discriminating) oral dose to rats of CP-163,625-BV was demonstrated to be 50 mg/kg bodyweight. As a result of mortality among rats dosed at 500 mg/kg bodyweight, in accord with EU hazard classification CP-163,625-BV will require labelling with the risk phrase R22 Harmful "if swallowed", in accordance with Commission Directive 93/21/EEC.