7,8,9,10-Tetrahydro-8-(trifluoroacetyl)-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

28 Days

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January - 07 June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2,2-trifluoro-1-{5,8,14-triazatetracyclo[10.3.1.0²,¹¹.0⁴,⁹]hexadeca-2,4,6,8,10-pentaen-14-yl}ethan-1-one

EC Number:

810-682-9

Cas Number:

230615-70-0

Molecular formula:

C15H12F3N3O

IUPAC Name:

2,2,2-trifluoro-1-{5,8,14-triazatetracyclo[10.3.1.0²,¹¹.0⁴,⁹]hexadeca-2,4,6,8,10-pentaen-14-yl}ethan-1-one

Test material form:

solid: particulate/powder

Details on test material:

Buff coloured solid or pale brown powder depending on which batch
Storage conditions: room temperature. in the dark

Specific details on test material used for the study:

Pale brown powder
Storage conditions: room temperature in the dark
Batch number: 42698-116-10

Test animals

Species:

rat

Strain:

other: Crl:CD ® (SD) BR

Details on species / strain selection:

The 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 or test system and environmental conditions:

A total of 40 (20 males and 20 females) Sprague-Dawley Crl:CD ® (SD) BR strain rats were obtained from Charles River (UK) Limited. Margate, Kent and were accepted into the study. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for seven days during which time their health status was assessed. At the start of treatment the males weighed 139 to 176g, the females weighed 121 to 162g, and were approximately five to seven 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. Mains drinking water was supplied.
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 controls were set to achieve target values of 21 ± 2°C and 55 ± 15% respectively.
The animals were randomly allocated to treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Animals were allocated to treatment groups as follows:
Treatment Group Dose Level (mg/kg/day) Treatment Volume (ml/kg) Concentration (mg/ml) Animal Numbers
Male Female
Control 0 4 0 5(1 -5) 5 (6- 10)
Low 15 4 3.75 5(11 -15) 5 (16 - 20)
Intermediate 45 4 11.3 5 (21 - 25) 5 (26 - 30)
High 150 4 37.5 5 (31 - 35) 5 (36 -40)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered daily, for 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 Arachis oil BP.

Vehicle:

arachis oil

Details on oral exposure:

Once daily, by gavage, using a stainless steel dosing cannula attached to a graduated syringe for twenty-eight consecutive days. Dosing will be performed at a similar time each day wherever possible.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material formulations were sampled and analysed within three days of preparation. A range of standard solutions were prepared covering the concentration range 0 to 0.15 mg/ml and analysed. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, by gavage for 28 consecutive days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control: 5 males and 5 females

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

5 males and 5 females

Doses / concentrations 3

Dose / conc.:

45 mg/kg bw/day (actual dose received)

Remarks:

5 males and 5 females

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

5 males and 5 females

No. of animals per sex per dose:

5 animals per sex per dose (10 total per dose)

Control animals:

yes, concurrent vehicle

Details on study design:

For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Results show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4°C in the dark.
Samples were taken of each test material formulation and were analysed for concentration of CP-548,507 at Safepharm Analytical Laboratory. The results indicate that the prepared formulations were within ± 8% of the nominal concentration.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Examinations

Observations and examinations performed and frequency:

Clinical Observations:
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. All observations were recorded.

Functional Observations:
Prior to the start of treatment and on Days 2, 9. 16 and 26. all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.
Behavioural Assessments:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait Hyper/Hypothermia
Tremors Skin colour
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behaviour Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation

Functional Performance Tests:
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each
day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The scoring system used is outlined in Appendix I. The following parameters were observed:
Grasp response Touch escape
Vocalisation Pupil reflex
Toe pinch Startle reflex
Tail pinch Blink reflex
Finger approach

Bodyweight:
Individual bodyweights were recorded on Day 0 (the day before the start of treatment) and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

Food Consumption:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption:
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations:
Haematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy
on Day 29. Animals were not fasted prior to sampling.

Haematology:
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices
- mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Cresyl blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by Hepato Quick' and Activated partial thromboplastin time (APTT) was assessed by 'Preci Clot' using samples collected into sodium citrate solution (0.11 mol/I).

Blood Chemistry:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na-i-)
Potassium (K+)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Pathology
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)

Pathology:
On completion of the dosing period all animals were killed by intravenous overdose of sodium pentobarbitone (Rhone Mérieux, Dagenham, Essex, UK) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Organ Weights:
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Liver
Brain
Ovaries
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus

Histopathology:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin:
Adrenals
Oesophagus
Aorta (thoracic)
Ovaries
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary
Brain (including cerebrum, cerebellum and pons)
Prostate
Caecum
Rectum
Colon
Salivary glands (submaxillary)
Duodenum
Sciatic nerve
Epididymides
Seminal vesicles
Eyes
Skin (hind limb)
Gross lesions
Spinal cord (cervical)
Heart
Spleen
Ileum
Stomach
Jejunum
Testes
Kidneys
Thymus
Liver
Thyroid/parathyroid
Lungs (with bronchi) #
Trachea
Lymph nodes (cervical and mesenteric)
Urinary bladder
Muscle (skeletal)
Uterus
All tissues were despatched to Precision Histology International , One Eyed Lane, Weybread, Diss, Norfolk, UK for processing (Principal Investigator: J P Finn). The tissues shown in bold from all control and 150 mg/kg/day dose group animals were prepared as paraffin blocks, sectioned at nominal thickness of 5 um and stained with haematoxylin and eosin for subsequent microscopic examination. The liver and spleen from all 15 and 45 mg/kg/day dose group animals were also processed. Since there were indications of treatment-related changes, in the liver, colon, bone marrow, mesenteric lymph nodes and thymus examination was subsequently extended to include similarly prepared sections of these tissues from all animals in the 15 and 45 mg/kg/day dose groups. Microscopic examination was conducted by the Study Pathologist. All findings were entered into the ROELEE Pathology computerisation system for tabulation and report production.

Sacrifice and pathology:

On completion of the dosing period, all surviving animals were killed by cervical dislocation and immediately subjected to an internal and external macroscopic examination. Animals that died during the range-finder were also necropsied. No tissues were retained.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs developed in females treated with 150 mg/kg/day from Day 25. By Day 28 these included hunched posture and red/brown staining of the external body surface together with incidents of tiptoe gait, diuresis, diarrhoea, and ptosis. Increased salivation was detected up to ten minutes after dosing from Day 6 onwards but this was considered not to be indicative of systemic toxicity. There were no clinically observable signs of toxicity detected at 45 or 15 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

There were no 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 females treated with 150 mg/kg/day during Week 1. Males were also affected but the severity was far less than that of the females. Bodyweight gain recovered thereafter but a group mean reduction was evident for 150 mg/kg/day females during Week 4. Animals of either sex dosed at 45 or 15 mg/kg/day were unaffected.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Reduced dietary intake was observed for animals of either sex treated with 150 mg/kg/day during the first week of the study, the effect more pronounced in females than in males. Food consumption recovered in males but was reduced during Week 4 for the females. Food efficiency was also reduced but this was confined to 150 mg/kg/day females during Week 1 only. No such effects were detected at 45 or 15 mg/kg/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No intergroup differences were detected.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Total leucocyte count, specifically in the lymphocyte fraction, was elevated for 150 mg/kg/day females compared with controls. Males from this dose group and animals of either sex treated at 45 or 15 mg/kg/day were unaffected.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood Chemistry:
Females treated with 150 mg/kg/day showed statistically significant reduction in total plasma protein, albumin and glucose. A statistically significant increase in plasma aspartate aminotransferase was also detected in this sex. No such effects were detected for males treated with 150 mg/kg/day or for animals of either sex treated with 45 or 15 mg/kg/day.

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):

A statistically significant reduction in absolute and relative thymus weight was detected for 150 mg/kg/day females compared with controls. An increase in relative liver weight was also evident in both males and females treated with 150 mg/kg/day, the effect extending to 45 mg/kg/day males. No such effects were detected for 45 mg,/kg/day females or for animals of either sex treated with 15 mg/kg/day.

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related changes were observed:
LIVER: Centrilobular hepatocyte enlargement was observed in males treated with 150 and 45 mg/kg/day. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and, in the absence of associated inflammatory or degenerative changes, is generally considered to be adaptive in nature.
COLON: Vacuolation of lamina propria cells was observed for two females treated with 150 mg/kg/day.
MESENTERIC LYMPH NODES: Higher grades of severity of histiocyte vacuolation were seen for females treated with 150 mg/kg/day.
THYMUS: Lymphoid atrophy was observed for females treated with 150 mg/kg/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Oral administration of the test material, CP-548,507, to rats for a period of twentyeight consecutive days at dose levels of up to 150 mg/kg/day resulted in treatment-related effects for animals of either sex dosed at 150 mg/kg/day and for males dosed at 45mg/kg/day. No such effects were detected for females treated with 45 mg/kg/day or for animals of either sex dosed at 15 mg/kg/day and the "No Observed Effect Level" (NOEL) was, therefore, considered to be 45 mg/kg/day for females and 15 mg/kg/day for males.
The changes seen in males treated with 150 or 45 mg/kg/day involved liver histopathology and associated organ weight changes. These, together with the slight reduction in bodyweight gain seen during Week 1 were considered to be adaptive responses and did not represent "serious damage" to health, as defined by the criteriagiven in the EC labelling guide of Commission Directive 2001/59/EC. For this reason, 150 mg/kg/day may be regarded as a "No Observed Adverse Effect Level" (NOAEL) for the males. The NOAEL in females is regarded to be the same as the NOEL for that sex, 45 mg/kg/day.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

3

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

4

Dose descriptor:

NOEL

Effect level:

45 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

1

Critical effects observed:

yes

Lowest effective dose / conc.:

45 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

2

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

colon

Treatment related:

yes

3

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

4

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

thymus

Treatment related:

yes

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material, CP-548,507, to rats for a period of twenty-eight consecutive days at dose levels of up to 150 mg/kg/day resulted in treatment-related effects for animals of either sex dosed at 150 mg/kg/day and for males dosed at 45mg/kg/day. No such effects were detected for females treated with 45 mg/kg/day or for animals of either sex dosed at 15 mg/kg/day and the "No Observed Effect Level" (NOEL) was, therefore, considered to be 45 mg/kg/day for females and 15 mg/kg/day for males. The changes seen in males treated with 150 or 45 mg/kg/day involved liver histopathology and associated organ weight changes. These, together with the slight reduction in bodyweight gain seen during Week 1 were considered to be adaptive responses and did not represent "serious damage" to health, as defined by the criteria given in the EC labelling guide of Commission
Directive 2001/59/EC.

Executive summary:

150 mg/kg/day may be regarded as a "No Observed Adverse Effect Level" (NOAEL) for the males. The NOAEL in females is regarded to be the same as the NOEL for that sex, 45 mg/kg/day.