Reaction mass of (2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide and (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 October 2006 - 6 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EU and/or OECD guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

EC Number:

700-041-0

Molecular formula:

C11 H20 N2 O2

IUPAC Name:

(2S)-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; (2S)-2-[(4S)-2-oxo-4-propylpyrrolidin-1-yl]butanamide

Test material form:

solid: particulate/powder

Remarks:

white powder

Details on test material:

- Name of test material (as cited in study report): ucb108628-1
- Stability under test conditions: not indicated
- Storage condition of test material: room temperature, in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Strain: Crl:CD (SD) IGS BR
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: the males weighed 141 to 163g, the females weighed 124 to 152g
- Fasting period before study: not applicable
- Housing: in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12:12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test material formulations were determined and show the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately +4ºC in the dark.

VEHICLE
- Concentration in vehicle: 3, 30 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of ucb108628-1 in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test material formulations were diluted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
The standard and sample solutions were analysed by HPLC using the following conditions:
HPLC : Agilent Technologies 1050 or Varian prostar 325, incorporating autosampler and workstation
Column : Luna C8 5μ (150 x 4.6 mm id) or luna C185μ (150 x 4.6 mm id)
Mobile phase : Acetonitrile:0.1% orthophosphoric acid in water (45:55 v/v)
Flow-rate : 1 ml/min
UV detector wavelength : 205 nm
Injection volume : 10 μl
Retention time : ~ 2.5 or 4.3 mins
Column temperature : 40°C

Homogeneity: The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
Stability: The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.
Concentration verification: The test material formulations were sampled and analysed within three days of preparation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily treatment

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/kg/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of the 14 day range-finder (0 and 1000 mg/kg bw/day)

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, immediately post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and at weekly intervals thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was recorded for each cage group at weekly intervals throughout the study and expressed as g food/animal/day.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes. In addition, water intake was measured during the third week of treatment and recorded daily for each cage group.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all surviving animals
- Parameters examined: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices (i.e. mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC)), Total leucocyte count (WBC), Differential leucocyte count (i.e. neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic),
Prothrombin time (CT) and Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 28
- Animals fasted: No
- How many animals: all surviving animals
- Parameters examined: Urea, Calcium (Ca++), Glucose Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat),
Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and on Days 6, 13, 20 and 27, all animals were observed for signs of functional/behavioural toxicity.
- Dose groups that were examined:
- Battery of functions tested: Functional performance tests (motor activity and forelimb/hindlimb grip strength) were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: On completion of the dosing period all surviving animals 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.
See table below.

ORGAN WEIGHTS: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus

HISTOPATHOLOGY: Yes (see table below)

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. All data was summarised in tabular form. Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were four deaths at the high dose (Group 4). One male (Number 34) and one female (Number 40) were killed in extremis on Day 6 and one female (Number 39) was found dead on Day 9. For animal welfare reasons and to preserve the integrity of the study a decision was taken to reduce the high dose from 1000 mg/kg/day to 500 mg/kg/day with treatment at the lower dose commencing from Day 11. Following this reduction one male (Number 31) was killed in extremis on Day 13. There were no further unscheduled deaths.
Findings were confined to high dose animals and developed between the first and second week of treatment with transient episodes of increased salivation, hunched posture, tiptoe gait, respiratory distress and abdominal distension observed. These findings were accompanied among males by instances of dehydration and lethargy; and in females by diarrhoea and soiled fur. From Day 14 findings only excessive salivation seen for a short period following dosing were observed.

CLINICAL CHEMISTRY
High dose males showed a statistically significant increase (p<0.01) in alanine aminotransferase when compared to controls.

NEUROBEHAVIOUR
Detailed behavioural assessments at Day 6 revealed isolated instances of respiratory abnormalities and ataxia in high dose males and, on Day 13 one of these males displayed signs of tiptoe gait. High dose females showed signs of laboured respiration at Week 1 and Week 13 accompanied in one female on Day 6 and 13 by tiptoe gait.

ORGAN WEIGHTS
High dose males showed a statistically significant increase in absolute liver weight and liver weight relative to terminal bodyweight.

GROSS PATHOLOGY
- Interim deaths: Macroscopic examination of these animals revealed gaseous distension of the stomach and intestines in both males. White nodules on the liver, small spleen and small epididymides were also identified in one male. The female terminated on Day 6 had gaseous distension of the intestines with examination of the female found dead Day 9 revealing darkened kidneys and liver together with reddened lungs and salivary glands. Histopathological examination of male Number 31 revealed gastric and liver changes that may have contributed to the poor condition of this animal. For female Number 40 focal epithelial ulceration of the trachea and associated mixed inflammatory cell infiltrates would have led to moribundity. No other significant pathology was identified.

HISTOPATHOLOGY: NON-NEOPLASTIC
Centrilobular hepatocyte enlargement was observed in relation to treatment for rats of either sex dosed at 1000/500 mg/kg/day, and also at 150 mg/kg/day, but not for animals of either sex treated with 15 mg/kg/day. Zonal hepatocyte enlargement is occasionally seen among control animals as a spontaneous change. 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.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: See above.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The repeated oral administration of ucb108628-1 by gavage for a period of up to twenty-eight consecutive days at dose levels of up to 1000/500 mg/kg/day resulted in deaths and treatment-related changes at the high dose with a minimal treatment related finding at 150 mg/kg/day.
During the first week of treatment there was an unexpected deterioration in condition of animals receiving 1000 mg/kg/day culminating in early termination of one male and one female on Day 6. Following the death of a third animal on Day 9 dose level was reduced to 500 mg/kg/day. Perhaps, because of a delayed response to treatment a further animal died on Day 13. Thereafter, there were no further deaths and the general condition of high dose animals improved. The aetiology of the premature deaths is unclear, macroscopic examination revealed no obvious causes and although hispathological examination revealed contributing factors in two animals there were no definite indications of cause of death. On this basis and in view of the recovery of high dose animals following the reduction in dose level it can only be assumed the deaths were a direct result of treatment with test material at 1000 mg/kg/day.
The remaining noteworthy findings were limited to high dose animals following the reduction of dose level. These changes involved transient and isolated instances of increased salivation following dosing, similar isolated instances of abnormal respiration and ataxia detected during behavioural assessment in the final week of treatment, minor disruption of food consumption and blood chemistry all of which while being associated with treatment were considered not to be of toxicological significance.
High dose animals of each sex showed an increase in liver and kidney weight and while the significance of the kidney changes is dubious the increase in liver weight was likely associated with the blood chemical finding of increased alanine aminotransferase for high dose males, and microscopic findings, which included centrilobular hepatocyte enlargement observed in rats of either sex treated with 1000/500 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 adaptive in nature. Hepatocyte enlargement was also evident in each sex at 150 mg/kg/day, but not for animals of either sex treated with 15 mg/kg/day. The minor change observed at 150 mg/kg/day was not indicative of serious damage to health as defined in the EU labelling guide of Commission Directive 93/21/EEC. This dose level can therefore be regarded as a "No Observed Adverse Effect Level" (NOAEL).