not costly, and are sustained long enough to make the necessaryobservations. This model is based upon the thought that atrialfibrillation requires heterogeneity of repolarization, that distributionof vagal fibers is heterogeneous in the atria, and that when stimulatedby elevation of systemic arterial pressure, atrial fibrillationwill persist.Dogs were anesthetized with morphine–chloralose because thispermits nearly intact autonomic control. Systemic arterial pressurewas elevated approximately 75 mm Hg with a constant infusion ofphenylephrine, 2 mg/kg/min. The right atriumwas paced for 20min at40 Hz. Atrial fibrillation was sustained in dogs receiving phenylephr-ine but terminated within seconds in normotensives. When dogs inatrial fibrillation were given dofetilide, all converted to sinus rhythmwithin seconds, whereas atrial fibrillation was maintained in dogsdenied dofetilide. In conclusion atrial fibrillation may be maintainedfor at least 40 min following cessation of rapid atrial pacing in dogswith hypertension produced by phenylephrine infusion, and all dogsconverted to sinus rhythms in response to dofetilide.

doi:10.1016/j.vascn.2008.05.019

Determination of LD50 and assessment of drug induceddevelopmental toxicity in zebrafish

Patricia McGrath, Wen Lin Seng, Catherine Willett, Karen A.Augustine (Phylonix Pharmaceuticals, 100 Inman Street, Suite 300,Cambridge, Massachusetts 02139, USA)

Using our ZETAX™assay, we determined LD50 and assesseddevelopmental organ toxicity using drugs provided by Bristol–MyersSquibb Pharmaceutical Research Institute. The goals of this studywereto use zebrafish embryos as a model to assess median lethal dose(LD50) and to assess overall body morphology and liver, intestine, andheart toxicity following drug treatment. Zebrafish were exposedcontinuously to compounds from 24 h post-fertilization (hpf) to120 hpf and mortality was assessed daily. Zebrafish were initiallytreated with 5 concentrations one log apart: 0.01, 0.1, 1.0, 10 and 100 µ.Body, heart, liver and intestine morphology were assessed micro-scopically. A teratogenic index was generated and compounds wereranked by relative toxicity. Data was evaluated for strength inpredictivity and accuracy based on vivoteratogenicity. The assayexhibited good (N70% b80%) specificity and excellent (N80%) sensi-tivity; there was a 75% success rate in identifying non teratogeniccompounds and a 100% success rate in identifying teratogens. Theaccuracy for predicting relative teratogenic potency of the compoundswas excellent with a 92% success rate. In summary, the results of theLD50/Visual Assessment Pilot study demonstrate that the zebrafishembryo assay format (ZETAX™) is a valuable model for conductingpredictive teratogenicity studies.

doi:10.1016/j.vascn.2008.05.020

Zebrafish: An automated high content screening platform forsafety pharmacology

Teresa P. Barros, H. Reynolds, S. Clarke, A. Roach, W. Alderton, S.Berghmans (VASTox plc, 7330 Cambridge Research Park, Cambridge,UK)

The zebrafish is an established model system for the study ofhuman diseases due to numerous advantages including anatomical,physiological and genetic similarity to man. In addition, large stocks ofanimals are easy to maintain and yield large numbers of embryos.These develop rapidly, ex utero, into small larvae, which fit into 96-well plates, making zebrafish amenable to high throughput screening.

Furthermore, the larvae tolerate DMSO up to 2% and require only smallcompound amounts which are easily absorbed allowing compoundsafety assessment to be undertaken in vivo at a much earlier stage ofthe drug discovery process than has been previously possible. Vastoxhas pioneered the use of zebrafish in safety pharmacology byestablishing assays to assess adverse drug effects on vision, hearing,cardiac function, gut motility and locomotor activity. We have nowdeveloped a high throughput, high content screening platformallowing automated in vivo functional readouts of the cardiac function,locomotor activity and gut motility. High quality digital videoobservations of both larval behaviour and internal organ systemscan be captured and stored in a high throughputmanner. The platformutilizes advanced custom-made software for data analysis whichallows analysis of large bodies of data. Our zebrafish liability assayshave now been automated greatly increasing the throughput to 650compounds/month/FTE at a single concentration, for the locomotoractivity and cardiac function assays, and 250 compounds/month/FTEat a single concentration for the gut motility assay.

doi:10.1016/j.vascn.2008.05.021

Evaluation of cardiovascular and ECG parameters in the normal,freely moving Göttingen Minipig

Miriam Stubhan, Michael Markert, Karin Mayer, Thomas Trautmann,Anja Klumpp, Brian Guth (Boehringer Ingelheim Pharma GmbH & Co.KG, Biberach an der Riss, Germany)

Introduction: Minipigs are being used increasingly in safetypharmacology and toxicology. However, there are few reference dataavailable from conscious, freely moving minipigs, particulary for leftventricular pressure. The aim of this study was to provide normalvalues for cardiovascular and ECG parameters in the GöttingenMinipig. Methods: 7 trained Göttingen Minipigs were instrumentedwith a telemetric device (ITS). Aortic blood pressure, left ventricularpressure, heart rate, LV dP/dtmax, body temperature and ECG (lead II)were monitored continuously over 24 h. The experimental designwasin accordance with our safety pharmacology studies. For dataacquisition we used NOTOCORD Hem 4.2. Data were summarizedevery 10 min as median values±SD (≥400 beats per median value).Results: Heart rate, systolic (mean value 123 mm Hg) and diastolic(mean value 88 mm Hg) arterial blood pressure as well as leftventricular pressure (mean value 111 mm Hg) were stable over the24 h monitoring period. Heart rate averaged 57 bpm during daytimeand increased (mean peak value 122 bpm) only at feeding after 7 h.Measurement of ECG intervals resulted in the following mean values:PR interval 128 ms, QRS interval 56 ms, RR interval 995 ms.Uncorrected QT was 335±26.4 ms. Discussion: We found stablehemodynamic parameters with a low intrinsic heart rate in theGöttingen minipig. Feeding, however, was associated with a markedincrease in heart rate. This effect was sustained and should be takeninto account when designing studies.

doi:10.1016/j.vascn.2008.05.022

Ferrets: A new cardiovascular telemetry model for earlycompound development screening

Stephanie Adams, Gerald Bricker, Brad Main (Eli Lilly and Co., 2001Main St, Greenfield, IN 46140, USA)

Our goal was validation of a conscious ferret model as replacementfor dog or monkey cardiovascular (CV) telemetry studies. Ferrets offermany benefits, as the ferret's cardiac ion channels are similar to those

150 Journal of Pharmacological and Toxicological Methods 58 (2008) 147–178

in humans and those used for in vitro cardiac electrophysiologystudies. The heart of the ferret is large making it suitable forinstrumentation while their small body size reduces compoundrequirements.20 ferrets were instrumented with a pressure cannulain the descending aorta and electrocardiogram leads placed in anapproximate lead II. Ferrets were exposed to 30 or 100 ug/kg singledoses of dofetilide. Additionally, CV responses to milrinone andtorcetrapib were analyzed. Blood levels were measured to compareexposure in ferrets and dogs. Results: Surgical instrumentationremains functional N18 months with high quality ECG's. Ferrets haveless signal noise compared to monkeys. Hemodynamic responses tomilrinone or torcetrapib, were similar to dogs. Ferrets treated with30 ug/kg dofetilide caused a 23 ms increase in QTcf while 100 ug/kgcaused a 68ms prolongation of QTcf. These changes with dofetilide aresimilar to the changes observed in dogs. Conclusion: Our data confirmthe ferret as a reliable CV model. The ferrets have proven to be assensitive as dogs and monkeys to predict changes in CV parameterssuch as heart rate, BP, and QT. Our evaluation of this model supportsusing ferrets instead of dogs or monkeys in early screening paradigmsand provides a reliable predictor of CV pharmacology expected in latestage development.

doi:10.1016/j.vascn.2008.05.023

Neurobehavioural assessment in pre-clinical species: Rodents,dogs, mini-pigs and monkeys

Alessandra Giarola, Elena Moscardo, Nicola Fasdelli, Marcello Tonto-donati, Roberto Dorigatti (GlaxoSmithKline SpA, Via Fleming 4,Verona, Italy)

Identifiable in pre-clinical species, there is therefore a continuousneed to improve the prediction performance in this area refining themethodologies used. A behavioural test was first introduced by Irwinas a psychotropic screening procedure in mice. Following modifica-tions Functional Observational Batteries (FOBs) were developed forrats and more recently adapted in the most common non-rodentspecies. When assessing neurobehaviour it is important to carefullycontrol and standardise all factors involved in the test (the animal, theobserver, the observation battery and environmental conditions) tomake the data collected objective and reproducible, and thereforepredictive of safety liabilities. In our laboratories, detailed neurobe-havioural observation batteries have been developed for use in mostpre-clinical species: rodents, dogs, mini-pigs and non-human pri-mates. The rodent FOB assesses home cage and open field activities,stimulus reactivity and neuromuscular functions. The dog and mini-pig FOBs have been developed based on that used for rodent but applydifferent levels of handling. The monkey FOB is considered the mostsuitable assessment when effects on higher-level cognitive andbehavioural processes are to be assessed. In conclusion, it is possibleto perform a reliable neurobehavioual assessment in all themajor pre-clinical species, using a standard observation battery, but theprocedure has to be adapted and optimised to the species.

doi:10.1016/j.vascn.2008.05.024

Evaluation of ethanol interaction potential using preclinicalmodels of rodent behaviour and motor coordination

J. Scatchard, N. Addley, T. Knight, R. Newman (Global SafetyPharmacology, Pfizer, Sandwich, Kent, U.K.)

Ethanol has the potential to interact with drugs to alter their effectsby altering the metabolism of the agent, and/or in the case of centrally

acting agents, by direct interaction in the brain. Metabolic interactioncan usually be identified from knowledge of the drug's structure andquantified using human or rat liver microsome preparations. There-fore, we focussed on a CNS model of ethanol interaction which mayoccur through a variety of known central mechanisms (e.g. benzo-diazepines at GABAA site) tending generally to cause impaired motorcoordination and sedation. A number of possible models are available,such as sleeping times, specific tests of motor coordination (rotarod,beam walking) and measurement of locomotor activity. Moresophisticated tests (memory and learning, spatial performance,tolerance) were beyond the scope of the study. Due to unfavourablein house experience with sleeping times and rotarod studies in rats,the two studies evaluated were beam-walking (reliable and repro-ducible but resource intensive) and locomotor measurement (detectswide range of effects, high throughput). The agents chosen forvalidation were diazepam, haloperidol and amitryptiline, all of whichare well documented to interact clinically with ethanol. All agentsclearly had a degree of interaction with ethanol in both study types,although data from the locomotor study was more variable andshowed less dose relationship than that from the beamwalking study.Therefore, beamwalking using rats is a useful model for assessment ofCNS related drug/ethanol interaction.

doi:10.1016/j.vascn.2008.05.025

Original FOB method adapted to safety pharmacology

Sophie Hoh-Begon, Sabine Hennequin, Michéle Beaughard, GerryKaijser (Department of Toxicology and Drug Disposition, PreclinicalDevelopment Center, Organon, Riom, France)

Introduction: The original Functional Observational Battery (FOB)has been adapted for ICH S7A guidelines. In addition to classicalneurobehavioral observations and interactive measures, this FOB hasbeen optimized by inclusion of motor activity, rotarod and painassessments, usually performed as follow-up tests. Another character-istic of this FOBconsists in expressingquantitative resultsper itemorperfunctional domain using severity scores. Methods: In a grid ofobservations, quantitative or qualitative data (descriptive, binary,ranked scores) are selected for each item. All raw data are automaticallyrecoded in a unique severity score that normalizes individual data of allmeasures to 1-to-4 scale (Mifob®). Then, severity scores are summedper functional domains: Behavior (B), Excitability (E), Neuromuscular(N), Convulsive (C), AutonomicNervous System (ANS), Sensorimotor (S),and Activity (A). Results: Pharmacological validation showed typicalchanges in domains, temperature (T), coordination (Co) or pain (P)related to each reference compound. Caffeine induced changes in B, A,Co, T; Chlorpromazine changed B, E, N, C, S, A, P, T; ImipraminemodifiedE, N, ANS, S, T; Diazepam induced B, E, S domains troubles; Clonidinechanged B, E, N, ANS, S, A, Co, T, P. Discussion: Use of unique quantitativeseverity score is an interestingmethod that gives a global overview. Eachcompound can be identified by a distinct neuropharmacological profileof functional domains. In addition, insertion of follow-up tests in FOBprovides ethical and scientific improvements.

doi:10.1016/j.vascn.2008.05.026

Graphical presentation of Irwin test data as an aid to interpretation

Paul Moser, Roger Porsolt, Vincent Castagné (Porsolt & PartnersPharmacology, 9 bis Rue Henri Martin, Boulogne–Billancourt, France)

Variants of the Irwin test are among the most widely usedprocedures within the context of safety pharmacology. If carried out

151Journal of Pharmacological and Toxicological Methods 58 (2008) 147–178