the use of transgenic animals in the european...

Preface

This is the report of the twenty-eighth of aseries of workshops organised by the Euro-pean Centre for the Validation of Alterna-tive Methods (ECVAM). ECVAM�s maingoal, as defined in 1993 by its ScientificAdvisory Committee, is to promote the sci-entific and regulatory acceptance of alter-native methods which are of importance tothe biosciences and which reduce, refine orreplace the use of laboratory animals. Oneof the first priorities set by ECVAM was theimplementation of procedures which wouldenable it to become well-informed about the

state-of-the-art of non-animal test develop-ment and validation, and the potential forthe possible incorporation of alternativetests into regulatory procedures. It wasdecided that this would be best achieved bythe organisation of ECVAM workshops onspecific topics, at which small groups ofinvited experts would review the currentstatus of various types of in vitro tests andtheir potential uses, and make recommen-dations about the best ways forward (1). Inaddition, other topics relevant to the ThreeRs concept of alternatives to animal experi-ments have been considered in severalECVAM workshops.

The Use of Transgenic Animals in theEuropean UnionThe Report and Recommendations of ECVAM Workshop 281,2

T. Ben Mepham,3 Robert D. Combes,4 Michael Balls,5 Ottavia Barbieri,6Harry J. Blokhuis,7 Patrizia Costa,8 Robert E. Crilly,3 Tjard de CockBuning,9 Véronique C. Delpire,5 Michael J. O�Hare,10 Louis-MarieHoudebine,11 Coen F. van Kreijl,12 Miriam van der Meer,13 Christoph A.Reinhardt,14 Eckhard Wolf15 and Anne-Marie van Zeller4

3Centre for Applied Bioethics, University of Nottingham, Sutton Bonington Campus,Loughborough, LE12 5RD, UK; 4FRAME, Russell & Burch House, 96�98 North SherwoodStreet, Nottingham, NG1 4EE, UK; 5ECVAM, JRC Environment Institute, 21020 Ispra (VA),Italy; 6Dipartimento di Oncologia Clinica e Sperimentale, Università di Genova, IST/CBA,Largo R. Benzi 10, 16132 Genoa, Italy; 7Institute for Animal Science and Health (ID-DL),Department of Behaviour, Stress Physiology and Management, Edelhertweg 15, 8200 ABLelystad, The Netherlands; 8Instituto di Biologia Molecolare, Via Pontina KM 30.600, 00040Pomezia, Rome, Italy; 9Department for the Study of Animal Experiments, University ofLeiden, 2301 CB Leiden, The Netherlands; 10Breast Cancer Laboratory, LICR/UCL, 67�73Riding House Street, London W1P 7LD, UK; 11Laboratoire de Biologie Cellulaire etMoleculaire, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352Jouy-en-Josas, France; 12RIVM, 3720 BA Bilthoven, The Netherlands; 13Department ofLaboratory Animal Science, Utrecht University, 3508 TD Utrecht, The Netherlands; 14SwissAlternatives to Animal Testing (SAAT), 8614 Bertschikon-Zurich, Switzerland; 15Lehrstuhlfür Molekulare Tierzucht, Feodor-Lynen-Strasse 25, 81377 Munich, Germany

ATLA 26, 21�43, 1998 21

Address for correspondence: Dr T.B. Mepham, Centre for Applied Bioethics, University of Nottingham, SuttonBonington Campus, Loughborough, Leics., LE12 5RD, UK.

Address for reprints: ECVAM, TP 580, JRC Environment Institute, 21020 Ispra (VA), Italy. 1ECVAM � The European Centre for the Validation of Alternative Methods. 2This document represents theagreed report of the participants as individual scientists.

The workshop on The Use of TransgenicAnimals in the European Union was held inSouthwell, Nottinghamshire, UK, on 7�11April 1997, under the co-chairmanship ofBen Mepham (University of Nottingham,UK) and Miriam van der Meer (University ofUtrecht, The Netherlands). It was held incollaboration with the Fund for the Replace-ment of Animals in Medical Experiments(FRAME; Nottingham, UK) and the Centrefor Applied Bioethics (CAB) at the Univer-sity of Nottingham, and was organised byBen Mepham (CAB), Rob Crilly (CAB), BobCombes (FRAME) and Anne-Marie vanZeller (FRAME). There were 16 participantsfrom six Member States of the EuropeanUnion (EU).

The principal aim of the workshop was toformulate a set of guidelines to assist regula-tory authorities in the EU in decidingwhether to permit and/or how to regulateresearch involving transgenic animals. Thisreport summarises the workshop discussionson the current status of transgenic animal

research, and proposes a number of recom-mendations for the appropriate and har-monised control of the use of transgenicanimals within the EU.

Introduction

The technique of transgenesis involves theintroduction of functional genetic material(DNA) into the germline of organisms. Thefirst �transgenic� animals, produced in1980, were mice (2, 3). A transgenic animalis an animal which has been geneticallymodified by the stable incorporation, byusing artificial gene transfer, of exogenousDNA into its genome, in order to introduceor delete specific characteristics of the phe-notype. A transgene construct can comprisea complete gene sequence derived from adonor organism, an in vitro synthesisedsequence, or a combination of both. One ofthe most recent definitions of transgenicanimals is that suggested by Beardmore

22 T.B. Mepham et al.

Table I: Methods for producing transgenic animals by introduction of foreignDNA into the mammalian genome

Technique involved Remarks

Pronuclear micro- technical simplicity; low success rate; applicable to a wide injection (introduction range of species; most widely used; unpredictable effects due to of expressed gene) random transgene integration

Embryonic stem (ES) cell substitution of a functional gene with an inactive gene; manipulation (introduction germ-line competent ES cells have been isolated in mice; of expressed gene, or gene ES-like cells identified in other species, including primates, inactivation by homologous but totipotency remains to be establishedrecombination)

Cre-lox technique preferred method with more control over resulting phenotype; time-consuming

Viral vectors complex; largely restricted to avian species

Cytoplasmic injection less efficient than direct pronuclear microinjection

Primordial germ cells chimaeric animals result

Nuclear transfer large potential for genetically modifying livestock

Spermatogonial transplantation into recipient testesmanipulation

(4): �organisms containing integratedsequences of cloned DNA (transgenes),transferred using techniques of geneticengineering (to include those of gene trans-fer and gene substitution)�. In this report,discussion is confined to consideration oftransgenesis in vertebrate laboratory andfarm animals.

Production of transgenic animals able totransmit genetic modifications to their off-spring requires germline transformation.There are several techniques for the produc-tion of transgenic animals (Tables I and II);the three most commonly used methods,pronuclear microinjection, embryonic stem(ES) cell manipulation, and the cre-lox tech-nique, are shown schematically in Figures 1,2 and 3, respectively.

There are several potential and actualapplications of transgenic animals (3): a) inbasic research; b) as a source of organs forxenotransplantation; c) as disease models; d)in the production of therapeutic proteins(that is, as bioreactors); e) in agriculture (forexample, the manipulation of livestock pro-duction traits); f) for vaccine testing; and g)in toxicity testing. Transgenic animals pre-sent unique opportunities for medical, agri-cultural and fundamental research, and as ameans of producing valuable pharmaceuticaland nutrient products. Their use hasincreased dramatically in recent years, and isset to rise at an even more rapid rate. Forexample, in the UK, there was a 525%increase in the use of transgenic animalsbetween 1990 and 1996 (5). This increaseshould be considered against a background

of a modest decrease in the overall use of lab-oratory animals during the same period oftime. While transgenic animals might allowreduction and refinement in animal use viamore-precise gene targeting in breeding pro-grammes, these objectives are threatened bytransgenic procedures which could promotegreater animal use, a greater variety of appli-cations, and an increased likelihood of ani-mal suffering (6�10).

There are also intrinsic ethical concernsrelating to the transfer of genes betweenspecies, especially when human genes areinvolved (with the potential for their pro-gressive transfer into laboratory animals[11]), and when animal organs are used inhuman medicine. In this regard, it is of inter-est that members of the general publicwithin Europe are becoming increasinglysceptical about the use of animal biotechnol-ogy (12). This situation has prompted someto call for further ethical debate (13). On 21May 1996, the EU Group of Advisers on theEthical Implications of Biotechnology(GAEIB) published an opinion on the geneticmodification of animals (14). One of the prin-cipal conclusions was that, while geneticmodification of animals might contribute tohuman well-being and welfare, it �is accept-able only when the aims are ethically justi-fied and when it is carried out under ethicalconditions�. There was a consensus at theworkshop in support of all the opinionsexpressed in the GAEIB report.

Current legislation on animal experimen-tation, such as Directive 86/609/EEC, wasintroduced before the full implications of

ECVAM Workshop 28: transgenic animals 23

Table II: Consequences of techniques used in transgenesis

Technique Remarks

Gene addition achieved by all methods

Gene knock-out targeted inactivation of host gene by embryonic stem cellmanipulation

Random insertion of mutations achieved by all methods

Inhibition of gene expression for example, prevention of translation by hybridisation of anti-sense RNA with mRNA

Figure 1: Pronuclear microinjection

microinjectionto transgenesolution

embryo transfer topseudopregnantrecipient

test fortransgene

test fortransgene 50%hemizygotes

test fortransgene

25% homozygous transgenics

G2 (generation 2)

G1 (generation 1)

wild-type

G0 (generation 0)

hemizygote

collection ofembryos


Figure 2: Embryonic stem (ES) cell method

transgenesincorporated intoES cells by �genetargeting�

ES cells injectedinto blastocysts

blastocyst transferto pseudopregnantrecipients

isolation of ES cells

collection of blastocysts

homozygous transgenics

heterozygotes

germ-line chimerawild-type

G1 (generation 1)

G0 (generation 0)

G2 (generation 2)


Figure 3: Cre-lox technology for targeted homologous recombination oftransgenes

Crerecombinaselines up withlox P sites inpairs andremoves theDNA betweenthem

chosen cell type

Cre gene introducedinto one cell type

gene to be targeted

all other cell types

no Cre protein made

gene product made

gene product not made

targeted generemoved

⇒lox P

⇒lox P

⇒lox Pcre gene

cre gene

cre genecre gene

⇒lox P ⇒lox P

⇒lox P ⇒lox P

⇒lox P

⇒lox P

⇒lox P


transgenesis were recognised. Consequently,one of the principal objectives of the work-shop was to review the current situationwith regard to the development and applica-tion of transgenic animal technology, withthe aim of providing a list of recommenda-tions to key regulatory authorities and, inparticular, DGXI/E/2, which is responsiblefor the administration of Directive86/609/EEC. The potential benefits and ani-mal welfare implications of animal transgen-esis were discussed, together withpossibilities for implementing reduction,refinement and replacement (the Three Rs)strategies wherever feasible (15). Generaland specific concerns about the effects oftransgenesis on animal welfare are discussedlater in the report.

Transgenic Animal Disease Models

More than 3000 human genetic diseases areknown, and there is much interest in study-ing their fundamental causes so that effec-tive treatments and somatic cell genetherapies can be devised. Specific inbredmouse strains, which inherit spontaneouslyderived phenotypes, have provided usefulmodels for investigating the pathogenesis ofhuman diseases. Nevertheless, several prob-lems are associated with studying naturallyoccurring human genetic diseases by usinganimals: a) animal strains showing particu-lar disease symptoms are often difficult toobtain and expensive to maintain; b) theirspecific genetic defects can be as difficult toidentify and characterise as those of theirhuman counterparts; and c) affected animalsoften differ from unaffected controls ingenetic factors additional to the gene inquestion.

Advantages

Over the last decade, many transgenic andknockout mutant mouse strains have beencreated as disease models (16). Models existfor neuropsychiatric, cardiovascular, pul-monary, oncological, inflammatory andimmunological diseases, as well as for study-ing mechanisms and disorders of humanmetabolism, reproduction and early develop-ment. These models are documented intransgenic databases, such as TBASE (17) orInduced Mutant Resource (IMR). So far,mainly single-gene conditions have been

studied, but potential applications of trans-genic mice include the analysis of polygenicdiseases.

Scientific limitations

One of the major problems in modelling ahuman disease condition is that the pheno-typic effects of a mutation may vary depend-ing on the genetic background in which it isexpressed, due to the presence of specificalleles at modifying loci in different inbredanimal strains. It is therefore important toselect the correct genetic background, as wellas the correct mutation(s), to produce anoptimal model of any particular human dis-ease.

It is apparent from an analysis of sometransgenic disease models that the actualbenefits of using the model are rarely com-pletely equivalent to the potential benefits,and that the decrease in aspects of animalwelfare might be disproportionate to anybenefits gained. The currently availabletransgenic models for cystic fibrosis (CF)illustrate this point. None of the strains isideal, with either the genotype and/or thephenotype of the mouse failing to accuratelymodel the human condition (18). For exam-ple, in the case of the Edinburgh CF mousemodel, the transgenic animals have a normallife-span, a normal body weight gain, and aphenotype which is generally non-lethal.Nevertheless, to model the human disease asclosely as possible, it is likely that the micesuffer to some degree. In addition, one of themajor potential benefits of the work, genetherapy, remains elusive. It might, however,be feasible to study the molecular effects ofthe genetic lesion at the cellular level, tounderstand the mechanistic basis of the dis-ease, and to develop other forms of therapy.

There are several limitations in relation tothe usefulness of the current approaches todeveloping transgenic disease models, partic-ularly since many diseases are multifactorial.Problems persist when extrapolating dataobtained by using such transgenic animals tothe disease condition in humans.

Transgenic Animal Models inToxicology

The majority of transgenic animal strains intoxicology have been used for genotoxicityand carcinogenicity testing. In the case of


genotoxicity, current methods for detectinggene mutations are restricted mainly to invitro assays. In vivo approaches have princi-pally involved the analysis of chromosomaldamage in a single tissue type for muta-genic/genotoxic effects, which has made theirapplication limited, particularly when othertarget tissues are involved. The rationale forusing transgenic animals was to develop anassay that would detect a mutagen/genotoxinin vivo in a range of different tissues, includ-ing germ cells.

The transgenic approach to genotoxicitytesting

Commercially available transgenic mousemodels include Mutamouse® and Big Blue®,which contain the Escherichia coli lac Z andlac I transgenes, respectively (19). The trans-genes are cloned in bacteriophage lambdavectors that are integrated in the genome.Following the treatment of the transgenicmice with a test chemical, the integrated bac-teriophage vectors are rescued from the totalgenomic DNA by in vitro packaging. Mutantphage, with disrupted lac genes, are recog-nised by their ability to grow on susceptibleE. coli host strains and by the colour of theresulting plaques.

Agents which are strong mutagens aredetected with a high degree of accuracy, butthe ability of these assays to correctly detectnon-carcinogens requires further investiga-tion (20). Also, failure to detect compoundsthat cause predominantly large deletions hasbeen reported (21), probably due to the factthat only DNA fragments of a specific lengthare efficiently packaged, so that large dele-tions or insertions will probably not bedetected. To overcome this problem, trans-genic mice have been generated that containa plasmid-based lac Z system, in which largedeletions are detectable (22).

Carcinogenicity testing

The chronic rodent bioassay uses a largenumber of animals (400�500 per compound),and is prone to generate spurious data athigh dose levels (23). The principle underly-ing the use of transgenic animals for carcino-genicity testing is that the presence of anappropriate transgene will not directly pro-voke tumours, but will establish a high pre-disposition to carcinogenesis. Since theemergence of a malignant clone requires sev-

eral additional genetic changes in affectedcells, the time needed for this to occur isshortened. This predisposition to carcinogen-induced tumorigenesis, without a concomi-tant increase in spontaneous tumour rate,might not only allow shorter times for expo-sure to the test compound, but also a consid-erable reduction in the numbers of animalsrequired relative to the conventional bioas-say.

Three different types of transgenicstrains have been employed in the genera-tion of transgenic mice for carcinogenicitytesting: a) the Eµ-pim-1 transgenic mouse,containing the activated pim-1 oncogene(which has a low spontaneous tumour rateand appears to be very sensitive to carcino-gen-induced tumorigenesis by genotoxiccarcinogens that target the lymphoid sys-tem [24]); b) those containing an activatedoncogene (v-H-ras, c-H-ras) or an inacti-vated tumour suppressor (p53) gene (25,26); and c) mice with an inactivated DNArepair (XPA) gene (27).

However, transgenic animals bearing sin-gle cancer-associated mutations might pro-vide misleading information. In rodent cells,single mutations may lead to a transformedphenotype but not be sufficient to causetransformation of human cells. The trans-genic models may consequently be oversensi-tive to additional carcinogenic events, suchas exposure to carcinogens, and thus over-estimate human risk.

Insufficient studies have been conductedto assess the suitability of using transgenicmice models in carcinogenicity testing asalternatives to the chronic two-year rodentbioassay. However, assessment may befacilitated when the results of a recentlyinitiated international collaborative valida-tion study become available. Consensushas, nevertheless, been reached at theInternational Conference on Harmonisa-tion on a recommendation that the chronicmouse bioassay be replaced by an assaybased on transgenic mice for the regulatorycarcinogenicity testing of pharmaceuticals(28).

One important potential problem withtransgenic models for carcinogenesis isthat the effects of mutations in certaingenes, including tumour suppressor genesand oncogenes, might be influenced byspecies variation. As a corollary, mice bear-ing specific oncogenic mutations might not


necessarily be sensitive to the same sec-ondary events as occur in human carcino-genesis.

Transgenic Farm Animals

Currently, microinjection is essentially theonly method which can be used for producingtransgenic farm animals. The applications ofthese animals fall into three broad cate-gories: a) as bioreactors; b) for xenografts;and c) in animal production (29�31).

Bioreactors

There are two approaches to generatingbioreactors, here defined as transgenic ani-mals producing pharmaceutical proteins.The most effective approach is to express aprotein in the mammary gland by using apromoter from a milk protein gene to directexpression. An example is the production ofthe ovine β-lactoglobulin promoter for use inexpressing a variety of human proteins withpharmaceutical applications, such as α-1-antitrypsin (AAT). Thus, expression isdirected to the mammary gland of the lactat-ing mammal, commonly a sheep, cow, goat orpig, and the human protein is secreteddirectly into the milk (32). In the secondapproach, the desired therapeutic protein isproduced in non-mammary body fluids, suchas blood. To date, this approach has onlybeen used in pigs for producing haemoglobin(33).

Xenotransplantation

A key element in producing transgenic farmanimals to provide organs (xenografts) fortransplantation into human patients is pre-venting rejection of the transplant throughactivation of complement factors belongingto the human immune system. This objectivehas been pursued, for example, by producingtransgenic pigs expressing genes coding forhuman complement inhibitory factors, suchas decay-accelerating factor (34).

Production traits used for transgenes

The following livestock production traits arecurrently subject to manipulation by trans-genesis: a) growth and body composition; b)improvements in the quality and yield ofwool; and c) increased disease resistance(35�39).

Consequences of Transgenesis forAnimal Welfare

Three factors that may negatively influencethe health and welfare of transgenic animalshave been identified by van Reenen & Block-huis (40, 41): a) reproductive and otherbiotechnological interventions; b) mutations;and c) expression of the transgene. Thesefactors are discussed below, followed by anassessment of their impacts in relation tospecific applications.

Reproductive and other biotechnologicalinterventions

Studies involving certain species (for exam-ple, sheep and cattle) have shown that invitro procedures employed both before andafter microinjection (in vitro culture, embryotransfer) might lead to increased gestationlength, body weight, incidence of dystocia,and perinatal loss and anomalies, relative toin vivo (artificial insemination) procedures(41�43). Moreover, there is evidence thatmicroinjection, irrespective of successfulintegration of the foreign DNA, increasesembryonic and fetal losses in manipulatedmouse embryos (44, 45). The culling of micefor embryo recovery, and other surgicalinterventions used in generating transgenicanimals, also compromise welfare.

Mutation effects

Following microinjection, foreign DNAoften becomes integrated within or near anendogenous gene, thereby creating a new(�insertional�) mutation and causing a lossof host gene function. Integrated microin-jected material is sometimes associatedwith chromosomal translocations, and withother rearrangements leading to develop-mental defects. While insertional muta-tions can be of either a dominant or arecessive nature, it is assumed that detri-mental insertions usually result in earlyprenatal deaths, and hence are unlikely tobe detected (46). Implicit in the unpre-dictability of both the integration site ofthe foreign DNA and of the number of DNAconstructs which are successfully inte-grated, is the notion that each transgenicfounder animal is unique in terms of bothgenetic make-up and the nature of anydefects resulting from insertional muta-tions. Gene targeting in ES cell manipula-


tions might reduce the uncertainty to adegree, by directing integration to a partic-ular site.

Expression of the transgene

The extent to which transgenic animalsexpress harmful consequences from expo-sure to foreign proteins and/or expression ofa transgene is dependent on the following,interrelated, factors: a) the biological proper-ties of the resulting protein; b) the tissue(s)in which transgenes are expressed; c) theroute of secretion of the gene product; and d)the level of transgene expression. Thus, interms of the severity of potential risks, anyparticular transgenic animal model is situ-ated at some point on a more or less contin-uous spectrum between, at one extreme, acondition in which inert proteins are synthe-sised at a low level in a limited number ofspecific tissues insulated from the blood-stream and, at the other extreme, a conditionin which a biologically highly active proteinis synthesised in large amounts in many tis-sues with abundant access to the blood-stream. An example of the latter extremewas the notorious Beltsville pig, which suf-fered from a range of pathological conditions(47).

Additional problems can be caused by theincorrect or unpredicted expression of thetransgene. This can be due to pleiotropiceffects of the gene itself, or may result fromepistatic effects � interactions with endoge-nous genes and their gene products (48). Thelocation of a transgene in a chromosome(that is, a position effect) can also affect geneexpression (49), especially if it is integratedclose to endogenous DNA control regions,such as enhancers. As a result, enhancedtransgene expression, no expression, orexpression in an inappropriate cell type ortissue, may occur.

Several strategies are being developed toimprove the control of transgene expression.Thus, it might be possible to eliminate posi-tion effects by incorporating insulators,which are portions of DNA which act asboundaries, preventing interference with thetransgene by endogenous sequences (49).Furthermore, the use of complete promoters,or the presence of control regions withinintrons and other untranslated regions,could enhance the control of transgeneexpression. Inducible promoters have beendeveloped which could restrict transgene

expression to those cells containing aninducer molecule (50).

The cre-lox system also allows tissue-spe-cific expression of transgenes. This methoduses the bacteriophage P1 recombinaseenzyme and its lox P target sequences (Fig-ure 3; 51). Lox P sequences are recognisableby the bacteriophage P1 Cre recombinaseenzyme which, although capable of beingactive in eukaryotic cells, is not normallypresent. The transgene sequence is con-structed from the DNA coding sequence ofthe gene to be targeted together with flank-ing lox P sequences. The modified DNA isintroduced into ES cells, in which homolo-gous recombination occurs to replace thetarget host gene with the modified genecopy. This genetically modified ES cell lineis used to develop a transgenic mousestrain. A second strain of chimaeric mice,which exhibit tissue-specific expression ofthe Cre recombinase, is produced by intro-ducing the coding sequence for Cre intospecific cells in late stage embryos or inearly adult animals. When the two strainsof mice are crossed, homologous recombina-tion occurs between the two lox P sites,under the influence of the enzyme, but thisoccurs only in those tissues expressing therecombinase within the resulting hybridmice. This results in elimination of theintervening gene sequence, leading to spe-cific gene inactivation. In those cells unableto express the enzyme, no such recombina-tion occurs, and the gene is expressed.

Systematic effects

In addition to welfare issues relating to themodification itself, subsequent housing,husbandry and production systems (that is,systematic effects) can affect the well-beingof a transgenic animal (7). For example,pigs generated as sources of organs forhuman transplant surgery, or cows kept forproducing human pharmaceuticals in theirmilk, will need to be maintained understrict hygienic conditions. This couldincrease the risk that transgenic animalswill be deprived of certain environmentalconditions necessary for accommodatingnormal behavioural needs (for example,proper bedding, rooting material and exer-cise space), and hence welfare might bereduced (52). However, provided that thebehavioural needs of transgenic animals aretaken into consideration sufficiently, opti-


mal care and hygiene could improve theirlevel of health and welfare.

Welfare implications of disease models

Animals generated in such a way that theydevelop a human disease raise a distinct setof welfare issues. Reduction in animal wel-fare is intrinsic to the objective and is there-fore inevitable while, for other applications,animal suffering, where it occurs, might beseen as incidental. The development oftransgenic animal disease models has led tosome unexpected effects, such as glomerulo-sclerosis in the growth hormone (GH) model(53). It is sometimes difficult to distinguishsuch effects from the symptoms of the dis-ease being modelled. Models are also beingdeveloped to study the control of blood pres-sure (54), and animals of such strains couldwell suffer from cryptic effects.

The replacement of conventional animalmodels of disease by improved transgenicmodels reflecting more accurately thehuman disease, could conceivably benefitanimal welfare via a reduction in the numberof animals needed to achieve statistically sig-nificant results. Furthermore, it should bepossible to generate animals that have anumber of salient features of the disease butdo not develop the full disease condition.However, this objective has so far receivedvery little attention.

Welfare implications of mutagenicity testing

There do not appear to be any well-docu-mented studies of animal welfare in theMutamouse and Big Blue transgenic strainscurrently in widespread use for mutagenicitytesting. The numbers of animals used rou-tinely in genotoxicity testing could bereduced by the use of a transgenic rodentassay that would enable the detection ofmutations in the whole animal in every tis-sue, following exposure by different routes toa potential genotoxin. A reduction in animalnumbers will be achieved more readily, how-ever, if protocols involve detecting mutationsin a wide range of tissues using the mini-mum number of animals consistent withachieving statistical significance. Also, theability to investigate the induction of muta-tions in germ cells might obviate the need touse currently available methods for detectingheritable mutations, which require largenumbers of animals.

Welfare implications of carcinogenicitytesting

The use of transgenic rodents for carcino-genicity testing could also lead to an overallreduction in the large numbers of animalsthat are used in the conventional rodentbioassay. In addition, the duration of suchtests would be shortened, resulting in anoverall decrease in suffering.

However, there have been reports ofadverse health effects which go beyond thosedue directly to the testing procedure itself.Thus, high mortality rates have beenreported for c-neu and c-myc strains (25),and v-H-ras mice are prone to papillomadevelopment following minor skin abrasions(55).

Welfare implications of using transgenicanimals as bioreactors

Data on transgenic bioreactor farm animalssuggest that potential welfare problemscould be related to the actions of biologicallyactive proteins after transgene expression(35, 47). For example, ectopic expression ofputative mammary-specific expression hasbeen observed in rabbits carrying humanerythropoietin (hEPO) genes (56), sheep har-bouring human AAT genes, and mice carry-ing the human GH (hGH) fusion gene. In thecase of potent transgene-derived proteinslike hEPO and hGH, ectopic expression wasassociated with severe detrimental effects onanimal health. Moreover, ectopic expressionof even a putatively non-detrimental protein,such as mouse whey acidic protein in sheep,could have contributed to unusually highmorbidity rates (57). In addition to ectopicexpression, proteins may leak from milk intothe bloodstream or, in some cases, exertdetrimental effects locally in the mammarygland (58, 59).

Welfare implications of using transgenicanimals for xenotransplantation

There appear to be no published data relat-ing to the welfare implications of generatingtransgenic pigs for the provision ofxenografts. The principal concerns relate tothe housing conditions of these animals, par-ticularly where pigs are maintained understrict disease-free (gnotobiotic) conditions toavoid any transfer of disease to patients. Inthe UK, these issues are about to beaddressed by introducing a code of practice


on the husbandry of such animals as part ofthe Government�s response to the NuffieldReport (52).

Productivity promotion

Addition of transgenes encoding elementsaffecting growth, such as GH, to sheep andpigs have generally had detrimental effects.For example, the Beltsville pigs sufferedfrom lethargy, lameness, uncoordination,exopthalmus, gastric ulcers, severe synovitis,degenerative joint disease, pericarditis andendocarditis, cardiomegaly, paraketosis,nephritis and pneumonia (47). Such severeproblems are thought to be the result of thecontinuous elevation of circulating GH lev-els, but attempts to control expression (bythe use of inducible promoters) have not yetbeen successful. Limiting transgene expres-sion to a particular region of the animal isanother strategy for reducing welfareimpacts. For example, insulin-like growthfactor 1 expressed in the skin of transgenicsheep stimulated wool production withoutany apparent welfare problems beyond thoseinherent in the process of transgenesis (52).

Animal welfare might also be improved bythe addition of genes encoding disease resis-tance. However, to date, only one example oftransgene-derived disease resistance hasbeen demonstrated (avian leukosis virus inchickens [60]), and this was associated withother health problems (61).

Monitoring welfare

The animal welfare concerns identifiedabove indicate the need for a substantialeffort to monitor the health and welfare oftransgenic animals in a systematic manner.Although there is an extensive literature onindices of animal welfare (62), the lack ofconsensus on their relevance to transgenicanimals might hamper progress. This sug-gests the need to identify a minimum num-ber of indices covering a broad spectrum ofbiological responses (for example, ethologi-cal, physiological and pathological). More-over, to assess risks of impaired welfarethere may be merit in monitoring molecularbiological variation (for example, by measur-ing transgene products and RNA in varioustissues).

Since each founder animal generated aftermicroinjection is unique, the effects of trans-genesis need to be evaluated on a case-by-

case basis. This requires half-sib compar-isons (transgenic versus non-transgenic), inboth sexes, of the progeny of founder animals(41, 63). As founders are hemizygous for thetransgene, breeding founders with wild-typeanimals will result in the transgene beingtransmitted to 50% of the offspring. Todetect any deleterious effects on welfare dueto insertional mutations, another necessarystep will involve breeding homozygous trans-genic animals from half-sib x half-sib, orhalf-sib x founder, matings. Although theremay be a tendency for early disposal of non-transgenic animals, it is imperative whenstudying welfare implications to maintaincontrol animals in sufficient numbers toensure reliable statistical analysis.

The first and foremost strategy to avoidharmful consequences of transgenesis mustbe systematic screening of all aspects of ani-mal health and welfare; this will form thebasis of critical decisions (for example, a deci-sion not to breed homozygotes, or to refrainfrom enhancing transgenic expressionbeyond a certain level). The availability ofES cells would both increase the number ofpotential applications and reduce the risks toanimal health and welfare.

Before generating transgenic animals,insights into potential problems may begained by using alternative techniques tointroduce the gene product into the animal.This may involve treatment with the exoge-nous protein, or its homologous analogue(where immunological consequences can beavoided), or the addition of transgenes tosomatic tissues of the adult animal.

Public Acceptability and the Role ofEthical Analysis

There seems to be a significant lack of publicsupport for animal transgenesis. For exam-ple, in a European Commission poll on atti-tudes to biotechnology (64), it was reportedthat nearly 50% of Europeans consideredthat, if they were able to, they might notallow production and use of certain forms oftransgenic animal. Moreover, a majority ofrespondents opposed farm animal transgene-sis. This situation was endorsed in a morerecent European poll, in which a minorityapproved of using transgenic animals (12).Some believe, however, that scientists, asguardians of scientific knowledge, possess


the only correct view, while the public are ill-informed or misled. Others argue that con-siderations within the scientific communityare considerably influenced by self-interestand professional loyalties. Public concerns,however, go beyond issues of pain, sufferingand the welfare conditions of animals.

Criteria for public acceptability

Several criteria determine the public accept-ability of animal transgenesis. First, there isthe need of the scientific community forsound methodology, combining optimalvalidity and sensitivity with full accountabil-ity for scientific work designed to achieve setobjectives. Some argue that the applicationof transgenic technologies is incompatiblewith the Three Rs concept. Thus, it isclaimed that welfare problems encounteredin the early phases of the development ofnew transgenic strains are often overlooked(65), and that animal welfare considerationsare secondary to commercial criteria.

A second criterion is the need to show ani-mals respect. The housing, husbandry, han-dling and use for experimental purposes oflaboratory animals might be said to be fur-ther violations of their species-specific life(their �telos�), as can substantial alterationsto the genome.

Other key ethical concepts about whichthe general public and scientists often dis-agree are �naturalness�, �integrity� and�intrinsic/inherent value� (66�70). Thus,while the public might perceive animals aslinking humanity to nature, some scientistsmay tend to consider laboratory animals astools to manipulate and exploit.

A further criterion for public acceptabilityrelates to the possible adverse effects of ani-mal transgenesis on future generations andthe environment. One way of addressingthese concerns is to invoke the precautionaryprinciple, which states, broadly, that oneshould not proceed with a new process unlessconsideration has been given to the worst-case scenario (71). If scientists do not antici-pate potential problems, it is likely thatpublic views will continue to contribute to,and possibly distort, the outcome of anydebate, thereby increasing polarity (72).

A final criterion concerns respect forlifestyle and religious orientation. Determin-ing one�s own lifestyle depends on the avail-ability of relevant information, for example,via unambiguous product labelling. In the

case of genetically modified products, such asfoods and pharmaceuticals derived fromtransgenic animals, the situation regardinglabelling has yet to be resolved.

These differences in values, views andlifestyles can be said to constitute different�world views� (73). Clearly, there is no rightor wrong position concerning these worldviews but, in democratic societies, radicaltechnological change, such as the widespreadproduction and application of transgenic ani-mals, should only be introduced with explicitpublic consent.

The decision-making process

All groups in society have a role to play indemocratic decision-making procedures,which should be based on rational ethicaland scientific arguments. While several ethi-cal schemes exist to aid assessment of thecosts and benefits of research proposalsinvolving animal experimentation, thesewere developed before the full implications oftransgenesis were recognised.

As part of an ECVAM/FRAME/CAB pro-ject, the UK Institute of Medical Ethics(IME), and Canadian and Dutch ethicalschemes (74�76), have been evaluated fortheir abilities to deal with proposals involv-ing transgenic animals. The schemes wereapplied to several different transgenic modelsystems, such as Immortomouse (77), andthe CF (78) and GH (79) mouse models. Theresults of the study suggest that transgenictechnology raises a number of unique issueswhich are not addressed by the application ofany of the ethical schemes. For example, theIME and Dutch schemes consider the suffer-ing imposed on animals solely in terms of theprocedures and housing conditions. Thisresults in an inability of the schemes toaddress issues relating to animals specificallydesigned to suffer, such as disease modelswhich spontaneously develop a pathology inthe absence of any scientific procedure.

Furthermore, none of the three schemesdistinguish between the generation and sub-sequent use of transgenic animals. For exam-ple, in establishing a transgenic animal line,the surplus animals required, and the possi-ble random side-effects of the transgene onthe host, need to be considered separatelyfrom surgical interventions.

In terms of the benefits likely to accruefrom the use of transgenic disease models,questions relating to the importance and


complexity of the genetic component (multi-factorial and polygenic diseases), and prob-lems linked to the production of a phenotypein relation to a genotypic condition, are notspecifically addressed by the schemes. Thenext stage of the collaborative project will,therefore, be to further develop the existingschemes so that the above questions andissues specific to transgenic animals are ade-quately considered. As an alternative, itmight prove necessary to develop a novelscheme to cope with the unique problemsencountered.

One possibility is to promote the use ofethical frameworks to aid reflection. Theterm �ethical� is used here in the broadsense of concerning �what we should do�,and thus encompasses risk, cost�benefit,welfare/suffering, and intuitive responses.Such frameworks should attempt toencompass the concerns of both the scien-tific community and the general publicwhich, as shown earlier, may differ consid-erably.

An example of such a framework is the�Ethical Matrix� (80), based on the princi-ples employed by Beauchamp & Childress(81) for medical ethics. The aim is to con-struct a comprehensive framework of ethi-cal issues which is both philosophicallycoherent and comprehensible to the lay-public. The three principles involved (cor-responding to the major ethical theories ofutilitarianism, deontology, and Rawlsian�justice as fairness�) are claimed to formthe basis of the �common morality� or�commonsense ethics�. For example, in thecase of experimental animals, respect forthe three principles of well-being, auton-omy and justice translates into respect for�animal welfare�, �behavioural freedom�and �telos�, respectively. Decisions regard-ing final ethical acceptability depend, how-ever, on the manner in which the ethicalmatrix is applied and, in turn, on the user�sworld view.

The use of a common framework through-out the EU would standardise the way inwhich decisions regarding ethical acceptabil-ity are taken, while allowing local differencesin attitude to be expressed. It would alsoimprove the accountability of regulatoryprocesses by making policy decisions trans-parent and comprehensible to a wider soci-ety, in turn promoting public debate andeducation.

Concluding Remarks

The technique of animal transgenesisappears to offer the prospect of considerableadvances in biomedical science and biotech-nology. Moreover, in some cases, the use oftransgenic animal models could lead torefinement and reduction in the numbers ofanimals used in experiments. There is, how-ever, a substantial risk that the currentintense interest in developing novel trans-genic strains will, in fact, result in an overallincrease in experimental animal use. Itshould also be remembered that many addi-tional animals are required during the gen-eration of new transgenic strains (forexample, surrogate mothers and vasec-tomised males). It is therefore cruciallyimportant that measures are taken to ensurethat the production and application of trans-genic animals is subject to close monitoringand control. The technique of transgenesisalso raises serious ethical concerns, since it ispossible to induce irreversible and oftenpotentially far-reaching alterations in thegenetic constitution of animals, for example,producing strains which express humangenes, or which, in the case of disease mod-els, are designed to suffer.

Urgent attention needs to be given toestablishing committee structures through-out the EU to harmonise the regulation ofthe production and use of transgenic ani-mals. National ethical and scientific commit-tees would be charged with the task ofinterpreting relevant EU Directives andmonitoring the activity of local (institu-tional) ethics and scientific committees. Thelatter committees would be responsible forensuring that individual research proposalsconform to agreed ethical principles. Institu-tional committees would also ensure that allpossible measures were taken to protect thewelfare of transgenic animals, workingtogether with any existing statutory bodiescharged with regulating laboratory animalexperimentation. In this way, implementa-tion of comprehensive measures to protectand continuously monitor the welfare oftransgenic animals should be guaranteed.Measures taken might include application ofaccurate gene targeting techniques, storageof cryopreserved transgenic embryos, andincreased application of in vitro methods forcharacterising new gene constructs. Theseobjectives would be facilitated by establish-


ing an international database (accessible onthe Internet), which would include informa-tion on: a) animal welfare; b) the validationand regulatory status of transgenic animalmodels for toxicity, carcinogenicity andmutagenicity testing; and c) the extent towhich the stated objectives and potentialbenefits of the work were achieved. Suchdetails should be used to assist with the eth-ical and scientific assessment of furtherresearch proposals.

It seems clear that existing regulationsand legislation for animal experimentationdo not adequately provide for the develop-ment and varied applications of geneticallymodified animals. It is hoped, therefore,that the recommendations of this work-shop will contribute to the formulation ofan appropriate set of harmonised regula-tions which take account of the importantpublic and scientific concerns raised bythis form of technology. A possible schemefor regulating the production and use oftransgenic animals in the EU is shown inFigure 4.

Summary and Conclusions

General considerations

1. The recommendations of the EU Groupof Advisers on the Ethical Implicationsof Biotechnology on the ethics of geneticmanipulation of animals are endorsed.

2. The objectives of this workshop reportare to build on the analysis of the EUGroup of Advisers, by providing more-specific recommendations for the regula-tion of procedures involving transgenicanimals in the EU, with the aim ofensuring inter alia that levels of animalwelfare are maintained at the highestpossible, while permitting the continuedlegitimate use of transgenic animals.

3. Attempts should be made to reduceunnecessary duplication of develop-ments in animal transgenesis, withoutunduly compromising the competitivenature of scientific investigation.

4. Surveys of public opinion within the EUsuggest that special consideration isrequired for the production and use oftransgenic animals within national andinternational legislation.

5. Existing controls on the production anduse of transgenic animals in some Mem-ber States of the EU may be inadequate,and EU legislation needs to be har-monised and rationalised since it is toovariable between Member States.

6. As current EU legislation (Directive86/609/EEC) does not explicitly refer totransgenic animals, a distinction needsto be made between the different(prospective) applications of animaltransgenesis, as this could have impor-tant implications for the acceptability ofcertain procedures.

7. Certain uses of transgenic animals (forexample, the use of higher non-humanprimates) should be considered, in prin-ciple, to be unacceptable.

8. Technological opportunity should notdrive the production and use of newtransgenic strains in the absence of anygenuine social need.

9. Any proposed work involving transgenicanimals needs to have a clearly statedpurpose so that its justification can beassessed.

10. It is important not to allow a narrowcost�benefit approach to assessmentwhich limits consideration of otherimportant ethical factors, such as: a)respect for cultural values; b) responsi-bility for the protection of the physicalenvironment; and c) the preservation ofspecies integrity and diversity. Certainschemes for ethical assessment andreview already exist, and it is recom-mended that their suitability in the con-text of transgenic animal proceduresshould be further investigated.

11. While some may consider the progres-sive introduction of increasing num-bers of human genes into transgenicmodels to be of minor concern at pre-sent, the technology exists, or couldsoon be developed, to allow this toattain a level of �humanisation� whichmany would consider unacceptable.This matter should be kept under veryclose review at national and interna-tional levels.

12. The labelling of products derived fromtransgenic animals should allowinformed consumer choice.


Figure 4: A possible scheme for regulating the production and use of transgenicanimals in the European Union (EU)

EuropeanCommission

Harmonised guidelines

New EU directive

National committee

Local committee

Ethical frameworkincorporating cost�benefit

analysis

Approval(limited or full)

Rejection/suggested

alternatives

ProposalApplicant

Production of transgenicanimal strain

Prospective benefits:valued products andprocesses and valid

methodologies

continuingassessment

(incorporatingcost�benefit

analysis)

Conditions:

� embryocryopreservation

� tissue banks� precise gene

targeting, etc.

Authoritative advicepublic/scientific

Database


13. Even where proposals for the use oftransgenic animals are acceptable inprinciple, the existence of a valid alter-native non-transgenic method, whichmeets the stated objectives, shouldresult in the rejection of the proposal toproduce and use transgenic animals.Consideration should be given to analternative procedure, at a late stage ofdevelopment, if it is likely to become rea-sonably and practicably available in thenear future.

Proposals following prima facie approval ofa proposal to use transgenic animals

14. It should be recognised that the geneticmodification of animals has the poten-tial to cause unexpected detrimentaleffects in progeny, which are some-times severe.

15. Approval of an application for a pro-gramme of work with transgenic ani-mals should be reviewed periodically,with regard to the alleged costs to theanimals involved and the benefits tosociety.

16. Independent, competent and continu-ing assessment of the health status ofanimals should be undertaken, and thisinformation should be freely availableand used to assess future applications.

17. Existing databases in the public domainshould contain up-to-date informationregarding the effects of transgenic proce-dures on animal welfare, to includedetails of: a) microbiological status; b)breeding, health and behavioural prob-lems; c) specific problems encountered ingenerating new transgenic strains; d)the actual benefits to be derived fromthe programme of work; and e) details ofany new alternative methods.

18. Full records of all relevant informationrelating to scientific and welfare statusshould be maintained at each researchestablishment for inspection for two fullgenerations of the natural life-span ofthe transgenic animal strain when keptunder optimal husbandry conditions. Inthe absence of adverse effects, the modelshould be considered equivalent to anon-transgenic animal model, but onlyfor purposes of implementing relevantlegislation.

Production and use of transgenic animals

19. More research is needed into: a) thebasis for differences in gene expressionin vivo and in vitro; b) methods for facil-itating the screening of transgenicembryos; and c) improved methods forgene targeting and inactivation. Suchmethods should be used to increase theuse, when appropriate, of cell culturemethods before, or instead of, the use ofwhole animals.

20. To maximise scientific benefit and ani-mal welfare, studies involving transgenicanimals should be conducted by teams ofindividuals with relevant experience in,for example, molecular biology, genetics,pathology, cell biology, and animal careand welfare.

21. It is important to ensure that housingand transport requirements appropriatefor specific transgenic animal strains arestrictly applied, to minimise adverseeffects on their welfare.

22. Measures should be taken to avoidunnecessary deleterious side-effects instocks of transgenic animals, for exam-ple, by maintaining breeding colonies inthe heterozygous state and subsequentlyproducing limited numbers of animalshomozygous for the prospective trans-gene for use only when strictly required.

23. Transgenic animal models could bestored as cryopreserved embryos insteadof in breeding colonies, in which theymight exhibit deleterious side-effects.The advantages and disadvantages oflocal, rather than centralised, embryobanks should be investigated.

24. To promote refinement of procedures, allpersonnel handling transgenic animalsshould be required to attend trainingcourses which stress the problemsunique to, or frequently encountered in,such work.

Specific considerations relating to animalsused as basic research/disease models

25. Clarification of the concept of �models�(for both disease and basic research) inthe context of animal transgenesis willassist both scientists and regulators inassessing the relevance and feasibility ofresearch proposals.


26. In view of the fact that substantialnumbers of new transgenic strains canbe produced, great care should betaken to select the most promisingmodels. Account should be taken of: a)the need to identify critical factorswhich are indispensable for establish-ing the relevance of a model; and b)any existing or possible alternativemethods of studying the human dis-ease in question, or for developingtherapies (for example, analysing clini-cal samples and disease conditions, theuse of specific cell lines, and geneexpression in vitro).

27. Models should mimic as closely as possi-ble the human condition being studied(for example, in terms of aetiology,pathology and molecular mechanismsinvolved), although it may not always benecessary for exact parallels to exist.Furthermore, attempts should be madeto model the earliest stage of the diseasewhich is required to produce effectivetherapies, without necessarily producinga model which exhibits all symptoms ofthe disease.

28. When the levels of suffering experiencedby a transgenic animal are not consid-ered disproportionate, continued studiesto improve the model in question mightbe justified, but only where the amountof anticipated suffering remains withinacceptable limits.

Specific considerations relating to animalsused in toxicity and carcinogenicity testing

29. When developing transgenic animalstrains for toxicity studies, it is impor-tant to bear in mind the specific purposefor which the model is intended (forexample, whether for routine regulatorytesting or for specialised mechanisticstudies).

30. The use of transgenic animals in toxicitytesting could lead to reduction andrefinement in existing in vivo test proto-cols (for example, with respect to therodent bioassay for carcinogenicity).

31. Although the use of transgenic animalsmight reduce and refine existing in vivotoxicity and carcinogenicity assays, thisshould not be regarded as a reason todiminish efforts to use and further

develop currently available in vitromethods (see point 32, below).

32. To answer specific questions and to char-acterise particular mechanisms of toxic-ity, as well as for routine toxicity testing,panels of cell lines should be derived fromany available genetically modified ani-mals and from clinical samples.

Specific considerations relating totransgenic farm animals

33. Relevant measures of animal well-beingshould be identified during the earlystages of a programme to generate newtransgenic farm animals. The systematicuse of these measures should involve: a)the use of general indices, such as thoserelating to behaviour, immunologicaland disease status, and measures ofgrowth and reproductive efficiency; andb) more-specific indices, chosen accord-ing to the anticipated consequences ofthe expression of a particular transgene.To assess the extent of any adverseeffects of the transgenic procedure, suffi-cient numbers of appropriate controlswill be required, for example, by retain-ing non-transgenic siblings alongsidetransgenic offspring of the same founderanimals.

34. It might be deemed appropriate to grantofficial permission for using transgenicfarm animals only for a limited period,pending the results of continued welfaremonitoring. The period for which theresulting �provisional licence� was to beissued would depend on the species ofanimal in question and the conditionsunder which the animals were to bekept.

35. The maintenance of adequate records onthe absence of adverse effects in trans-genic offspring before they are releasedfrom automatic protection under animalprocedures legislation might be espe-cially important in the case of farm ani-mals, in view of the greater likelihood oftheir being released into the environ-ment for routine use.

Other conclusions

36. Improved dialogue is needed betweenscientists and the general public con-cerning the scientific rationale and


perceived ethical justification forapplying transgenic techniques to ani-mals.

37. The independent assessment of propos-als to use transgenic animals should beundertaken in ways appropriate to thenature of the application. Some issuesinvolving transgenic animals are ofsuch fundamental and/or generalimportance as to require regulation byEU or national committees. Other,more-specific, issues dealing with par-ticular programmes of work, are moreappropriately considered by local orregional ethical and scientific assess-ment, or by other systems (such as theHome Office Inspectorate operating inthe UK).

38. The composition of ethics committeesshould: a) allow both the general pub-lic and scientists to be confident thattheir views receive proper considera-tion; and b) genuinely reflect the rangeof viewpoints within society, while tak-ing into account the need to reach aconsensus.

39. The accountability of ethics committeesshould be ensured by rendering the deci-sion-making process as open and trans-parent as possible (while recognisinglegitimate provisions for commercialconfidentiality). Consideration should begiven to the use of a widely acceptedpublished framework, such as thattermed the �ethical matrix�, to facilitatethis process.

Recommendations

The above list of conclusions has been usedto derive a series of key recommendationsdesigned to assist regulatory authorities informulating legislation for the productionand use of transgenic animals throughoutthe EU.

1. Agreement should be sought through-out the EU concerning the degree andlevel of continuing legal protection tobe afforded to transgenic animals. Aspart of this process, and as current leg-islation does not specifically covertransgenic animals, regulations for thecontrol and use of such animals

throughout the EU should be har-monised and rationalised. If necessary,existing legislation should be modifiedor replaced.

2. Adequate and clear statistics on thenumbers, species, types, production,breeding and uses of transgenic animalsshould be collected and published atleast bi-annually by each EU MemberState, and by the European Commissionto promote transparency and account-ability.

3. A list should be compiled of proceduresinvolving transgenic animals which,while technically feasible, would not beconsidered ethically acceptable underany circumstances.

4. There should be a broad and systematicethical assessment of all proposals forwork involving transgenic animalswhich have not been excluded by apply-ing recommendation 3, above.

5. Principles concerning matters of funda-mental and general importance aboutthe use of transgenic animals should beagreed at EU and national levels. A localinstitutional ethical and scientific com-mittee should ensure that each individ-ual research proposal conforms to theseprinciples.

6. When an alternative, non-transgenic,method is at a late stage of development,and is likely to be reasonably and practi-cably available in the near future, itshould be given consideration as areplacement for the proposed use of atransgenic animal method. For example,appropriate cell culture methods shouldbe developed and used wherever possiblebefore, or instead of, using whole ani-mals in transgenic research and applica-tion.

7. Legal approval for any research involv-ing transgenic animals should beregarded as contingent on the outcomeof independent and continuing assess-ment of the health and welfare of theanimals involved, and of the benefitsbeing derived from the resultsobtained. Such information should berecorded in a database, be publiclyaccessible wherever possible, and be


used in the appraisal of future researchproposals.

8. To maximise scientific benefit and ani-mal welfare, all studies involving trans-genic animals should be conducted byteams of individuals covering the fullrange of relevant expertise in, for exam-ple, molecular biology, genetics, pathol-ogy, cell biology, and animal care andwelfare.

9. All personnel directly involved in usingtransgenic animals should be required toattend training courses which emphasiseissues and problems unique to, or fre-quently encountered with, such animals.

10. Approval of any research proposalsinvolving transgenic animal models ofhuman disease should be based primar-ily on a requirement for the model toparallel as closely as possible the humancondition being studied. The relevance ofall such transgenic models should bekept under continuous review, andshould not be assumed because of anysuperficial similarity with the diseasebeing modelled, or solely on the basis ofthe importance of this disease.

11. During the production of new transgenicanimal disease models, efforts should bedirected toward modelling the earlieststage of a disease which will allow theobjectives of the research to be achieved.

12. When transgenic animals are releasedfrom protection under the terms of labo-ratory animal legislation, such as trans-genic farm animals kept for agriculturalproduction, the maintenance and centralstorage of adequate records of the breed-ing and location of such animals, evenduring their routine husbandry, shouldbe required.

13. Where biomedical applications of trans-genic farm animals require that they arekept in quarantine, or specific pathogen-free or gnotobiotic conditions, the maxi-mum possible provision for thebehavioural needs of the animals shouldbe guaranteed.

14. While the application of bioreactor andxenograft technology might offer signifi-cant medical benefits to society, the pro-duction of foreign biologically activemolecules within the transgenic animals

concerned might reduce their standardof welfare, and monitoring for anyadverse effects should be mandatory.

Acknowledgements

Thanks are due to Mr & Mrs B.H. Annett(AMA Services, London, UK) and to Mrs M.Smith (FRAME, Nottingham, UK) forundertaking organisational aspects of theworkshop. We thank Dr R.J. Lysons (HomeOffice, London, UK) who was invited toattend the workshop as an observer and whocontributed information and advice for thereport. The assistance of Dr C.G. van Reenen(Institute for Animal Science and Health[ID-DL], Lelystad, The Netherlands) in pro-viding information on transgenic farm ani-mals is also gratefully acknowledged.

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