Refi Ning Procedures for the Administration of Substances

WORKINGPARTY REPORT

Rening procedures for theadministration of substances Report of the BVAAWF =FRAME=RSPCA=UFAW JointWorking Group on Renement

Members of theJoint Working Groupon Renement: D.B.Morton (Chairman), M.Jennings (Secretary),A. Buckwell, R.Ewbank, C.Godfrey, B.Holgate, I. Inglis, R.James, C.Page,I. Sharman, R.Verschoyle, L.Westall &A.B.Wilson

Contents

Preface 2 3.6 Intraperitoneal 19 1Introduction and aimsof thereport 2 3.7 Intratracheal 20 3.8 Intravaginal 20 2Generalprinc iplesof `good practice’ 3 2.1 Planningand preparation 3 3.9 Intravenous and intra-arterial 21 2.1.1 Experimental aims 3 3.10 Oral routes 25 2.1.2 Theroute 3 3.10.1 Inclusionin an animal’s food orwater 25 2.1.3 Thesubstance 3 3.10.2 Dosingdirectly into 2.1.4 Theanimal 5 the pharynx 27 2.1.5 Thetechnique 7 3.10.3 Oral gavage 28 2.1.6 Staff and training 7 3.11 Osmotic minipumps 30 2.2 Technical preparation 3.12 Respiratory routes 31 andaftercare 8 3.12.1 Wholebody exposure 31 2.3 Generalre® nem entfor 3.12.2 Noseonly =Snout only all routes 9 exposure 32 3Re®nem entfor individual routes 3.12.3 Mask exposure 33 and procedures 13 3.13 Subcutaneous 34 3.1 Intra-articular 13 3.14 TopicalÐderm al 35 3.2 Intracerebral (intracerebro- 3.15 TopicalÐoc ular 36 ventricular) 14 3.16 Footpad 37 3.3 Intradermal 16 3.17 Uncommonroutes 38 3.4 Intramuscular 16 4Special considerations for wildanimals 38 3.5 Intranasal 18 References 39

Correspo ndenceandre q uestsfo rreprintst o:ProfessorD.B.Morton,DepartmentofBiomedicalSciences & BiomedicalEthic s, TheMed icalScho ol,UniversityofBirm ingham,Edgb aston,Birm inghamB152TT, UK

Accepted 14July 2000 # LaboratoryAnimals Ltd. LaboratoryAnimals (2001) 35, 1 41 2 Joint Working Groupon Renement

Preface viously beenc ollated and werenot available inthe literatu re. Thisworkshop report is the® fth intheseries It ishoped th at thereport willbe widely producedby theJoint WorkingGroupon circulated inan international forum,and that Re®nem entorgan ized by theBri tish Veter- therec ommendations willbe adopted as inary AssociationAnim al Welfare Founda- currentgood practice. tion(BV AAWF),theFundfor the Replacementof Animals inMedical Experi- ments(FRAME),theRoyal Society for the 1Introductionand aims ofthereport Preventionof Cruelty toAnimals (RSPCA) and theU niversitiesFederation for Animal Theproceduresin volved inth eadministra- Welfare (UFAW).Theworkshopsare based on tion ofsubstancesto animals can have a theimportant principlet hat whenever signi®cant effectonthewelfare oftheani mal animalsare usedin laboratories ,minimizing and thescient i®c value of theresults Ðif any pain and distress shouldbe as important carried out incorrectlythenboth can be an objectiveas achieving theexperimental compromised. Re®ning adm inistrationpro- results.Thisis essen tial for humanitarian cedurestherefore provides opport unitiesfor concerns. It isalso necessary inord erto improving both welfare and science.The satisfy thebroad legal principlesi nnational welfare bene®ts are widely recognized; the and international legislation,and toproduce scienti®c bene®ts resultfrom better quality reliable and reproducible scienti®cdata. data obtained from morecareful lyprepared Them embersof eachWorking Party are experiments, usingless stresse d,more`nor- drawn from thescient i®ccommunity, from mal’ animals (Manser 1992, Vogel 1993, industry, academiaand animal welfare orga- Poole1997 ). nizations. Someoftheorganizat ionspartici- Theadm inistration of substancesis a very pating (for example,th eRoyal Society for the broad topicÐmanydifferent kinds ofsub- Preventionof Cruelty toAnimals) are stance are administered by many different opposedto theuse of animals inexperiments techniquesan droutesand for avariety of that may causethe anim als pain, suffering, purposes.T hemethodsinvolved are descri- distress orlasting harm,and this particular bed inanumber ofpublications (e.g.Paget& workshopwas a dif®cult onefor anyonein Thomson1979, Kirk &Bistner 1985, Poole suchapositionto be involved withbec ause 1987, Rollin& Kessel1990, Waynforth & ofthenature oftheproc eduresdiscusse d. Flecknell1992, Tuffery 1995, Wolfensohn& (Note: participation inthis Workshopdoes Lloyd 1998)and guidelineson g ood practice not indicate endorsementby theSoc iety of have recently beenprod uced by theLabora- researchrequi ring theuse of theproc edures tory Animal ScienceAssociation (1998).This described.) However,the c ommonaim isto reporti sintended tocomplementt heexist- reduceanimal sufferingwhereverit occurs ing literature by identifyingpotential prob- and thereport is intended tohelpac hieve lemswithindividual methods and proce- this,partic ularly ifread inconjun ctionwith duresan dfocusingon how t hesec an be otherrec entreportson the rec ognition, re®ned to reduceadverse effectsand develop measurementand alleviationof painordis- best practice.Them ost commonlyused tressin anim als. routesin t hecom monlaboratory speciesare Thispart icular workshopalso proved coveredwith notes on add itional methods or extremely dif®cult toreport, partlybecause otherspec ieswh ereappropriate. The ofthebreadth of thesubject andthenum bers emphasisison tec hniquesc arried out in ofprocedureswe set out tocover.In addition, biomedical researchand testing but someof althoughmany ofthetec hniquesare descri- theprin ciplesalso have clinical application bed brie¯y elsewhere, thesort ofdetailed inveterinary medicine. information required regardingpotential Thereport shouldprovide usefuli nforma- problemsand solutions,and about ways of tion for anyoneplannin gorcarrying out furtherre® n ing techniques, had not pre- procedures,or whohas todeal withth eir

LaboratoryAnimals (2001) 35 Administration of substances 3 consequences.General principlesof `good animal, thepossibl eeffects ofthedosi ng practice’whenadm inisteringasubstanceby techniqueon th eanimal and theexpec ted any route are set out inSec tion 2,together frequencyofdosing. Thechoiceoftechnique withrecommendationsfor re®nem ent and, whererelevant ,thesite selectedare both withregard tothesubstanceadministered, also in¯uencedby suchfactors. equipmentand techniques.Section 3then Someroutesand techniquesare more describes speci®cre®nem entswithi nindivi- stressfulthan othersand theleast severe dual routesand techniques.Thereport consistent with theexperi mental aims concludeswith a section addressingspec ial shouldbe selected(seeTable 2). considerationswhenadm inisteringsub- stancestowild animals. 2.1.3 Thesubstance Thereport i sbased onth epublishedlit- Thereare constraintson the form ulations erature wherethi sisavailable and ont he that can be usedfor any particular route. consideredexpert opi nionof theworking Both thesubstanc e and thedosing vehicle group and theirc olleagues. must be appropriate for theroute, thespeci es and thepurpose of theexperim ent. Detailed 2Generalprinciples of `goodpractice’ informationon fac tors tobe consideredis available int heliteratu re(see for example, Whenadm inistering asubstancetoan Sanderson1959, Spiegel&Noseworthy1963, animal by any routetheai mshouldbe to TheMerck Index 1968, Claasen 1994, achieve`best practice’, sincemistakes at any Waynforth 1995, Reynolds 1996)and the = stage can causeavoidable suffering and or followingbrief summary isintended only as waste animals’ lives. Best practicedepends astarting point. onm inimizingoravoiding adverse effects, minimizingthenumber of animals used,and maximizing thequality and applicability of Physicochemicalproperties :Thephysic o- = results. chemical propertiesof thesubstanc eand or its vehiclem ay adversely affect animals. 2.1Planning and preparation Theseinc lude: theform ulation,sol ubility, viscosity, pH, biocompatibility, purity, Thelikeli hood ofdif®culties arising is stability, standardization and microbial reducedby thorough preparation. Con- contamination.Backgrounddata onsuc h tingencyplanssh ould always be prepared in propertiestog etherwith any extraneous case problemsdooccur. A`checklist’ for effects(e.g. irritancy),shouldalways be planningproceduresis given inTable 1. investigated. Theexpected effect may be altered by concentration anddosevol umeso 2.1.1 Experim entalaims thisshouldbe checked too. Ausefulrefer- Thescienti®c aimsmust be metby theroute encedocumentis the Dictionary ofSub- and administrationreg imeselected, soit is stancesand theirEffects (Richardson 1993, important tocheckthis® rst. Thelikely Claasen 1994). pattern ofresultsshould be considered, together with theway they relatetothe Solutions and solvents :Wheresubstanc es study’s goals and what willsubsequen tly be are dosed as solutions,water for injectionor donewith the result sandwiththeanim als. physiological salineare thecommonest and At this stage itisimportanttoconsidernot simplestsolvents .For water-insoluble com- only whethertheexperim ent can be done but poundsa suitable organic solventm ay be also whether,given thelikely effects onth e employed. Ideally this shouldlack pharma- animals, it sh ould be done. cological effects, be stable underc onditions ofuse,non-toxi c,non-irritant and non-sen- 2.1.2 Theroute sitizing. Theviscosity shouldbe suitable for Thech oiceofrouteisdeterm inedby the easeof injection. Thesolvent m ust remain purposeof theexperiment, thespeci esof the ¯uid at thetem peraturesat whichit willbe

LaboratoryAnimals (2001) 35 4 Joint Working Groupon Renement

Table1 Checklistwhen planning procedures

Experimental aims Whatare you trying to achieve scientically? Will theadministration regime selectedmeet the aims of theexperiment? Consider not just whetherit can bedone but whetherit should bedone and whether there is a betterway of doing it?

The route Is theadministration routesuitable forthe substance? Doesthe proposed route have ahigh severity rating? Would aless severeroute achieve thesame aim? Is it suitable forrepetitive doses?

Thesubstance Are you certain you know whatyou areadministering? Will thesubstance have any adverseeffects on theanimal andare there any background data on these?If so, have thenecessary preparations been made? Could thenature of theformulation alterthe expected effect? Doesthe substance need to befreshly prepared? Will theconcentration anddose volume alterthe expected effect? Arethere any additional concerns regarding thephysico-chemical propertiesof thesubstance or associatedsolvents, e.g. osmolarity? Canthe volume bereduced? Canthe frequency of administration bereduced? If thesubstance is toxic can thedose be reduced? Is thesubstance likely to beirritant? Arepilot studies needed,e.g. to ascertaina toleratedand =or effective dose?

The animal Arethere any problems with theparticular individuals, species or strain? Is theanimal easily stressedby handling? Is it themost appropriatefor the study in terms of thesefactors? Canthe animal betrained to cooperatewith theprocedures? Doesthe animal needtime to acclimatize to theprocedures? Is ananaesthetic, sedative or analgesic required. Would it reducestress or confound the experiment? Haveyou donea pilot study fortolerated and effective doselevels in thestrain used?

Thetechnique Whatare the scienti c problems (e.g. rst-passmetabolism in theIiver afteroral or i.p. dosing, degreeor rateof absorption, local effects)? Whatare the technical problems (e.g. whatis thecorrect way to hold ananimal to allow insertion of agavagetube with minimum distress)? Will thetechnique itself have any effecton theanimals? Arethe severity limits =humane end-points clearly dened? Whatre nements can beintroduced to overcome any adverseeffects? Is apilot study necessary? Haveyou checkedon referencesand sources of expertise in otherorganizations?

Staff Dostaff have thenecessary licences? Arestaff competent in thetechnique andtrained to dealwith any untoward effects? Who arethe best members of staffto carry out theprocedure, when considering both the handling of theanimals andthe procedure? Areenough staffavailable to restrainand dose the animals andto monitor them post-administration? Arestaff aware of theseverity limits andhave thedelegated authority andskill to kill animals if theseverity limits areexceeded?

usedan dideally have asuf®cien tly high Insolublesolids orim miscible chemicals boilingpointto allowheat sterilization if may be dosedas suspensionsor emulsionsin required. Unfortunately noideal solvent suspendingagen tssuchas gumtragacanth or existsÐmany are suitable only for particular methyl cellulose,Lissapol 2, Sorpol2 or routesand thoseavailable must be evaluated Tween2.(Note that material suchas Tween 2 todecide whichismost appropriate. and Cremophor 2 can causeanaphylact ic-like

LaboratoryAnimals (2001) 35 Administration of substances 5 reactionswhen adm inistered intravenously Se x:Somestudiesm ay requireonly males to dogs.) orfem alesand therem ay be signi®cant Formulationsshould be usedas soonafter wastage of theunwanted sexas aresult. The preparationas possible,or within t heperi od justi®cationÐ scienti® c, economic ortradi- de®ned as satisfactory for maintenanceofthe tionalÐfor usinga singlesex should always stabilityand quality oftheformulation. be questionedtotry toreducesuc hwastage. Themotivationm ay initiallyhave beent he welfare implications, for exampletheease of Rateofabsorption :Theexpec tedpharma- group housingfem ale animals suchas mice. cologyortoxici tyof asubstancemay be However,it may be possible toadapt the in¯uenc edby its rate ofabsorption whichis husbandry system sothat male animals can inturn in¯uenc edby: (a) itsphysical also be group housed(Jenn ings et al. 1998). propertiesÐsuc has molecular conformation, charge, particlesi ze, crystallineform,lipid Habituationandtraining :Many laboratory solubility, salt form,rate of disintegration of animals willhave had little ornoexperience thesolid dosage form,thedeg reeto which ofbeinghandled orrest rainedbefore arriving thechemical isionized at theabsorbing at aresearchestablishmentsoonthe ® rst membrane; and(b) theroute ofadministra- occasionth eyare pickeduptheyare likelyto tion(see Waynfort h1995 for moredetails ). be apprehensiveand frightened. Thisnot = Pilot pharmocokinetics metabolismst udies only makes theproc edurem oredistressi ng may be valuable (or essential)before long- for theanim al and moredif® cult for the termadm inistrationto con®rm the dose is doser, but isalso likely toincreaseany stress- reachingthe target organs. related background variancein the results . The®rst stage thereforesh ould be toget the Extraneous factors :Extraneous factorsc an animals accustomedtobeing handled intheir also have an effectonth eexpected pharma- newsurroundings. M any speciesbecom e cologyortoxici tyof asubstance. These familiar withindivid ual people,so it is include variations int heen vironmental helpfulfor thestaff whowill be carrying out temperature, thenum ber and =ordensity of thedosi ngtodevelopthis familiarity animalshousedt ogether, theirdietary status beforehand. Theim portanceof thisfor dogs and thetim eof dosing inrelation toadiurnal and primates isrec ognized but otherspec ies rhythm usually controlledby thelight =dark suchas ratsand micewill al sobene® t. Dur- cycle(Friedman &Walker 1972, Weihe1973, ing handlinganimals can thenbe accus- Rao 1986, Wollnik 1989, Waynforth 1995). tomedtobeing heldinthepositionfor dosing sothat any stressis m inimized whend osing doestake place. 2.1.4 Theanimal Wheresubstanc esare administered infre- quently but ona long-term basis (e.g. dietary Speciesand strain :Whenselect ing the toxicology inrodents )handling thean imals speciesand strain ofanimal, welfare factors duringroutinedaily husbandry willhelp shouldbe consideredas wellas theneed t o reducestresswhen subsequen tly dosing satisfy scienti®c goals. Importantpoints to them.Ingeneral, staff should be encouraged considerare whetherthereare any contra- tohandle animalsas muchas possible.T here indications about usingany individual spe- are exceptionst othis general rule, e.g. with ciesor strain ofanimal, whetherth eanimal neonates, orifhandling islikely tocom- isappropriate for therout eand thedosing poundthe effec tsofprocedures. compound, whethersatisfactory husbandry Somespeciesof animal can be trainedto can be provided and whetherthe anim al is cooperate with theh andlerd uring handling easy tohandle. Somestrains ofcommon and dosing and thiscan furtherreduc edis- laboratory speciesare moredocil eand easily tress. Training-by-reward (positive reinforce- handled and it may be bene®cial toselect ment)can encourage animals toparticipate suchastrain toreduceunnecessary stress. inthe ad ministrationproc edure, e.g. pri-

LaboratoryAnimals (2001) 35 6 aoaoyLaboratory

Table 2 Welfare impact of dosing routes Animals Welfare Route No. of doses Restraint Comments impact (2001) Intra-articular S Anaesthesia Can damage the joint; sterility essential; use once only Intracerebral S Anaesthesia Technically dif cult in neonates. Death can result from poor technique, volume or 35 nature of material, or from rejection by dam Intradermal R M Good technique essential to ensure injection is not subcutaneous Intramuscular R M Irritant effects can be a serious problem. Possible damage to nerves. Avoid injecting into fascial planes or blood vessels. Effects of large-dose volume and tissue damage are hidden. Care with adjuvants. Consider injecting into different sites for successive administrations Intranasal R M Not easy to ensure complete dose is in the nostril. Adverse effects unlikely, but care with uids Intraperitoneal R M Irritant substances cause severe problems. Easy to misplace dose into an organ but not easy to know it has happened. Not recommended for animals larger than rodents Intratracheal S Anaesthesia Can result in death if substance is administered wrongly or material is irritant Intravaginal R M Can be dif cult to retain substance in vagina Intravenous, non-rodent R M Bolus effects on central nervous system (and probably heart) are relatively common Intravenous, rodent R M Might need to warm animal to dilate vein; this should be done with care. Rapid injection can result in a bolus reaching the central nervous system or other organ and being fatal Intravenous R Tethers, jackets Aseptic technique critical. Surgery may be required. An expanding eld; with new infusion techniques being developed Oral: Joint In food=water R None Dose may vary with food=water intake. Dosing in food causes little stress but unpalatibility can restrict intake; possible distress from thirst. Knowledge Working of feeding behaviour important Capsule R M Easy administration in dogs and primates and failure is very unusual. May

be used to aid administration of tablets Group Gavage R M Accurate placing of tube essential. Failure should be rare but death can occur in rodents if tube misplaced. Restraint can be stressful for primates non Osmotic pumps Continuous Anaesthesia, Avoids multiple injection e Re then none nement Administration of substances 7 . s e c

r mates willpresent an arm (Reinhardt1991, u o

s 1997). e r Onrare occasions, for exampleone-off e t a i

r dosingwhent hereis not timetoaccustom p o l

r theanim al tohandling orto theproc edure, it y a r p i t e p e r

n v may be best toconsidersedating theanim al a a e m e h r s t s e

b i before dosing. However,sinc ethisinitself l e e r w b d l a f o a n willrequire both restraintand adosingpro- f r s a a p m t i m s

s cedure, thead vantages and disadvantagesof n a e e e t l c a b s n b l n

f e sedation must be carefully weighed. e o o a a c t o i r t t e t n s p n a p d n b t o e e e s u i m r s e s t d s b o a a n c e t f

e 2.1.5 Thetechnique u l s u s s n l d e t l o i a t a n a d s t w b n a i t Therem ay be practical dif®cul ties with m = r a m e i a g l n i r d t i u h n n a a i e i n r q c

a v individual techniqueswhic hcould compro- i e i n r d A i I m u f n b n j n . a . o l h o r h s a d s

r misethescienti®c goals orthewel fare ofthe c t c u t e f a i e f n e g y n t c t t i n o l h b n

i e animals and theseneed to be recognized in i t i a t r t u i t a c p r n s s o a t i e u p w s e r advance,and avoided.For example,there u a o s e r r t e r e i e e h r o d T r r b t n c b e

r may be alimitonthe m aximumdosevol ume . a i a u n a l t h i c n e r c i t r h n a i ; h a i a

s c t e ordegreeof absorption ofasubstance, ora m f c w d a e g o o s r t s s n r y t a i g r f u r a

s o procedure(suc has oral gavage)may demand n o e a m b e t d i l g v r u s a s , u s

n e r q considerable technical skill. d e s y i i e v e a s e a i l n n r b e r l i s o h d u y m

s It isimportant tostudy atechniquebefore o a d c t d e r e c l e e e c f T t e u t v t u c

r carryingit out and toobtain information i o . o u p e q l s u u c i s b e h s e e u n t

c = s f y

a from theliteratu reand orfrom othersexpert r h y h s c r t u e s s o d c a u a a f j a e e a m h c

t inthe tech nique. Particular factors tocon- r n e o t f i e t e t r t o s s o i s e b t a l

s e l sider are whether: thetec hnique,as opposed t d l u u l u t u e a n o l q f m u e i i c e i v i f n r u u s n g a i

t tothesubstanc e,willhave any effectonthe s i n s c o e r r o s a n s s a t h r a d e a a s m c t p f r r p

i animal; theequipm entto be usedisthemost e h e e t e d o l y D S R C R T n g h n a

t appropriate; thefrequencyofadministration n a i , a s e f e c

o could be reduced;and theen d-pointsare ˆ d n S , y a ) t t ; s i

e s clearlyde®ned and humane. r g k v , b e s i n i s i s v u r r s s Atthisstage,considerwhethe rthereis any a e s a t s o v l r e l i n d n e h i

a o a way thetech niquesor proceduresc ould be - t t h c d h a n t T c , n e o o s .

s further re®ned. e r p n t n e g t e r o t , e r n r a u i s e s k o s t e d c e ˆ g s h u k e n a t b e n R j c i c o i o r i u l r ; o 2.1.6 Staff and training w u r N T S M D M g ( e q p n h i , d t s t e e Thebest way toensurem inimal discomfort o r f n h i i t d o a u f r t f for animalsduring proceduresi stocarry q t c o o s e a r e e d p r theseout competentlyandef®cient lyÐthe c e o l i n m b i r a e i

e severity ofevena simpleproc edureis y e r m p i a h e t n f p i m increased ifit ispoorly performed. It is the e x o i m R R R R R R a t e r i , s b s

l responsibility ofall facilitiesto ensureth at n r e u a a e f h f t n m g e

s animals are only held, restrained,dosed i r u n e a i h e a r p g r m and monitored by thoroughly trained and n y u t n l e o u a d t s o o n d . s t ( n o competentstaff. Thereshould be enough y o g d n . s . p i k e e e x g s s a g . s

: competentstaff available at allstages . r e a a e n t t o l i b s c : y r r n m s a ) t e a o d

a o Theselec tion ofstaff tocarry out proce- u r c e l p : c s m n n t o a l l o s r y u o o ) a m a p b a r e i i e c i

t e duressh ould takeinto accounttheir skill in t t u o s n d o m m t h e i n t a a i i n l a s l l l l T e r a t a t g o o

: handling theanimals competentlyand sym- a a a a r p d s i u s n h c c m h h i i i a M o c r e p r r n n e t s p p b ˆ W I I o

( pathetically,as wellas inperform ingthe o L o e o o u c R S T T M N S technique.Staff training and adequate

LaboratoryAnimals (2001) 35 8 Joint Working Groupon Renement supervisioninthe learn ing stages isvery available tohelpand answerquestion sif important. Theessen tial components necessary. (In theU K,supervisionisgen- include: erally required underth epersonallic encing system and shouldbe discussedwith the Animalhandling :Training int hehandling local HomeOf®ce Inspector. ) ofthespeci esto be dosedi simportant since sensitivean dcompetenthandlin gand Maintaining competence :All staff should restraint minimizes animal distress.It also maintain theirown level of competenceor helpsensure con® denc eand dexterity in allocate theworkto apersonwho is c ompe- carrying out theproc edurewh ichfurther tent and usesthe tec hniqueregularly. Thisis reducesanimal distress. particularly applicable tothosewho only performa procedureoc casionally orona Useoftraining aids :Videosand educa- small number ofanimals. Consistenthigh tional computersoftware provide auseful standards can be ensuredby procedures resource(seeZin ko et al.1997),preferably always beingcarried out by experiencedani- watchedorworked through withan appro- mal housestaff ratherthan by scientists who priately experiencedperson.Preserved or are lessskille dinsuc htechniques. Where recently killedanimalsand animal models, thisisnot an option,re-trainingmay be suchas theKokenrat and rabbit, can be used necessary andvideosan dmodelsare parti- toassist study of anatomyand theprac ticeof cularly usefulfor this purpose.T herem ay technique. Particularly usefulare models also be aneedfor further supervision. developed by MoredunIsolators Ltd (Pent- Inthe U Kattendanceonan accredited land SciencePark, BushLoan, Penicuik, trainingcourse,toget herwit hsupervision Scotland EH26 OPZ). until competent,isa necessity,with additional training for surgical and otherspec ia- Practicewith inanimate objects : Objects lized techniques. Practicingtechniqueson suchas fruits can be usedto helpgain living animalsprotectedunderUK law isnot familiarity inhan dling andusingneedles, allowed. syringes and otherequipm ent,e.g.try satsumas for subcutaneousinjec tions. Awareness:Staff shouldhave adetailed knowledgeofwhat isbeing doneto animals Observationand assistance :Thetrainee intheir c harge and whenit isbein gdone. shouldobserve and assist inthe relevant Theyshouldkn owth eend-points and techniquesas muchas possible priorto car- severity limits oftheprojectand be able to rying out aprocedure. Thisis an essential recognize iftheseare exceeded. Theyshould part oftraining, and its importancecannot be also have thedelegated authorityand skill to over-emphasized. It may also be necessary killanimalsifnecessary. Informationon who and helpfult ovisit establishmentswith togo tofor advice(experiencedlaboratory expertiseina particular technique. animal scientists, animal technologists, the veterinarian attachedtotheestablis hment) Supervision :Onceauthorized tocarry out a shouldbe easilyavailable toall staff who proceduret he`trainee’ should initiallyassist need it. and thencarry it out underthe direct super- visionof an experiencedmember ofstaff 2.2Technical preparationand aftercare until suf®cient experti sehas beengained to Beforec ommencing dosing :Before sub- avoid mistakes.Supervisorssh ould explain stancesare administered toanimals for the fully what they are doing and pointout how ®rst time,apilot study witha small number and wherethi ngscan go wrongtohelpt he ofanimals shouldbe consideredif thereis traineeavoi dproblems.Theyshouldenc ou- any risk ofadverse effects. Always: rage thetrain eeto ask questions.Even when thetraineeh as becomefamiliar withthe Make sureyou are doingwhat you think technique, thesupervi sorshould always be you are doing,i.e. checkthat both the

LaboratoryAnimals (2001) 35 Administration of substances 9

substanceand its vehicleare stable and dosingsubstance, equipmentan dtechnique) pure;chec kthelabels onthe subst ance that can have signi®cant effectsonth ewel- and thecages oranimalstoensuret he fare oftheanimal andthequali tyofscienti®c correctsubstanceisgiven tothec orrect data and theseare identi®ed below. animal at thec orrectdose. Ensurethe nec essary equipmentis to hand,cleanor sterileif necessary,and workingproperly. Thesubstance Con®rm the ti meplan andexperimental protocolto ascertain thenum beringof (i) Irritancy=pH:Thesubstanc emay cause animals ineachdoseg roupan dthet iming irritationand/ orulc erationof skin, ofany additional investigationsrel ated to mucousm embranes orm uscles,or dosing (e.g.urinec ollectionor blood destroy tissueloc ally (e.g. theendothe- sampling) and have thenec essary recep- liumof blood vessels).Thism ay not tacle(s)ready and pre-labelled. be easilyvisible depending onth eroute Checkthesex,ag e,weightand condition of administration,but may cause ofeachanimal. seriouswelfare problems.Irritationm ay be aparticular problemwit hthe Animals shouldal ways be approachedand intraperitoneal,ocular, inhalationand handled ®rmly,quietly andsympathetically, intratracheal routes. and reassureddurin gtheprocedure. Only the minimumrestraint necessary shouldbe Always checkbac kground data onthe used. substanceand its effects. Therapeutic Consider whetheran anaesthetic,sedative data may helpassess the local effect oranalgesic isrequired and whetherother of asubstancepriort odosing. procedures(e.g. weighingor palpation)can be Solutionsfor injection shouldideally carried out at thesam etime.Ing eneralani- be closeto neutral pHsin cehigh or malsshouldbe weighedbefore dosingso that lowextrem esare not tolerated by thecorrectdosecan be calculated for the tissues.Waynforth and Flecknell weight, but ifhandling themtwic eislikely (1992)recommendaworkingrange of tobe stressfulthey shouldbe weighedand pH4.5± 8.0. Theorder of degree of dosed at thesam etime. toleranceof pHfor different dosing routesis: oral > intravenous > Afterdosing :Animalsmust be closely intramuscular > subcutaneous. monitored after dosing. Staff must always be Note that thepHal oneisan insuf®- prepared todeal withboth expected and cientguide toirritant potential since unexpectedadverse effects. Therem ust be irritancyalso dependson t hec on- adequate arrangementsto coverthe possibi- centrationan dionizationpoint (pK) lityof animals becomingdistressedovernight of thecompound. orat weekends. Ifanimals die as aresultof Someeffects, e.g. acuteskin or theprocedurea post-mortem examination endothelial damage, willbe apparent shouldbe carriedout toascertainthecauseof eveni fthesubstanc eisapplied to death and toavoid thesituationrec urring. recentlykilled animals socheckthis ®rst, thenc onsider assessingthe Repeatdosing :Repeat dosingshould be effectonan anaesthetized animal. carried out at approximately thesam etime (ii) So lub ility:Particulate matter may be eachday toavoidvariability associatedwith presentin the m aterial being injected or circadian rhythms. thesubstanc emay fall out of solution resulting inform ation oflarge particles. 2.3General re nement for all routes If administrationis intram uscular this Thereare someopportunitiesfor re®nem ent can be very painful andcan prevent withmost procedures(e.g. withrespect to the absorption.

LaboratoryAnimals (2001) 35 10 Joint Working Groupon Renement

Ideallyusebioc ompatiblestable Stabilityof substancescan be solutions. improved by microencapsulation. Before dosing, checkwhetherth e Thisc an also be usedto separate formulationi saqueous oroil based incompatibleorreacti vesubstan ces, andwhetherit isstable orlik ely to toprovide controlledand sustained fall out of solution.If thereis any release,or tomask bitter tastes chanceof particulatematter being (Melnick et al. 1987). presentorof thesubst ancecoming (vii)Temperature :Injection ofcold sub- out ofsolutionthen a ®lter (e.g. stances,e.g. straight from therefrigera- micropore)can be included inth e tor, can caused iscomfort and shock. injectionl ine.However, not ethat test material can be ®lteredout or Warm substancesto room,orbetter adsorbed onto the® lter and thedose still, body temperature immediately reducedas aresult. before administration. (iii) Biocompatibility :Substancesthat are not biocompatible may causet issue Useofantiseptic solutions damage. (i)Athickhair coat preventscontactof the Use in vitro techniquesto identify antiseptic withthe skin. Excessive adverse effects, e.g. haemolytic and application ofantiseptic solutionsto cytopathiceffects, ifappropriate. theskin may causeloss of body heat through evaporation, especially insm all (iv) Viscosity:Highly viscousli quids may mammals. causediscom fort and are dif®cult to injectrequiringalarger needlesize. Part thehair, orit may be necessary toclipthehair, from thei njectionsite Try not touseviscous substanc es. toallow antiseptic preparationsto workeffect ively. Usethese prepara- (v) Ste rility:Contaminated substancesc an tionswith care. causeinfec tion and causeirritati onat thesite of injection.Thisc an lead toself- mutilationand, at worst,the deat hofthe Injectio ns animal. Needle-free injectors are becoming more widelyavailable and theirusefor adminis- All substancesfor injection shouldbe tering substancesto animals shouldbe sterileÐautoclavingandmicro®ltra- encouraged. tion are usually themost practicable ways ofachievingthis. Useasept ic (i) Needle s:Insertionof large needlescan be techniquesthroughout inorder to painful and causeun necessary and minimize theri sk of introducing excessivetissue dam age. pathogensint othebod yat thetim eof dosing. Askinanaesth etic, e.g. EMLA(Astra Pharmaceuticals Ltd, HomePark, (vi) Quality, standardizationand stability of King’s Langley,Hertfordshire, UK) thesubstance :All ofthesefac tors can cream (Flecknell et al. 1990) can affect experimental results. reduceth epain of needleinsert ion. Checkthecompoundis pure.Avoid EMLAneedstobe applied 30±45 min contamination withoth ersubstances priort oinjectionto have full effect. ormicroorganismsand avoid varia- It isi mportanttobalancethesize of tion eitherin batchconstituentsor in needlen ecessary for easeof injection thephysic al and chemical form of the ofthesubstancewithth at whichwill substance(e.g.differingsalts).Note causem inimal pain,i.e. usesharp thebatc hnumber for future reference. needlesof thenarrowestgauge con- sistentwithef®ci entac curate dosing

LaboratoryAnimals (2001) 35 Administration of substances 11

Table3 Needlesizes for administrationof substances

Species Intradermal Subcutaneous Intramuscular Intravenous Intraperitoneal

Mouse 27G 25G 27G 26 28G 25 27G Rat 27G 25G 25G 25 27G 23 25G Guineapig 25G 23 25G 25G 25 27G 23 25G Hamster 25G 25G 25G 25 27G 23 25G Rabbit 25G 21 25G 25G 23 25G 21 23G Ferret 25G 21 23G 23 25G 21 25G 21 23G Dog 25G 21 23G 21 23G 21 25G 21 23G Cat 25G 21 23G 23G 21 25G 21 23G Large primate 25G 21 25G 23 25G 21 25G 21 23G Small primate 25G 23 25G 23 25G 21 25G 21 25G Sheep 25G 19 23G 21G 19 21G 19 21G

Adapted fromWolfensohn and Lloyd(1998)

(seeTable 3).Thegauge dependson thesyringe, withdraw theneed lea thetough nessof thetissuesto be short distanceandredirectit. The penetrated (especiallyskin)whichin pain receptorsare intheskin andso turn dependson th especiesan dthe this isless painful than withdrawing site. Then eedlebore isdetermined theneed leandre-injecting.After by thevisc osityof theform ulation injection withdraw theneedleslowly andthespeedof administration;the but ®rmly. needlelength depends onthedepthof injection. (iii) Le ak age :Fluid may leak from thesit eof Change needlesbetween an imals to injection reducing accuracyof dosing. avoidtransfer of infection. Ensurea sharp needleisalways used. It may be better toinjectat adistance Considercat heterizing thevein if from theen try site usinga longer repeat dosingovera shortperiod. needleand topushth eskin toone side before entry sothat askin seal is (ii) Accuratepositioning and injection formed. It helpsto massage thesite te chniq ue:Severeadverse effectscan be after injection todisperseth esub- caused ifneedlesor gavage tubes are strate, and also toapply pressuret o incorrectly placed, e.g. an intramuscular thesite after injection. doseinjec ted intravenously can be fatal. Tissuesc an be damaged ifinjectionsare (iv) Bleeding afterinjection :Sitesof injec- made clumsily. tion may bleed especially after intra- venousdosin g. It isessential tostudytheanatomyof theanim al,e.g.thejoints for intra- Apply gentlebut ®rmpressure with a articular dosing; theleng thofthe swab untilthebleed ingstops. Wipe oesophagusfor gavage,thepositionof tracesof blood away toprevent nervesfor intramuscular dosing. excessivelic king orgnawing at the Accuratemeasurementof depthof injection site. insertionc an be made usingstops or marksonn eedles/tubes. Insertn eedles =tubes ®rmlybut gently Volum es=Sizesofcapsules and depressplung ergently.Oncea needlehas beeninserte dwithdraw (i) Large volumes :Administrationof large thesyringe plungert oensuret he volumescan be dif®cult inprac ticeand needlehas not entereda blood vessel can causeadverse physiological effects, unintentionally. If blood has entered compromisinganimal well-being.

LaboratoryAnimals (2001) 35 12 Joint Working Groupon Renement

Table4 Guidelines:maximum volumes for commonroutes of administrationin the common laboratory species

Routesand volumes

Oral (ml=kg) ip iva im (ml=kg=site) scb idc (ml=site)

10 10 5 0.05 2 5 0.05 0.1

The essentialrequirement when determining dosing volumesis that the volume givenshould minimizeany discomfort or welfareproblem and should not causeany physiological or pathological change which compromises theexperiment. As far as an animal isconcerned thelower the dose volume thebetter Mostcurrent guidance givesdifferent volumes for different species but in mostcases there are no physiological reasonsfor doing so.This was borne in mind when drawing up thistable such thata single guideline volume wasconsidered to be applicable tomostspecies for any given route. When determining volumesfor less commonly used speciesand otherroutes the anatomical and physiological characteristicsof thespecies must always be considered

Notes: sc subcutaneous; ip intraperitoneal;iv intravenous;id intradermal;im intramusculariv a Bolus injection carriedout ˆ overa relativelyshort ˆ period of time(approximately ˆ one ˆ minute); sc b Volumeˆ depends on theˆlooseness of the skinof the animal (and thereforethe potential subcutaneous space). Multiplesites ˆ can be considered foradministration of greatervolumes, although when repeateddaily administration is intended, four sitesshould normallybe considered the maximum. Voume does not include Freund’sadjuvant administration. Freund’s limited to 0.1ml CFA =site; idc Volume depends on thicknessof skin which varieswith site and species.Maximum number of sites 6; im Thisis the volume ˆ fora single site.The useof more than one sitein more than one limb maycause multiple limb lameness;ˆ ˆip Thisis not acommon routein dogs, birdsand primates ˆ

Themaximumdose l evelin t ermsof Injectthesm allest possible volumeof ¯uidvolumeand sizesof capsulesand adjuvant nomoret han100 ml per site tablets varies witht heroute and the (usec oncentrated antigen). species.T heessent ial requirementis Choosean injection site whereloc al toensurethe volum egiven causes pathological responsesc auseleast minimumdiscom fort and doesnot pain,can be readily observed and resultin physiological orpathological treated ifnecessary (e.g. subcuta- change whichwould c ompromiseth e neous). experiment.Asfar as thean imal is Uselong booster intervals(at least concerned, thelower th edosevolum e 4 weeks). thebetter (seeTable 4). Take advantage oftheantibody memory response. Minimize animal numbers by sequential ratherthan simultaneous Useofadjuvants to enhanceantibody immunizationsofanimals. pro duction Chooseanim al speciesth at yield large amountsofblood (e.g. sheepand (i)Many adjuvants can be noxiousto ani- goats). mals and producesigni®cant pathologi- Plan ahead and be patient;successful cal lesions.Most information onth e production ofagood antiserumc an ef®cacy ofdifferent adjuvants isanecdo- take 6monthsor m ore.However, tal soitcan be dif®cult toselectthem ost note that animals usedt oraise appropriate (Jackson& Fox 1995, Animal antibodies should not be kept for Welfare InformationCenter 1997, inde®n ite periodsof timewithoutt he Leenars et al.1997, Palmer et al. 1997). justi®cation ofan authorized scien- Thefollowingrecommendations are ti®c need. reproduced from Palmer et al. (1997 ): Chooseth eleast harmful adjuvant. Neonatal=Young animals Neveruse Freun d’s completeadju- vant(FCA)twiceinthesam eanimal. (i) Reje ction: Neonatal=young animals may Inthe U K,useof FCArequires be rejectedorcanni balized by themother justi®cation to theHom eOf®ce. after handling.

LaboratoryAnimals (2001) 35 Administration of substances 13

Be familiar withthe m aternal beha- dif®cult iesrelatin gtothesubstanc e, the viourof thespec ies. techniqueand theproc edureas awhole. Usegloves whenhandl ing young Abrief summary ofeachtechnique, suf®- animals to mask odoursan davoid cientto identify theproc eduresdescri bed, is mis-mothering. Handle all theyoung given withfull descriptionsprovided only ina littersothat them othercannot wheresuch inform ation isnot easily avail- discriminate betweent hem. able inotherpublic ations (seePaget & If appropriate tothespec ies,rem ove Thomson1979, Kirk &Bistner 1985, Poole them otherbefore returningthe 1987, Rollin& Kessel1990, Waynforth & young. Put themin the fron tofthe Flecknell1992, Tuffery 1995, Wolfensohn& cage andmix withbeddingto mask Lloyd 1998). odd scents, thenreturn t hem other. Minimize disturbancewhenobser- 3.1Intra-articular vinganimals. Useofthetechnique Thismethod isusedfor local dosing into the Restra int joint space(normallythesti¯e joint)eitherto checkthe ef® cacy ofdrugs intendedfor (i)Restraintcan be as, oreven m ore, clinical treatmentof joint disease, orto stressfulthan theprocedurei tself, parti- model joint diseases. Thetec hniquehas a cularly for animals that are not usedt o clinical application inveterinary thera- being handled.Periodsof restraint in peutics(Kirk 1980). tubes orstocksare liabletocausestress. Thereare particular problemswith inhalation restrainttubes; iftheseare not Sum mary oftheprotocol thecorrec tsize and shapefor theanimals Thetechniquerequires the anim als to they can partly turn around, and are remain very still.Short-term anaesthesia for likely tobecomedistressedand may die. restraint ispreferred in al lspeciesand is essential for rodents, rabbits ordogs. Inlarger Usetherestrain tprocedurem ost species,inject ionsare doneusing loc al appropriate, i.e. least distressful,to anaesthesia at thesite ofneedleinsertion, theanim al and of minimumduration togetherwith adequate restraintorsedation (Poole 1987, van Zutphen et al. 1993, tostop theanim al moving at acritical Tuffery 1995). moment. Habituationand/ ort rainingof the Thehair overth ejointshouldbe clipped, animal may helpreducestress(see thesti¯ e jointpalpated toestimate the Section2.1.4 ). positionof thejoint spaceand theneedle Ensurestaff are skilled at getting (without thesyringe attached) inserted animals to relax whenbein gheld; throughtheanterior aspectofthejoint ,i.e. restraintshould be ®rm,yet sensi- throughoradjac enttothepatellar ligament. tive. Theneedleis then c onnected tothesyrin ge Ensureequi pmentsuchas restraint and thesubstanc einjected.Locationwithin tubes are thec orrectsize and shape thespac eisfelt as asudden forward move- for theanimal. mentof theneed leafter ithas penetratedthe Neverleave restrainedanimals skin and thecapsuleof theknee joint. In unattended. larger animals, synovial ¯uid should ¯ow back along theneedle. 3Renement for individualroutes andprocedures Potentialproblemsand re®nement Thetechniquec an be very painful and can Recommendations for re®nem entare pro- causedam agetoarticular surfacesif not vided for eachofthecommonroutes taking carried out withgreat care. However,proper into accountpotential problemsor attentiont oanatomyand aseptic technique

LaboratoryAnimals (2001) 35 14 Joint Working Groupon Renement shouldm inimize thein cidenceofadverse Asa scienti®c procedureon an indivi- effects. dual animal, thistechnique shouldbe usedonc eonlyandona singlejoint . Incorrectplacementof theneed lemay damage thesurrounding tissues and cause 3.2Intracerebral (intracerebro-ventricular) pain. Even whenthe needle is correctly inserteddamage can occur. Useofthetechnique It isessential tohave agood knowledge Thistechnique isusedto deliverph armaco- oftheanatom yofthejoint . logical agents tothec entral nervoussystem Usea needlesize as small and ®neas eitherwhere t heblood-brain barrierm ust be possiblewit hrespecttothespeciesand crossed,orto avoiddirectsystemic effects. thejoint ,e.g.26G 63=8"for rats. It is Intracerebral injection isalso usedto isolate unlikelytobe necessary touseneed les orassay microorganisms. greater than 21G evenin larger species suchas thedog. Sum mary oftheprotocol If theanim al moves,th eneedlewill Therouteisused in neonatal and adult damage sensitivet issuesin andaround the rodentsand neonatal chicks. Inadult rodents joint. Thiswill be painful and may cause astereotaxic frameisused for accuracyand lameness. tominimize tissued amage. Ensurethe anim al isim mobile when carryingout theprocedure. Usegeneral Neonatalrodents and chicks anaesthesia. If thisis not appropriate, Useasmall bore, lowvol ume(lesst han accustomt heanimal totherestraint 0.5 ml)plastic syringe witha27G needle. required soth at it relaxesand remains Draw theinoc uluminto thesyrin geand immobile duringinjection. checksm oothoperation of theplunger Monitorthe anim al for lamenessafter (avoidingaerosolg eneration)before picking completingtheprocedure.Gi vean alge- uptheanim al. sicsun lessthis willseriousl ycompro- Mice, rats and chicks shouldbe nomore misethe aim softhestudy. than 3days ofage. Holdtheanimal gently on an absorbent towel =pad ona ®rmsurfac e Failure toapply good aseptic technique withthe dorsal surfaceoftheanim al held will resulti nasepticarth ritis. Thisc an uppermost betweenthe gloved thumband causesevere lam eness,heat and swelling fore®nger. of thejoint and isextremely painful. Insertthe t ipof theneed lein tothebrain, Steriletech niqueis essen tial: swab the usually from thefront, at an angleof 45 , in skin withantiseptic,wearsterile gloves thearea of theanterior cranial fontanelle and usesterile syringes and needles. (top, midline)and gently depressthe plunger Injectedsubstancesm ay distendthejoint ofthesyringe toexpelthe dose. Returnth e capsulecausing pain. animal toits mother, checkfor adverse Avoid over-distensionof thejointwith effectsafter oneh our(to avoid toom uch excessivevolum esofsubstanceÐwh ere immediate disturbance)andmonitor closely possible,rem ovean equal volumeof at least onceperday post-injection for synovial ¯uid before injecting.The adverse effects ofthetechnique orthe maximumvolum ethat can be ad- inoculum. ministered(assum ing aspiration of an Thereis d ebate overwh etherth euseof equivalent volumeof synovial ¯uid) is anaesthesia for animals ofthisage wouldbe 15 mlfor rats (Waynforth&Flecknell appropriate, sincethis initself can cause 1992); 200 mlfor rabbits; 1mlfor dogs. distress whichh as tobe balancedagainst the stressof intracerebral injection. However, Them oret imesthe joi nt ispenetrate dby modernanaesthe ticscan be given safely by repeat dosingtheg reater therisk of competentpersonnel and topical anaes- introducing an infection and oftrauma. theticsshouldnot be aproblem.Theper-

LaboratoryAnimals (2001) 35 Administration of substances 15 ceptionof pain inneonat al animals has been gain experienceinhandling them thesubjectofconsiderable investigation and b efore startingprocedures.To perform advancein t helast fewyears. Insom espe- theprocedure hold theanim al gently cies,inc luding rats and humans, thed es- but ®rmly.It iseasiest todo this by cending inhibitory ®bres whichmitigate the restingthean imal onan absorbent feelingofpainand raise thepain thresholddo towelon a ®rmsurfac e. not developunti l2±3 weeksafter birth.Some Practisem inimal depressionof syringe neuroscientists nowbeli evethat neonatal plungersusi ngfragile inanimate objects animalswithim mature nervoussystem sare (suchas arti®cial sponges)wh ich morel ikely tofeel pain than older animals. mimic thetext ureand thepressure Thecurrent non-use of anaesthesia invery neededto expel¯ uids. young animals ispart of outdated wisdom Keepvolum esof inoculumin th e based on`sm all things donot feel pain’. It syringe small. Finec ontrol isnot then may also resultfrom usingmethodsof reducedby stretchingthe® ngerto restraint whichare relatively sopowerful reachtheplunger. that weeith erd onot realize theanimal is Incorrectplacementof theneedle, (e.g. struggling, orput thisdownt oaresponseto therestraint alone. insertionfrom therear ofthebrain, from thewrongang le,ortoo deeply) can kill the animal. Adult rodents Knowprec isely whereto inject the Astereotaxic frameisneeded t oensurethat animal and towhat depth.It may be therei snolateral movementof theneedle possibleto put stopson the needle or whichwould c ausetraum a. Anaesthesia is mark theneedlein som eway toassess essential for thisas isaseptic techniqueand depth. sterilem aterial. Injectionvol umesshoul d not exceed2% ofbrain volumeandshould Needlesizes g reater than26G cause always be given slowlyenoug htoavoid excessivedam age tothebrai nofneonatal increasingcerebrospinal ¯uid pressure. animals, andmay kill them. Penetrationof theven tricleis readily asses- Donot usen eedlesgreater than27G . sedby thesudden reduction of backpressure Avoid inoculating viscoussubst ances intheinjec tion line.C hronicplacem entof whichwould require larg erneedles. cannulae allowsin jections, orconti nuous Injectionof an excessivevol umecan intraventricular infusion,without needfor distend thebrain and skull. repeatedanaesthesia. Donot injectm orethan 20 ml in neonatal rodents. Potentialproblemsandre®nements Alternative methodsofobtainingtheresul ts Substanceswith hi ghproteinc ontent may required shouldalways be consideredbefore causethe death of theanim al. usingthisroute. Theprocedureis not easy to Always checktheproteinc ontent of the performand them ost commonad verse substancetobe administered andavoid effects resultfrom clumsytechnique. theintra-c erebral route iftheprot ein contentishigh. Expellingsmall volumesof inoculum gentlyfrom thesyringe isdif® cult. The Occasionally them otherrejec tstheyoung skull ofneonatal animals isvery thinand whenthey are returnedtoher.If thereis fragileandcan easily be damaged, as can blood leaking from theinjec tion site she thebrain, withhaem orrhage occuringat may eat them. thesi te ofinoculation. Movementof the Minimize disturbanceof thelitter after animal duringdosing willresult i nbrain inoculation.C heckafter half an hour, damage. onehour, and daily thereafter. Any Neonatal animalsrequirevery careful, animal showingunexpec tedneural gentle handling and it isessent ial to de®cits, e.g. ataxis orparalysis, within

LaboratoryAnimals (2001) 35 16 Joint Working Groupon Renement

the® rst 24±36 hshouldbe humanely Depresst hesyringe plungeras gently as killed as this ism ost likely to possible.C on®rm c orrectlocationby be due tofaultytechnique. Seealso thebleb that results from the¯uid Section 2.3 (Neonatal =Younganim als). injectionparting thecutaneouslayers. Thedensity ofthesk inm ay make needle 3.3Intradermal insertiondif®cult . Useofthetechnique Usethe sm allest and sharpestneed le Intradermal injectionis a technique com- that willpenetrate theskin (27G for monlyusedin stud iesof in¯ammation, sen- rodents; 25G for dogsand primates). sitizationand cutaneousblood-¯ ow Anintraderm al injection, evenwithout diagnostics, and inim munology. Often the in¯am mation,can caused istressif not objectiveis to administer aputativeantigen performedcorrectly. orin¯am matory mediator andlook for a Consider theuse of alocal anaesthetic reaction (oedema, swellingorredness)whic h cream (e.g. EMLAcream). might occurrapidly orafter aperiod oftime Minimize adverse reactionsby splitting from minutest odays. thedose over a fewsites (but seebelow) . Donot injectan individual site more Sum mary oftheprotocol than once. Theinjection ismade into theouterlayers of theskin,usually onthe bac k.Thedorso- Theresponsefrom multiple injection sites lateral skin shouldbe closely shaved with may coalesce. electric clippers(avoi dingany skin damage) Them aximumnum ber of sites should at least onehour before injection. Theneedle not normally exceed6. shouldbe heldal most parallel tothesk in Ensuresites are suf®cientl yfar apart to surface and advancedcarefully afewm illi- avoid coalescence.Consider usinggen- metresi nto theskin. If thereis asudden loss eral anaesthesia, particularly ifmore ofresistancetothepassage oftheneedle, thi s than 6sites are required. isusually due toitpassing subcutaneously. It Whenc arrying out multiple injections must be withdrawnan dre-introduced. usea statistically designedLatin square Rotating theneed leinthe inj ection site just system toaccountfor regional varia- after insertionand immediately priorto tionsin skin thickness. withdrawal helpsm inimize leakage ofinjec- 3.4Intramuscular ted¯uid. Asuccessfulinject ionwill result in theformation ofableb. Useofthetechnique Ingen eral, intramuscular injectionis usedas Potentialproblemsandre®nements aroute ofsystemicadministration.It is Sympathetic restraint isvery important. sometimesused in slow release studies Good technique isessential toensureintra- whereim plants oroily formulationsare dermal rather than subcutaneousadm inis- employed toprovide adepot from whicha tration. drug isgradually absorbed. It isal soused in testingvaccinesan dtoadministeranaes- Thevolumetobe injectedmay be large and thetics,particularly towildanimals via a thereis no natural spacetocontain it. dart gun. Injection volumeshouldnot normally exceed 100 mlpersite; 50 ml may be Sum mary oftheprotocol preferable insom einstances. Twopeople m ay be required, oneto holdthe It iseasy toinjectsubcutaneouslyinerror animal, theot hert oinjectthe m aterial. The instead ofintradermally. injection shouldbe made into asuitable large Good training isessen tial toensure musclem ass, appropriate tothepurpose of accurate injection. If indoubt clip or theproc edureand away from major blood shavesites c arefully toaid visibility. vesselsand nervesto avoiddamaging them.

LaboratoryAnimals (2001) 35 Administration of substances 17

Thiswill norm ally be intothelateral and thereis an ydanger ofthesubstan ces cranial thighm uscles(the vastus group) being irritant. ratherthan theham string musclesin th e hind limb. Theinjec tion site inbirds isthe Theinjec tion itself may causet hean imal thighor pectoral muscles;in ® shthe dorsal seriousdist ress. musculatureis used. Insuc hinstances,c ompletethe inj ec- Theanimal’s legshould be held®rmlyand tion as quicklyas possibleor, ifthepain theneedlei nsertedcarefully anddeliberately. reaction becomesviolent,withdraw the Thesyrin geplungershouldbe withdrawn needleto avoid unnecessary pain or slightlypriorto injectingtoensurethe needle mechanical damage. Theinjec tion isnot inablood vessel.T hec ontentsofthe shouldbe completed at another site syringe should thenbe expelledslowly, the whenthe pai nreactionhas subsided needlewithdrawn and thesite gently mas- and thean imal has beenc almed. saged. Re-assessthe nec essity ofusingth is routean dformulation.

Potentialproblemsandre®nements Nerves(e.g. thesciatic nerve)can be Intramuscular injectionsappear tocause damaged by incorrect placementof the morepain than injectionsat othersit es,both needle, orby areaction tothesubstance at thetim eand afterwards, as evidenced by administered. Traumanearto thesciatic temporary limping. Therei salso agreater nervec an lead toself-mutilationinrabbits. chancethat major nervetrun ks orm inor Thesubstancemay be inadvertently intramuscular nervesm ay inadvertently be injectedinto ablood vesselwithin the injured, as wellas injury tomuscletissue musclemass, and thereis also arisk of caused by thedistensi onin a closedfasc ial injectingthematerial intothefascial tissue compartment. ratherthan them usclemass itself. This Theroute shouldonly be usedif aless willintroduceexperimental variation. painful alternativeis im possible,or ifit is Movementof thepoint oftheneedle required clinically.Thepotent ial irritancyof whilst inthem usclecan alsocause asubstanceif injectedsubcutaneouslyor damage, e.g.whenwithdrawing theplunger intraperitoneally shouldnot be areasonfor tocheckfor penetrationofablood vessel. choosingtheintram uscular route whereit is Keepth eanimal very stillÐsensit ive equally likely tobe irritant.Before usingthi s restraint isessent ial, as isfam iliarity technique carefully considerwhat you are with anatomy, particularly ofthe trying toachieve,the li kelydamage, whether musclemassesand routesof nerves. therei san alternative, whetherthetech ni- Take great care toavoid nervesand que isa one-off, and whetherrepeat doses blood vesselswhenplac ing theneedle. willbe required. Thesciatic nerveis situated deepin the posterior(c audal)femoral musclem ass If irritant substancesare administered and along thelineof thebone, sofor intramuscularly theresul ting tissue preferenceinjectintothem usclemass necrosisat thein jection siteislikely tobe onthe anterior ofthethigh. Donot painful sincemusclesare continuallyused insertth eneedletoo deeply (beyond tomaintain posture.U seof adjuvants can 1.5 cminadult catsand2.5 cminadult compoundt heproblem as they provide an dogs). ongoing irritantfocus(seeSec tion 2.3). Donot useirri tant substances. Thevolum einjectedcan physicallydis- Clip theinject ionsi te sothat any local tendthem usclecausing swelling at the reactionscan be seen.In bi rds, the injectionsi te with associateddamage and feathersc an be dampenedwith an pain. Thisis aparticular problemfor small alcohol solutionandpartedÐdo not mammals. pluckfeathers (seealso Hawkins et al. Thevolum ethat can be comfortably 2001).Thisis partic ularly importantif accommodateddependson the spec ies

LaboratoryAnimals (2001) 35 18 Joint Working Groupon Renement

and thesh ape and size ofmuscles.D o callyupwards. Theori®ce of theadaptor not usethe t echnique for small animals shouldbe placedabout 1mmabove the (e.g.rats, miceor ham sters)unlessth ere nostril.Thepipette plungeristhendepressed are exceptional circumstanceswhen it sothat adroplet fallsinto thenostril .If the shouldbe usedwith extrem ecare. droplet failsto separatefrom thepipette it It may be better todivide larger can be touchedgen tly against thenasal ori- volumesbetween ,for example,hind ®cesothat it drains intothenost ril. and front legs, ortohave 4sitesand Twopeople are required todosedog s.One rotate around them.However,thi s holdsthe anim al ina sitting position,the must be balancedagainst thepotential other(th edoser)an glesthe dog’s head tocausem ultiple limblameness.Sites slightly (about 30 )above thehorizontal .For shouldbe separated by at least 4cm. application usea syringeor a spray pump Disperseaqueous substancesafter heldinthedoser’s otherhand and positioned injection by gentlem assage. 2±3 mminside then ostril,but not touching them ucosa. Thespray pumpisac tivated, With good restraint andafamiliar handler, usually onceortwic e,andthenrem oved. largemammals suchas sheepand cattle, Thedog’s head isheld sl ightly upwards for willnormallyremain still for injectionbut about 30 stoallow thetest material todrain pigs willnot. intothenasal passages.If avery small Usingabutter¯y needlewitha lengthof volumeisad ministered this may not be polythenetubing willm ake injection necessary. easieras theoperator can followthe pi g Anequivalent procedureis usedfor pri- roundthepenand injectat thesametime. mates, although for large OldWorld Feeding theanim al with somenuts primates, threepeople are required,twoto simultaneously willalso help. hold theanim al andonetodose. In® sh,i njectionc an dislodge scales. It may be necessary toconstructa restrainerfrom ablockoffoam rubber Potentialproblemsand re®nements orsponge.A `V’ shapelarg eenoughto Thetechniquei mposeslittle stressi fthe holdthe®shiscut inthesurfaceof the animal iswell t rained.Ifthe® rst attempt block. The`V’ islinedwith wettissues fails thetec hniquec an be repeated provided and thewhole bloc ksaturated with that thean imal remains relaxed and water(seealso Brattelid&Smith 2000). unstressed. Dosingis nearly always inaccurate since 3.5Intranasal thean imals often sneezewhen the dose is Useofthetechnique administered. Sneezingcan contaminate Intranasal administrationis used when otheran imals orst aff witht est materials. investigatingtherespi ratory tractas aroute Dosean imals away from otherst hat ofinfectionan dwhenst udyingpharmaceu- could be contaminated andallowfor ticals absorbed from them ucosa. potential inaccuracieswhen c alculating the dose. Sum mary oftheprotocol Iftheanim al movessom eof them aterial Thetest material isadministered by allowing may misst henasal ori®ce orthe pipett e small droplets from an automatic pipette or may jolt thenose and hurt theanim al. spray pumptofall intooneorboth nostrils. Train animalstoaccept restraint and Thetechnique can onlybe usedtoadminister dosing(e.g .withsaline )before com- small volumes(e.g. 50 mltorodentsand rab- mencing thestudy. Hold thean imal bits, 500 ml to dogs). very still,withthe ad ministration Smallanimals suchas rodents are devicevery closeto (i.e. within1 mm), restrainedby scruf®ng. Theym ust be still orwithi n,then ostril.Brief parenteral and relaxed withthe nostril spointingverti- anaesthesia =sedation may be preferable

LaboratoryAnimals (2001) 35 Administration of substances 19

whenad ministering asingledose or carefully (seeSec tion 2.3).Pilot studies whendosing small rodents. are advisable. Avery steady handisneeded for this technique. Practise thetec hniqueby Thisis a dif®cult techniquet operform dosing intoasmall containerand correctly, particularly insm all rodents weighing thedose del ivered. (Lewis et al.1966, Claasen 1994),because itisdif® cult toensuret hedose is adm i- 3.6Intraperitoneal nistered intotheperitoneal cavity rather thaninto theintestine,gut, urinary blad- Useofthetechnique der, muscleor otherorgan .Occasionally Intraperitoneal injection isused to adminis- blood vesselsm ay be damaged resulting in terrelativel ylargevolumesof soluble haemorrhage. substances,suc has anaesthetics, tosmall Good staff training isessential. To animalswhereth eseneed to be rapidly avoid puncturingtheabdom inal vis- absorbed and wheret heintraveno usor oral cera, introducethen eedlerapidly at an route isinappropriate. Thisisa common angle of 30 ,slightly left ofthem idline route for administering substancesto ®sh. umbilicus,about halfway betweenthe pubic symphysisand thexiphisternum Sum mary oftheprotocol (seeWaynforth &Flecknell1992).For Thetechnique involvesinjec tion through the rodents, thetechnique may best be abdominal wallintotheperit oneal cavity. It performedby holding theanimals with isdescribed indetail for rats inWaynforth thehead tilted downwards.Note that and Flecknell(1992 ). with thistechnique withdrawal ofthe For ®sh,intraperit onealinjec tionsare best plungerwill n ot usually be helpful as given antero-laterallytotheanus with the gutcontentsare tooviscousto be drawn depth ofpenetrationgaugedby maintaining a into theneedle. slight pressureon the syringe. Penetration shouldc easeas soonas the¯ uid injected Injectionof toolarge avolumeof substance startsto¯owfreely indicatingtheneedle’s willdistend theabdom enand causedis- presencein t heperi toneal cavity. comfort. Thevolumeofinjection dependson the Potentialproblemsandre®nements size oftheanim al andwhat itcan Thisroute shouldnot be usedrout inely accommodate.Use10ml =kgas aguide. (except for administrationof certain anaes- Repeated useof thistec hnique can cause thetics)as otherroutes are conveniently seriousstress because oftherestraint accessibleand usually preferredfor research required, thecumulative irritant effect, purposes. needledam age and thepot ential for Thetec hniqueis not recommended for injecting into abdominal organs. animalslarger thanroden ts, norfor pregnant Donot injectindividual animals more animalssincetheneedle can puncture the than onceperday. Thenum ber of gravid uterus. Neveruse this techniquewith subsequent injectionsneeds rigorous birds becausethe substanc emay gointothe justi®cat ion.Brief anaesthesiacan air sacs. make thetec hniquem orereliable and Substanceswhichare irritantcan be life lessst ressful. threatening whenadm inistered by this Whererepeat ed =continuousadm inis- route. Therec an be appreciablereaction in trationi srequired (for example, where theperitoneal cavity includingpain, for- thisi susedt oimitateambulatory mation of®brous tissueand adhesions. peritoneal dialysisi nhumans),con- Many non-aqueous solventsc auseswellin g sider whetheri twouldbe lessst ress- of edges of theliver lobes. ful toimplantanimalswith a Consider thepH, irritancy, toxicity and minipumpdosedel ivery system(se e compatibility of thesubstanc every Section3.11 ).

LaboratoryAnimals (2001) 35 20 Joint Working Groupon Renement

3.7Intratracheal necessary andwhetherit wouldbe satisfactory toadminister thesubstanceintranasally Useofthetechnique orby inhalationin stead. Intratracheal administrationis usedto study Thetechniquehas the potential todamage theeffects ofpoorlysolublesubstanc es therespirat ory tract and isnot appropriate for (e.g. dust and ®bres) orpoorly solubledrugs repeated administration.T racheotomy intheairways of laboratory animals. shouldbe consideredif this isabsolutel y necessary. Sum mary oftheprotocol Thetechniqueis usuallyusedwithhamsters, Puttingany foreign material directly into rats, guineapigs and rabbits. It involves therespiratory tract can causean adverse administrationinto thetrachea via theoral reaction. Irritant materials can causeser- cavity, orinto thelungsby tracheotomyto iousproblem s. allow for repeat dosing.Thereare anumber Be aware ofpHan dotherfac tors that ofreferencesdescribing thetec hnique inrats may affectirritancy. Avoidusingi rri- and rabbits (seeSedg wick1988, Waynforth & tantmaterials (seeSect ion2.3) . Flecknell 1992, Cambron et al.1995, Davies Thephysic al presenceofthesubst ance et al.1996)and primates (Morris et al. 1997). particularly as large volumesoramountsof Tracheotomyisnot coveredin t hisreport. solid can damage thepharynx orcause Theanim al shouldbe anaesthetized and local lungirritation. placedsupineon a padded table. Intubation Donot exceedm aximumvolum esof shouldbe carried out withan appropriately 500 mlfor rabbits and 40 mlfor rats. The sized tube (e.g. orogastrictube),the maximumpartic lesi ze ofsolids admi- unin¯ated cuffed endbeing guided into the nisteredshouldbe dependenton th eir tracheavia alaryngoscope.It isimportant to not impeding theairways oractingas a checkcorrectplacementby themovementof mechanical irritant. inspiredand expiredair through thetube. Thetest substanceisdrawn intoasyringe Thetongue can be damagedifnot handled whichi sthenattac hedto an intravenous carefully. Theph arynx =larynx can be catheter. Thisis th enplac edinsidethe damaged by thepassag eofthetube and endotrachealt ube (whichactsas aguide) and thisc an causelaryngospasm . insertedto thepoin tofbifurcation ofthe Take great care whenhandlingthe airways. Thiscan be felt quite easily. tongue and whenpassing tubes. Agood Analternat ivetec hnique whichcan be understanding of theanat omyof the usedfor rats, istoplacethean aesthetized animal isessential as isappropriate trai- animal insternal recumbency withthebot- ning(e.g. usingrecentlykilledanimals). tomjaw anchoredbeh indtheincisorsan dthe Lubricatethetube witha watersoluble topjaw raised by an elasticband placed sterilelubricant (Davies et al. 1996). A behind theincisors.T hism aintains awide local anaestheticspray can reducethe gape toallow accesst oand visualizationof chanceofinducing laryngospasm. thelarynx by meansofan adaptedcanine Monitor theanim al closelyduring the otoscope(Sedgwic k1988).A¯exible catheter recovery period and for upto3h can thenbe passed an appropriate distance thereafter. throughthe laryn xfor material tobe instilled. Shininga®breoptic light ontothenec k at thelevel of thelarynx helpsto trans- 3.8Intravaginal illuminatetheph arynx. Useofthetechnique Thistechniquehas limited applicationsin ce Potentialproblemsandre®nements itisusedtoimitate therout eofexposureto a Intratracheal administrationinvol vesgeneral pathogen(e.g. herpes,thrush )topharmaceu- anaesthesiaso before usingthis route think ticals orotherprod uctssuchas IUDs, tam- very carefully about whetherit isreally ponsor pessaries.

LaboratoryAnimals (2001) 35 Administration of substances 21

Sum mary oftheprotocol It may be usefulto dilate thevagina Viscous¯ uids and creamscan be adminis- with asuitably lubricated(and warm!, teredto rodents, rabbits, dogs and primates e.g. plastic)speculum.Thisshould be usingasterilesyringe insertedgently insertedgentlybetweenthe lips of the betweent helips of thevulva andadvanced vulva and thenceintothevagina. upwards and forwardsintothelum enof the Gentleclosureof thehandles of the vagina. Anoral dosing catheter cut downto speculumwil lcausedilation ofthe approximately10 cmcan be usedinstead ofa vagina allowingeasy administrationof syringe for rabbits and rodents. thedose by ®nger, tamponor tube. Wherea vaginal suppository istobe admi- Theanimal may pushth esuppositorybac k nistered, this isheldbetweenthe fore® nger out orincorrectplacementm ay resultin it and thumb, and insertedgentlybetweent he falling out. lipsof thevulva and thenceint othevag ina. Checkthean imal at intervalsafter Deeperinsert ioni sthenac complishedby returningher to herc age toensurethis pushingthepessary orsupposit ory forward hasnot happened(but remember, the intothevag inausingtheindex ®ngeror other ejectedsuppository may be swallowed). suitable probe, suchas asmooth plastic rod. Theanimal may not retain thewholedose ifthevol umeislarge, renderingthedosage Potentialproblemsandre®nements inaccurate. Substancesmay be absorbed throughth e Keepvolum essmallÐless t han3 mlin vaginal mucosa andthis may resultin dogs, lessthan 500 mlfor rabbits, less unintended systemic toxicity. than 50 mlfor rodents. Before dosingc heckavailable data on absorption rates toassesspot ential 3.9Intravenous and intra-arterial adverseeffec ts. Useofthetechnique It may be dif®cul t,particularly withsm al- Theintravenous route isoftenused in ph ar- leran imals, togain entry into thevagina maco-toxicological experimentsto mimic andtheurethral ori®cemay be enteredin therout eofexposuret odrug formulations, error.T hereis alsoariskofpenetratingthe blood-replacementproducts, nutrient solu- vaginal wall witha syringeorrod ,ifthe tions, infectious and diagnostic agents. It depthor an gleof insertionis inc orrect. ensurest hat maximumplasm aexposurei s Take great care wheni nsertingany- achievedas rapidly as possibleand avoids the thinginto thevag ina. It isvery impor- possibilityofpre-enterohepatic metabolism tant tohave athorough knowledgeof and elimination. theanatom yoftheanim al andin Theintra-arte rial route isused infre- particular thelength ofthevagina and quently, exceptto assesst hepossi ble con- theposit ionof theuret hral ori®ce. sequencesof accidental intra-arterial During insertion,stay onthe upper injection ofaformulation intended for (dorsal)surface soas toavoid the intravenousad ministration. urethral opening.It isim portant to appreciate therelative positionof the Sum mary ofprotocols cervix withint hepelvic c avity,and to advancethesyri ngesuf®cient lyfar Intravenous dosing :Adetailed description forward (e.g.8.5 cmin 6± 15 kgbitches) ofthisroute for rats isgiven inWaynforth for thesubstanc etobe retained inthe and Flecknell(1992)withadditional infor- vagina. mation onot hercom monlaboratory species Checkthe substanc eisnot inthe inPag et and Thomson(1979 )and Tuffery urethra by pullingbacktheplun ger (1995).Further information onm ethods as slightly tocheckfor thepresenc eof they are updated and re®ned is publish edin urine. Awiderc atheter isless lik ely to journalssuch as Laboratory Animals, The entert heurethra. Toxicologist and Hum an & Experim ental

LaboratoryAnimals (2001) 35 22 Joint Working Groupon Renement

Table5 Intravenousinjection (sites of injection,needle sizes and maximum volumes)

Species Typical catheter =needle size Usual(occasional) vein forinjection

Mouse20 25 g 25 27G65/8" Tail Rat 200 g 23G61" Tail Rabbit 2.5kg 21G61" Marginal ear Dog 6 kg 2125G up to 1" Cephalic (saphenous) Primate 2125G up to 1" Cephalic (saphenous) Bird 21 25G61" Brachial (jugular) Waterfowl=pigeon 23 26G61" Tarsal

The choice of infusion deviceis partly determined bythe volume to be infused, therate of administration and thespecies. Largerbore needlescan be used forthe heavier animals within aspeciesgroup Maximumvolumes should typicallybe 4%and no more than 5%of circulating blood volume Infusion volumesshould be based on kidneyfunction data;generally around 5%circulating blood volumesper hour, or 4 ml=kg=h To xic ology.Theprin ciplesof re®nem entare anaesthesia. Otherwelfare issuesrelate to similar tothoseth at apply whent akingblood restraint by tetheringsystemsorth euseof samplesand thereforeanother usefulrefer- jackets, restricted cageenvironmentsand encedocumentis the BVA(A WF) =FRAME= singlehousing. RSPCA=UFAW report onrem oval ofblood Infusiontec hniquesare an advancing ®eld (Morton et al. 1993). whichisoutside thescopeof this document. Inth emajority ofcasesof intravenous Thereare detailed descriptionsoftheprac - administration,substancesare introduced by tical aspectsofinfusionin the literatu re(see asingleinjec tion intoasuitable peripheral Gregory 1995, van Wijk 1997, Laboratory vein.Table 5gives recommended sitesand Animal ScienceAssociation 1998, Healing & volumesfor different species.Som edegree of Smith 2000). physical restraint isal ways required.

Intra-arterialadministration : Recommen- Potentialproblemsand re®nements dations regarding sampling ofarterial blood Withthisroutethereare many factors which (seeabove) may also be relevant toadminis- can compromisethe experim ental objectives trationof substancesby thisroute. and increasethe severit yof theprocedure. Rabbits and dogsare usedand restraint is always necessary. For rabbits asedative or Thesubstance neuroleptoanalgesic may be better than phy- Thebufferingcapacityof theblood allows sical restraint;dogs must be anaesthetized. slowad ministrationof solutionswithawide Theusual sites are thec entral artery ofthe range ofpH(between 2± 11 )providedthat ear inrabbitsandthefem oral artery for dogs. contact with blood vesselwalls is not pro- Needlesizesand administrationvolum esare longed.Formulationsfor injection must be similar tothoserecom mended for intrave- miscible withblood without precipitation. nousd osing. Injectionsshould be made under Substancesm ust not causehaem olysis or asepticconditions inthe sam edirection as coagulation, and not elicit degenerative or theblood ¯owand administered fairly slowly in¯am matory reactionsin blood vesselwalls overapproxim ately 30 s. orsurroundingperivasc ular tissues.

Infusiontechniques :Infusiontec hniques Somematerials can damage blood vessels (eitherinterm ittentorcontinuous) may be at thepoin tofinjection. usedas alternativest orepeated injections, Consider altering theform ulation but this optionsh ould be consideredvery (e.g. increasing thedilut ion)and =or carefully, sincethereare inherentwelfare alteringtheveh icle. If thisisnot problemsrelating tosurgeryÐim plantation possibleth enc onsider ¯ushingthe ofindwellingcatheter may requirea surgical equipmentand blood vesselwith saline procedurewhi chm ust be performedunder before and after administrationof the

LaboratoryAnimals (2001) 35 Administration of substances 23

test substancesusin gaT-connector It ispreferable toinjectsubstances witht wosyringes attached. moresl owlyover a period ofminutes. Thism ay requiread ditional restraint Thec onsequencesof intra-arterial admin- proceduresrath erthan manual restraint istrationof tissueirritati ngchemicals and soweigh the disadvantag esof this arterial-constricting substancesare poten- against thebene® ts oftheslower tially catastrophic. injection. Neveradm inistersuchmaterials by this route. Compatibilitytesting with blood isessent ial priorto dosing.Take Volum es care toensureth at thereis no risk of subsequentarterial haemorrhage on Adding large volumesof ¯uid tothebl ood completionof theprocedure. stream causeshaem odilution,increased Always dilute substanceswel l,usinga central venouspressure, alteratio nsin suitable formulation and ¯ushthe acid-base equilibrium and diuresis.It may artery with salineafter injection. also causepulm onary oedema. Therewill be additional problemsoftoxicity and Therem ay be unanticipated reactionsin haemolysis withnon-aqueous solutions. somespecies.For example,som ecompo- Be aware ofblood volumevalues for nentsof formulations(e.g. Tween80, differentspecies.To minimize adverse polyvinylpyrrolidine, someliposomes) effects, avoid increasing theblood - causeanaph ylactic-likereactionsif ad- volumeby moret han4% for rapid ministered i.v. todogs,but donot cause bolus injections(see Table 5)orinfusing suchreactionsin rod entsand man. at greater than4 ml =kg=h. Checkthe poten tial effect of thesub- stanceand its vehiclewi th background datainarange ofspecies. Injectiontechnique

Particulate matter may be presentin t he Iftheanim al movesduring thecourseof material beinginjectedorunsuitabl e theinjec tion thusd islodgingtheneed le material may precipitateout between from theblood vessel,part ofthetest liquid = preparation and injection. may be injected perivascularly and or Filter them aterial, e.g. by including a subcutaneouslyproducingaswellingofthe = ®lter inthein jection linebet ween skin adjacentto thevein artery. syringeand blood vessel( Note : ®lters Before carrying out theprocedure, can adsorb and retain test material.) always checkthat theanim al isadequately restrainedand that theneedleis If thesubstan cec omesout ofsolution ®rmlyaf®xed tothesyringe. Useof an wheninjec tedinto thetail vein,the tail over-the-needlec atheteror a butter¯y may blackendue tothrombosis. Lung needlewith a length of connecting oedema and=orembolism may also occur. tubing willreduc etherisk oftheneedle Ensurethe substan ceis adequately dislodgingfrom thesyringe. If the dissolved inan appropriate solventand animal movesc heckthe posi tion ofthe checkits compatibilitywithblood needle.If ithas comeout of thevein before starting theprocedure. withdraw itimmediately and repeat the injectionat adifferent site. Theeffec tof acompoundcan be in¯u- Usea local anaesthetic cream,e.g. enced by therate ofadministration. EMLA (Flecknell et al.1990),sothat Injectingintravenously and intra-arterially theanim al doesnot feel any pain, thus overa fewsec ondswi ll resultin transi- reducing therisk ofit moving. ently highlevels of thesubstanc eint he If aperivascular injection occursand if blood withpossibly toxic effects occurring thesolutio nisirritant ,counteractthe very rapidly. effect,e.g. by injecting buffered saline

LaboratoryAnimals (2001) 35 24 Joint Working Groupon Renement

and local anaestheticat thesit eas a make sureit hasvery goodthermostatic diluent. control. Observethe anim alscon- stantlyfor signsof distress. Them ax- Bad injectiontech niquem ay damage the imumwarm ing period for miceis vesselm aking subsequentinjectionsdif® - 5minand for rats 15 min. Asa guide, cult. nomorethan ®veanim als shouldbe Whenusing the ear veinm ake the® rst placedinthe box at onetime. injection as nearthe t ipas possibleso that theveinrem ainspatent. In rodents, giveinjec tionsnear th etip of thetail ratherth an at thebase since Infusiontechniques this isusually easieran dthereforem ore Sincethis involvessurgery, all there® ne- accurate. mentst hat apply tosurgical techniques,e.g. Rotate theinj ection sitesfor repeat appropriate useof anaesthesia, asepsis, doses. appropriate woundc losurem ethods and dressings, and recoveryenvironm ent, and Intravascular injection can resultin emboli post-operative analgesia shouldbe imple- (e.g. skin fragments)depositing inthelung. mented as appropriate. Somepotential Thisis rarely ofclinical signi®cance but problemsand suggestions for re®nem ent can affectthehistopathology. are given below,but referto theliteratu re Usea ®neneedleand injectwith for furtherdetails. minimal trauma. Surgical techniquesfor implantationof Locating thevein =warming theanimal catheters orinfusiondevic esc an trauma- tize theanimal and introduceinfection. Thevein m ay be dif®cult tosee. Ensurestaff are competentin the Trydilatingthevesselto renderit more anaesthesia, surgery and after-care visible by rubbingthesurface, warming required, including aseptic technique. theear ortail (e.g.inawarmingbox),or Reducepost -operative traumaby accli- usingcertain sedatives, e.g. Hypnorm 2 matizing animals tojackets for several (Janssen Pharmaceutica, Turnhoutse- days before surgery. baan 30, 2340 Beerse,Bel gium)inrab- Thinkabout howt hean imal’s beha- bits. Carefullyremoving thehair may viourwill be affectedby thec atheter- also improvevisibili ty. ization andexteriorization site before going ahead. Theanimal willoverheat ifintenseheat is usedfor too long. Constant orfrequent contactofan Any warmingtechniquem ust ensurea implanted catheterwitht heblood vessel carefully controlled maximumtem - wall can causeth rombo-embolism. perature. Warmingthetail aloneis less Accurate placementof thec atheteris likely tocausea problem,although essential,as isth einvolvementof an locally applied warmthdoesnot work experiencedveterinary surgeon.Make as wellas whole-body warming. surestaff have appropriate trainingand Donot usehair driers orheat lampsas are fully competentin the proc edures. they can overheat animals and cause Useof biocompatible materials is skin burns. essential. Wherea warming box isused, thet her- mostat onthe box can fail. Theanim als Technical problemsmay occurwith infu- may overheat leadingtounnecessary stress sionequipm ent, e.g. ifsmall volumesare and,inextrem ecases, death. given intermittently, thenpum psm ay be Thec hamber temperature shouldnot inaccurate. exceed 37 C.Calibrate and monitor the Ensurepum psare meshedin/ warmed temperature ofthebox carefully and upevery timethey are used.

LaboratoryAnimals (2001) 35 Administration of substances 25

Ensureadequate quality control proce- poundis required, e.g. incarc ino- duresare inpl acesothat thesel ected genicity testing. devicedeliversthe form ulation at the (ii)Oropharyngeal administrationof cap- correct volumeand rate of ¯ow. sules,pills or ¯uidsÐthis method is Sophisticated computer-controlled sys- usedwhena material isunpalatable or temsare nowavail able allowing remote whenm oreacc urate dosingis control ofpre-programmable pumping required. devices. (iii)Oral gavageÐthis tec hnique also Donot usem echanical pumping avoids palatibilityproblemsand isthe devicesfor miceas the¯owrate for most accurate method for administra- suchsm all animals cannot be ade- tion ofsubstancesinto thegastro- quatelycontrolled. intestinal tract.

Theuse of tetheringsystemsorportable Them ethod ofchoicewilldepend on the jacket systemscan be uncomfortable, species,the nutriti onal requirementsand restrict theanim als’ movementsor limit physiological state oftheanimals, thepur- thepossibili ties for social housing. poseof theexperi ment, and theaccuracy of Techniqueswhic hallow thepum pand dosingrequired. reservoirof dosingsolutionto be carried by theanim al are preferred since 3.10.1 Inclusioninananimal’s foodorwater tetheringisavoided. Usejac ketswhich Sum mary oftheprotocol allow social housingofsocial speciesor Thesubstanceisincorporated inthean imal’s redesignjack ets whereno suitable ones food orwater. Highly palatable substancesare exist. readilyconsumedwhenoffered inthis way, or Checkthe ® tof jackets and tethering willbe lickedfrom theendofadosingdevice attachmentsdail y. suchas asyringe.Whereanimalsare fastidious Usesubc utaneousport sfor discontin- eaters,it isessential tounderstandtheir uousinfusion ,sothat norestraintis feedingbehaviourin order todeterminethe necessary whenthe ani mal isnot being best way ofincorporating thesubstanc einto infused. thediet. Apilot study totest thesubstanc es acceptability, measuringfood consumption and body weightm ay be necessary. 3.10Oral routes Potentialproblemsand re®nements Useofthetechnique Thisistheleast stressfulm ethod ofadmin- Theoral route isc ommonly usedwh ensys- isteringasubstance,but it can alsobe the temic exposureis required and it isknown least accurate. Therem ay be problemsrelat- that thereis g ood absorption from thegas- ingtothephysico-chemical propertiesof the trointestinal tract and thereis little ®rst-pass substance,its palatabilityand thefeeding metabolism inthe liver. Intoxic ological behaviour of thespec ies.However, the ease investigationsit iscommonlyusedregardless ofadministration,tog etherwith t helow of®rst-pass metabolism.It isalso usedwhen levelof stresson the anim al, may outweigh studying alocal effectinthe g astrointestinal many ofthedisadvantages. tract. Substancescan be introducedinto an Propertiesofthesubstanceand its animal’s mouthor st omachby: palatability Thestabilityofthesubstanceorits homo- (i)Inclusionin food orwaterÐ this geneityinthediet may causea variety of method most closely mimicsthe practical and scienti®c problemsandalter its ingestionof substancesi nhumans pharmocokinetics. and isparticularly appropriate where Thesubstanc emay: bind toingredients in lifetimeadministrationof thec om- thediet; be altered by theheat oftheproc ess

LaboratoryAnimals (2001) 35 26 Joint Working Groupon Renement ifpelleted; react withother dietary compo- Donot feed pelletsthat are too hard. nentsÐfor example,with essent ial vitamins Checkthean imals’ teeth regularlyto oraminoac ids, renderingthem unavailable ensuret hereare nodental problems. totheanimal; break downto moreor less Thesubstanc emay have limited nutri- toxic derivatives(see Sect ions2.1.3 and 2.3). tional value but still constitute alarge Irritant substancesm ay damage the proportionof food. Thisupsets the norm al mucosa oftheoesophagusand stomach. dietary balance(e.g. vitamin,protein Adetailed knowledgeofthepropert ies levels).Inrodent studies, upto10% non- ofthesubst anceinrelat ionto the nutritional substancesm ay be added tothe dosing routeisessent ial. diet. Always checkbac kground datafor Be aware ofthenutriti onal require- indicators of likely adverseeffec ts on mentsand physiological state ofthe thespec iesto be used. animals (e.g. growing,pregnant orlac- Donot useirri tant materials. tating animals).Monitor themc losely for signsoflossof conditionor weight Thesubst ancemay be unpalatable. If it loss. imparts an unpleasant¯avour tothefood orwater, theani mals’ consumptionwill be reduced. If animals refuseto eat, it may be Speciesfastidiousness necessary towithold thenorm al diet. Somespecies(e.g. non-human primates) Careful preparation ofthecompound, readily detectdietary inclusions. Cats are e.g.by micro-encapsulation,may also fastidiousfeeders and it may be removem any of thepal atability dif®cult tohidesubstancesin t heirfood. problems. Pigs have ahighlydevelopedsense of taste Mask unpleasant ¯avours inwat erby and smellso similar problemsarise. Rats adding sugar (water bottles containing and micemay also refuset oeat novel sugar solutionsshouldbe changed daily substances. inorder tominimize microbial growth). It isessent ial tobe familiar withthe Anotheroption is to use¯ avoured (e.g. feeding habits and preferencesof indi- blackcurrant, orange)gelatin whichrats vidual species. and otheranim als appear tolike. Gela- Primates can be trainedinoperant tin hasa meltingpointofaround 60 C feeding techniques.T heyread ilydrink but asolidi®cation pointofaround fruit juicesso substancescan be 25 C.It can thereforebe usedto dissolved inthejuiceandgiven directly. dissolvewater solublesubstanc es(or Liquids can also be injectedintoagrape, substancesdissolved ina miscible an orange sliceorbanana. Powdered solution)whichare temperature sensi- solids can be thoroughlymixed witha tive. Thestoc ksolutionof acompound favouritetitbit; suchofferings shouldbe can be addedtogive a®nal concentra- small enoughso that theanimalswill tion that theanim al willcomfortably consumethemqui cklywithout taking and reliably eat. severalbites (Schrier1997) . Donot withold normal food for long Dogs eat food quickly sothey can be periodsand always consider them eta- dosedbefore feeding and thefood then bolic rate, age andconditionof the acts as areward. animals before doing so.

Thesubstancemay causedental problems, Accuracyofdosing e.g. feedingwetm ashto dogs for prolonged Thedoselevel and durationof dosing cannot periodsc an lead toaccumulation oftartar always be accurately controlled whenthe onteet h.Inrodents, too coarsea ground compoundis sim ply included inthedaily diet can causeg ingivitis, toothdec ay and diet. Theamountofsubstancec onsumed pharyngeal penetration. willvary withthein dividual animal’s food

LaboratoryAnimals (2001) 35 Administration of substances 27 and water intake.Waterintake isparticularly ing animals of social interaction. Bal- variable and dif®cult tomeasure. ancetherequirementto standardize Thereare particular problemswithth e betweentreatm entgroups ag ainst the feeding habits of certain species.O ldWorld desirability ofprovidingsocial = primates willstore food inth eirmouthpou- environmental enrichment. chesfor as longas 30 min, soitisdif®cult to Animals are handled lessfrequentl y say whenit has beenswallowed. Ham sters becausethere is noneedfor restraint.This willstore food incheekpouc hes(and intheir may make theconduct ofotherproc edures cage) making accurate dosage dif®cult. Rats moredif® cult. eat overseveral h ourswhereas dogs consume Overcomeany problemsresultingfrom theirfood very quickly. Thiscreates infrequencyof handling animals by problemsinassessin gdurationof dosing. asking for daily bodyweightm easure- Coprophagic animals presentpartic ular mentas part ofthetechnique orby problems,e.g.recycling unabsorbed material routinelyhandling thean imals during orits metabolites.(Thiswi ll always be a husbandry procedures. problemwhic heverrout eisc hosen.)

Inm ost speciesthe d ietary intake perunit 3.10.2 Dosingdirectly into thepharynx body-weight reduceswith age. Try presenting smaller amountsof food Sum mary oftheprotocol at regular intervals toensureac curate Thetechniquei scommonlyusedonly in dosesare consumed. Usehi ghlypala- cats, dogs, ferrets, farm animalsand birds, table food makingsureanim als are used although rabbits can also be dosedi nthis tothenew food andrecognize itas a way (seeTuffery 1995, Wolfensohn&Lloyd treat. 1998).Asuspended,enc apsulated,orsolid Vary theconcentrationind iet =water to amountof material isplacedontotheback of achievea steady intake as `mg’ or thetongue and aswallowingre¯ex induc ed `ml=kg’. Dosesshould be expressedin by strokingthethroat. Fluids can be instilled termsoftheweight of thebase (not of ontothe tongue orpouredin thelabial =cheek thesalt )given perunit body weightor pouchat theangleofthejaw withthe m uzzle body surfacearea. heldupward s.Toadministeracapsuleto a Usespecial containerswhic henable the primate orrat, put thec apsuleon the en dofa amountof diet consumedtobe more gavage tube and `blow’ itout (Lax et al. 1983). accurately measured. Theseh ave been Rats needto be trainedtoaccept thegavage described for feeding mice,rats, guinea- tube ®rst. pigsand hamsters(Poole 1987). Neonatesc an be dosed by allowingthemto suckthetest compoundas an emulsionfrom Therem ay be cross-contamination ofdiet PE50 plastictubing. Volumesof40±50 ml can = from spillageand ordust and this can be givent o10-day-old mice, body weight affectaccuracy. approximately 4±6 g.Thetech niqueis m ore Animals may needt obe separated for stressfulthan oral dosinginfood orwater = feeding;careful ventilation air¯ow because restraintisnecess ary. control may be necessary. Potentialproblemsand re®nements Otherwelfarepro blems Thesameproblemsoccuras in3.10.1 above. Inaddition: Animals may have tobe singly housedto assessdietary intake moreprec isely. Ifthesubst ancehas an unpleasant taste, Always questionthe nec essity for pre- thean imal may developa conditioned cisem easurementofdietary intake and responseand vomit before beingdosed. thusfor singlehousing. It may be Try making thesubstanc emorepalata- relativelyunimportantscienti®cal ly ble by ¯avouring it orby encapsula- but causeunnec essary stressby depriv- tionÐchoose the c orrectsize ofcapsule

LaboratoryAnimals (2001) 35 28 Joint Working Groupon Renement

for thespec ies.Treats can be given as a from theend of thegavage tube entert he reward after dosing. lungs, orinapparent, e.g. ulceration inthe stomach. Dosingm ay be inaccurate ifsubstancesare Donot administersubstanc eswhich spilled, spat out (immediately oras a causeg astric irritation. delayed response)orreg urgitated. Avoid direct contactofthetube with Encapsulationoverc omesthisproblem theoesoph ageal orstomach wall by but be aware that capsulesth emselves ensuringthec orrectlength ofgavage may break, releasing thec ompoundin tube. thewrong part oftheg ut. Avoid generationofdropletswhichmay Dosingac cidents,suc has choking, can be inhaledandcausesevere reac tions. It occur. isimportant tomake surethere is no Avoid choking by giving ¯uid insm all droplet onth eendofthetube as it is aliquots (5 mlfor dogs, 250±500 ml for pusheddown the oesophag us.Blot or rabbits),allowingthean imal toswal- wipeit withatissuean d¯ushthe tube lowbetween aliquots. with asmall volumeofwater after dosingso no irritant drops are inadver- Repeat dosingmay be morestressfu lfor tently released as thetube isremoved. theanim al andtheopportun ity for errori s increased. Compoundsm ay froth and blockthe Limit then umber ofrepeated doses trachea orcausea foreign body pneumonia given perday tobetween2 to4.This ifadministered into thelung sby mistake. often mimicstypical clinical usage and Take particular care toensurethe may thusbe amoreacc urate and gavage tube isinthestom ach.Consider appropriate method ofdosing. maximumvolum escarefully for com- Reducestressby training theanim al to poundsthat can froth. cooperate intheproc edure.

3.10.3 Oralgavage Thetechnique Sum mary oftheprotocol Thetechn ique has beendesc ribed indetail Ifthetube isin correctlyplaced, ifundue for all thecom monlaboratory speciesi na force isused, orif theanimal moves,th e number oftexts (Paget &Thomson1979, tube may penetrate thetrac heaor pass Poole1987, Waynforth &Flecknell1992, throughthe oesophagus or st omachwall Tuffery 1995).Insum mary,afeedingtube or into theth oracic orperitoneal cavity. gun ispassed into theoesophagus =stomach Subcutaneousabscesses m ay resultfrom and thesubstan ceto be dosed isgen tly infectiontrac king out from them ediasti- expelledat aregular controlled rateslow num,e.g. swellings intheaxillary region. enoughto deter regurgitation.Thetube is Repeated insertionof thetube for frequent withdrawngent lytoavoidre¯ex vom iting or dosing may causein¯ am mation and regurgitation. ulceration of theoesoph agus. Whena rigidgavage tube attachedtoa Potentialproblemsandre®nements syringe isbei ngused, negative pressure Thisis the m ost stressful,andtechnically may draw inair, ratherthan create a dif®cult, method oforal administration, vacuum,ifthetube has inadvertentlybeen althoughan experiencedand skilledtechni- insertedinto thelungs rath erth an the cian can make it seemdeceptivel yeasy. oesophagusor stomach. Keepth eanimal still and ensurethe Thesubstance best angle of thehead and body to facilitatedosing. Thisis very important Substancesmay be irritant.Theeffect s and requiresc orrectand sensitive may be apparent e.g. wheezing ifdroplets restraint.

LaboratoryAnimals (2001) 35 Administration of substances 29

Be familiar withthe anatom ical rela- Gags can causedisc omfort. tionshipsof theoropharynx and develop Onlyusegag sifthey make thetec hni- thenec essary highdegree ofskill before que easierto performan dlessst ressful commencing dosing toensureac curate for theanim al.Make sureany gags are placementof theg avagetube. designedtobe comfortable for the Always usethe c orrectsize (length animal. Tapering oneend m akes it and width)offeedingtube toensure easierto put into them outh. thedose enters the stom ach and not the Thean imal may regurgitate thedose or oesophagus.For example, indogs vomit.Thisisa particular problemwit h thec orrectlength correspondstothe ferrets. distancefrom then osevia theac romion Always expelthe c ontentsof thegavage oftheshoul dertotheten th costochon- tube at acontrolled rate and withdraw dral junctionÐcorrect placementi s thetube carefully.Monitor theanim al con®rmedby thesm ellofthegut carefully onceit hasbeen returned to contents. Inm icethetube should the cage. extendfrom thetip ofthenose to the last rib. Mark thelength inadvanc e. Make allowancefor youngorsm all individual animals. Withholding food Checkfor condensationwhic hmay be visible ifthetube has inadvertently Food isoft enwithhe ldovernight,for as beenplac edinthe trac hea. longas 18 h,inorder toemptytheanim al’s Usetubeswith a small smooth knob on stomachbefore dosing. Thisis alongtime, theend to preventpenetratio nof the particularly for ratsorotheranimals whose gut wall. Flexiblecatheters are prefer- normal feeding behaviour is`litt leand able torigidtubesand plastic can be often’an dwhosefeedin gtimeisat night. usedin stead ofmetal (but consider Thinkc arefully about thereasons for possiblereac tionsbet weenth ecom- witholding food. It israrely justi®edÐ a poundan dthetube ). recentstud yby Vermeulen et al. (1997 ) Consider lubricatingtubeswith petro- demonstratedthat thestomachofmale leumjelly orm edical paraf®n toease ratswasem ptyafter 6hwithout foodÐ passage. and requiresa good scienti®c reason If anyresistanceisfelt during insertion (i.e. iftheabsorptionof thec ompound oftheneedle, or if thereis any sign of varies depending onwhethertheanimal choking ordistress, withdraw thetube isfed orfasted) .If food iswithdrawn, and re-start theproc edure. water shouldalways be offered. Always monitorth eanimal for ashort Authoritymay be required from licen- period after returningit toits cage to cingauthoritiesorAnim al Care and checkfor anyadverse reactiont othe UseC ommitteesfor food withdrawal procedurean dtocheckwhetherre- together withprec isein structionsfor gurgitation orvom itingoccurs.If timeofnext feed. animalsdiefollowingoral dosing performa necropsy examinationt o ascertain thecauseof death and Young animals eliminatepoorte chnique as a Extremecare should be taken withyoung factor. animals. Thereare special considerations If you suspectyou have dosedi nto the withrespect t otheirsize, disturbanceof lungshold theanim al’s chestc loseto littersand timeofdosing after ingesting your ear andlistenfor `gurgling’ or milk. Dosingpre-weanedani mals is `rasping’sounds.If thed osehas gone dif®cult andshouldbe avoided ifpossible. into thelungs kill theanim al toprevent Gavaging rats lessthan 6days old shouldbe further suffering. avoided.

LaboratoryAnimals (2001) 35 30 Joint Working Groupon Renement

Prim ate s oftheback bone. Theinc isionshould be Oral gavage of OldWorld primates should positionedabout 1±2 cmcaudal totheesti- only be considered inc aseswh ereit is mated caudal endoftheminipumpwhenthis essential toensurethat thefull doseof adrug isin plac e.Apair of haemostats (straight reachesthe stom ach at thesam etime.It isa artery forceps)isinserte dthrough theinc i- straightforward procedure provid ed staff are sionunder the skin inacranial direction and highlyskilled and experienced inc arrying it thejaws on thehaemostats openedto make a out.Thestress of thetechn ique iscom- subcutaneouspoc ket for theminipump. The pounded by thestressof therestraint minipumpshouldbe inserted, delivery end required and staff m ust be welltrained and ®rst, into thesubcutaneouspoc ket.Theskin competentin this too. Lubrication ofthe woundis c losedwith wound staples (not tube willease its passage. nickel)orsutures.Appropriate peri-operative If at all possible,oral gavage shouldbe monitoring andcare must be provided. avoided and substancesshouldbe adminis- Someprotocolsmay requirethe rem oval or teredorally by otherm eans. replacementof them inipumpafter aspeci- ®edperiod. Thisshould al sobe doneunder general anaesthesiausingaseptic technique 3.11Osmotic minipumps and withanalgesia. Theoriginal incisionis Useofthetechnique opened,t hepumpremoved withforc eps,and Osmotic minipumpsc an be usedto allow thewound sutured. continuousd rug delivery overlong periods Somemanufacturers/distributersof mini- (Theeuwes& Yum1976, Nau 1985, Collins pumps(ALZA Corporation1990 )provide a 1987)and toavoid thestress of repeat dosing. video whichdemonstratesthebasic tech- niquefor subcutaneousim plantation plus otherappl ications,suchas intraperitoneal Sum mary oftheprotocol implantationan dconnection tocatheters for Commercially available pumpsare designed intravenousinfusion .Theseprovide infor- todeliver different durationsofdosing.A mation onprac tical surgery but do not steadyout¯ow of test material can be pro- addresswelfare issues. ducedfor upto2weeks.Mini pumpsare It ispossibl etosupplythe m aterial for commonlyusedin rat s, mice, guineapigs, dosingtoan establishmentand thenbuy the hamstersand rabbits, althoughthey can be animals alreadyimplanted. However,note implanted invirt uallyanyspecies.In m ice, that thetransport ofsurgicallyprepared and minipumpsare large relativeto thebody size dosed animals also has welfare implications oftheanim al, but are tolerated well,provided whichmust be takeninto account. theyare sited ina positionwhic hdoesnot interferewith the anim al’s movement.The Potentialproblemsand re®nements device isinsert edsubcutaneously, orocca- Themethod can have signi®can tanimal sionallyintraperitoneally, witha single welfare bene®ts overrepeated dosingby surgical procedure.Aseptic tec hnique, otherroutes wh ererestraint andinfusionor anaesthesiaand analgesia are necessary. injection may be required, but thismust be Analgesia should be administered as soon balanced against thestress of surgery. The as theanim al isanaesthetized. It willhave most likely adverse effectsofthetechn ique thenbec omeeffective by thetim etheanim al are thosedue totoxicity ofthecompoundor regains consciousness.Thehair should be drug containedinthe m inipump. gently removed from thedorsal area with® ne electric clippersan dtheskin cleaned withan Areactiont otheminipumpmay be seenif antisepticskin preparation.Askininc ision, its surface isabraded. Weight lossm ay about 1cmlong, shouldbe madeonthe occur. dorsumof theani mal midway betweenthe Handle pumpsvery carefully. When head and base ofthetail. Thepumpshouldbe monitoringbody weightin sm all ani- placedalong theaxis of thebody tooneside mals, remember totake accountofthe

LaboratoryAnimals (2001) 35 Administration of substances 31

weightofthem inipumpand thec om- shouldbe consideredin eac hcase (see pound. Any weightloss should not be Section2.3 ).Pilot studies are advisable to expected toexceedthat seenaft er assessand avoid potential adverse effects. anaesthesia alone. Inhalation administration ofsubstancesis described indetail by Phalen(1984 ),(seealso Thetec hniquenec essitatesanaesthesia Kennedy et al. 1989, Waynforth &Flecknell andsurgery both of whichare potentially 1992). traumatic procedures.M ost wounds shouldheal wellbut woundbreakdowns 3.12.1 Wholebodyexposure may occasionally occur. Thistechniqueshould only be carried Sum mary oftheprotocol out by experiencedand competentstaff. Animals are exposedto thesubstanceeither Traininginanaesthesia, aseptic surgical ina speciallydesigned inhalationch amber, technique, and peri-operative care, orwhereexposure is for many hoursper day including post-operative analgesia, is (i.e. closeto continuousexposure ),inc ages essential. withsim ilar dimensionstotheirn ormal Monitorthe anim al carefullyafter housing.T hecagesmay be made ofmesh(to implantation.It may be necessary to ensureci rculationoftheatm osphere)and housethe an imal onits ownfor 24 hfor therem ay be otherm inorm odi®cati ons. theskin toseal adequately. Potentialproblemsand re®nements Onlysmallvolumescan be administered Thereshould be littleorn omoredistress soth eformulation isoften very concen- from thetec hniquethan occursfor stock trated.It may precipitateinc ontactwith animals inholding rooms,provided standard body ¯uidsand thepum pcan become cages are used. However,any restrictionson blocked as aresult. available space, food orwater oron the nat- Check (in vitro )that precipitationwi ll ureof thephysical andsocial environment not occur. may causedistress, as may thenoise from ventilationor dosing equipment. 3.12Respiratory routes It may not be easy toregularly monitor Useofthetechnique animals ininh alation chambers. Therespiratory route isused frequentl yin Usec lear polycarbonate cagesto facil- toxicologyand isselectedwhenit istheroute itate observationof animals and/orplace ofhuman exposurefor thetest substance.It cages inoptimal relationto lightingand isalso usedto administer certainvaccines windows.Moni tor theanimals’ behav- and medicaments particularly wheneffec ts iourvery closely for signsof distress, ont herespiratory tractare required orwhen possibly usingvideo equipment. very rapid systemicabsorption isneeded.The Someinhalationc hamber designshave no range of experimental exposuret echniquesin litter trays soas not toimpedeair circula- animalsre¯ec ts thevariation ofhuman tion.T herats inlower levels m ay then exposure.T hiscan vary from afewsec onds receivet hefaec esand urinefrom rats (somevaccinesan dmedicaments)tovir- housedhigher in the c hamber. tuallycontinuousexposure, 24 hperd ay Thisdesig nofcage isunacceptable and 7days perweek (for airborne environmental shouldn ot be used. pollutants).Exposurem ay be: Inhalationc hambers are designedfor the (i)Wholebody purposeof theprocedure rather than the (ii)Noseonly comfort oftheanimals. The¯ oorspac e (iii) Mask may be reducedand theopportun ity for Thepotential adverse effects ofthesub- environmental enrichmentis restrict ed. stanceontherespiratory tract are commonto Look for ways ofimprovingthec omfort all, and factors suchas pHand irritancy of thecage and enrichingth eenviron-

LaboratoryAnimals (2001) 35 32 Joint Working Groupon Renement

ment. Returnanim alstoconventional 3.12.2 Nose only/Snout onlyexposure cages betweenexposures. Useofthetechnique Whereexposures l ast for severalh ours(e.g. Thistechnique isamoreprec isem ethod of 1±6 h)perday ,food and water are generally dosingthanwhole-body exposure.It is withheld. normally only usedwith rats, miceand Ideally, do not withhold food and hamsters. especiallynot waterfor morethan 6±8 h/day depending onth especies, Sum mary oftheprotocol particularly ifexposurei soverseveral Animals are placed inc lear polycarbonate days. Water shouldbe continuously conical tubes sothat thesnoutprot rudes available. from aholeat thepoint of thec one.Tubes are made ina range ofsizest omatchthe Thegeneration oftest atmospheresusually sizesof rodent tobe exposed.T heBatelle usesc lean,®ltered, dried air. Inthe l onger type tube includesa backstop witha hole term,thiscan resultin a lowrelat ive throughwhichthe rodent’s tail ispassed . humidityinth einhalationchamber and Thispreventstheanim al reversingout ofthe thusthe risk ofringtail inyoung rodents. tube. Ventsin the sid eofthetube allowthe Ensurehum idity isadequately con- animal toremain coolan dreducesthebuild- trolled ( > 40%)andthat theair supply upofwater vapour. cannot fail. Animals are sometimeswashed and dried Potentialproblemsand re®nements orotherwis ecleaned toremovet est mat- Thistechnique isinevitablystressfulbec ause erial deposited onthe fur after completionof ofthetube restraint.Deaths can and will exposure.T hisadd stotheiroverall stress. occurif thisisnot carried out competently. Ascertain whetherdeposition of test Tubes that are not thec orrectsize and material isreally aproblemin term sof shapefor theanim al may allow theani mal exposurean ddonot washanim als ifit toturn partlyaround.Theywill t hen isnot necessary. If animals do have to becomedistressedand may die orinhala- be cleaned thecl eaningagent shouldbe tionexposure will fail. carefully selectedsoas not tocause Theshapeof tubes isobviously critical, adverseeffec ts. soth econstruction of`in-house’ devicesshould not be attempted with- Food and water can be contaminated with out athorough appreciation ofrequire- thesubstanc e. mentsand preliminary testsnot Designfood and water containersto involvinganimals. minimize contamination and/or Ensurethe ® toftheanimal inthe cone measureth econtent oftest material in issuchthat it cannot turnroun d. food/water. Therem ay be noneedto Always monitor theanim als continu- exposeanim als withtheirfood /water ously whilstin thetubes toidentify and presentif exposureis only for short helpany that are indistress. periods. Placingarat ormousein a tube may seem Theapparatus usedto generate test atmo- simpleand requirelit tle training.How- spheresm ay be noisy,eitherin theaud ible ever,poor techn ique can resultin un - orult rasound range. necessary stressand eventhe deat hof Be alert tothispossibili tywhenasses- animals. sing animals’ behaviour. Measure Ensureall staff are trained and com- ultrasoundas wellas normal frequen- petentin the proc edure. ciesand do not usenoisy equipm ent morethan oncein24 h.Habituate the Animals may be restrainedfor longperiods animal tothech amber toavoid stressof eachday for manymonthsin som e anovelenvironm ent. toxicological studies.

LaboratoryAnimals (2001) 35 Administration of substances 33

Habituateanimals tothetubes to and nostrils.Atube goesthroug hthem ask minimize stress.T heperiod ofrestraint and intotheanim al’s mouthover the tongue. shouldbe builtupoverseveral d ays Thepositioningofthetube relativeto the before exposuresto test material begin; tongue can be checked by lookingdownthe however,even i ftheanim als become tube. Theremainderof theequipm ent usedto thet ubes, the®rst day ofactual includesa one-way inhalationvalve; apres- dosing can be very stressful. suresensor (th ism ay be necessary todetect Only keepani mals intubes for the inspirationso that apressurepack ,i.e. a minimumtim econsistent withsc ien- dosingpackwithair pressuredelivery, can be ti®c requirements. Thisshould not `activated’ coincidentwith inspirat ion);anda exceed4h.Reducetimeinthe tube by one-way exhalationvalve. Theprecisenature ensuringtheanimals that are ®rst in, ofthedosi ng,airsupplyand venting equip- are also ®rst out.Try tohave morethan mentwi ll vary withthe test material (i.e. oneski lled personto load andunload liquid, solid, gas),pressurepac k,accuracy of animals thereby reducingtheoverall dosingand theneedto exposethe nasal pas- restraint time. sages. Note :For metered doseinhaler (M DI)dos- Faecesand urineaccumulate inthe tubes ing for short-termexposure, an oropharyn- during exposure. geal tube may be usedinstead ofamask. This During long-term exposuresm onitor restson top of thelarynx enabling the and removefaec esand urine.Make sure respirationto be monitored. tubes are washedwellafter use. Potentialproblemsand re®nements 3.12.3 Maskexposure Therestraint required can makethis tech- Useofthetechnique niquepotential lyhighlystressful,especially Facemasks are usedfrequentlyfor inhalation whenit isfor long periods. exposurein d ogs andprimates. Thetec h- nique isbroadly (but not entirely)equivalent Wearing amask willbe unpleasant for the tosnoutonl yexposureof rodents. animal. It can causedisc omfort and/ormay leak. Make surethe m asks are designedfor Sum mary oftheprotocol comfort and acceptability. Checkthem Exposurecan be onceorseveral tim esdai ly regularlytoensurethey ®teachindivi- (up to5exposuresper d ay) and might be for a dual animal comfortably and securely. matter of secondsperoc casionor up toone Whereoroph aryngeal tubes are hourper d ay ina singlesession for several incorporated, smallerdogs willrequire weeksor m onths. shorterand narrowertubes. Animals must be restrainedand ifexposure Train animalstoaccept therestrain t isbrief (secondsor afewm inutes)itisbest to and thent hefacemask before thestudy do this manually.Dogs requiretwo people, starts. Thism ay take upto3weeksand onet ohold, oneto dose,although ifanimals adequatetimemust be allowed for this. are trained asingletech nician can suf®ce. Individual animals that do not adapt Large primates may requirethree people. For (i.e. that showdist ress)shouldnot be longerexposures dogs may eitherbe heldon put onstudy . thelap ofan animal technician orm ay be held insl ings whilst wearingthem asks. Thetest material, e.g. drypowders,c an Primates can be restrainedinchairs. Animals causedryin gofmucosal surfaces and result inslingsor ch airs must NEVER be left indiscomfort totheanimals. Inprim ates unattended, althoughoneperson m ay be able thisc an lead tobreath holdingand fainting. tosuperviseseveral well trained animals at a Inspecttheanim als’ oral cavity before time. eachexposureand reduceth edosean d Facemasksgenerally have arubber sleeve period ofinhalationexposure. Form u- toprovide an airtight ®taround themouth lateinaqueous ifat all possible.

LaboratoryAnimals (2001) 35 34 Joint Working Groupon Renement

Mechanical equipmentcan fail. tionst oadjuvants, particularly Freunds Checkequi pmentregularly. Always completead juvant,can have acom- ensurethe anim al willbe able to poundingeffectont heinject ion.For breatheintheeven tofan equipment example,such a reaction inthe skin failure. overt herespiratory musclesin the ¯ank ofrabbits can make breathing 3.13Subcutaneous painful. Carefullyconsiderthe n ature ofthe Useofthetechnique adjuvant and theprotocolwith refer- Subcutaneousinject ionis com monly used encetoliterature suchas Jacksonan d for theparenteral administrationof many Fox (1995),Animal Welfare Information substances.T heroute providesfor slow Center (1997)and Palmer et al. (1997 ). releaseavoid ing®rst-pass liverm etabolism SeeSec tion 2.3 for detailed recommen- and may be usedto imitate therout eof dations. administrationof apharmaceutical. Using skin antisepticstotry to`sterilize’ theskin may causeun necessary damageor Sum mary oftheprotocol disturbanceoftheskin’s commensal Thesite for asingleinject ionfor rodents and organisms. rabbits isnorm ally thesc apular region.In Normallythereis no needto cleanthe dogs and cats thepreferredsite isbehind the site, except perhapsin farm animals. neckoroccasionally inthe sub-lum bar fossa Ensuresubstanc esfor injection are onthe ¯ ank, orbehindtheneck. Inpri mates sterile(and warm)andusea newneedle theinjectioni smade undera fold ofskin on for eachanimal. theanimal’s back.Inbirds, subcutaneous injectionscan be giveninto themedial aspect Incorrectorfumbled insertionof the ofthethigh wherethere is usually ample needlec an hurt theanim al, orpunc ture a spaceand wheret heposition of theneedle blood vessel,or d eliverthe substanc e can be clearly seenif thefeathers are dam- incorrectly.If theanimal moves,an penedwi th alcohol.Thereshould be no unwantedintramuscular injection or needtopluckfeathers(Hawkins et al. 2001). punctureof ablood vesselm ay occur. This SeeWayn forth and Flecknell (1992),Tuffery isaparticular problemwi th dogs. (1995)and Wolfensohnand Lloyd (1998)for a Hold theanimal very still. Introduce descriptionof thetec hnique. theneedle point into thesubc utaneous spacewith a ®rmbut rapid movement. Potentialproblemsandre®nements Point theneedletowards theside of the neckwith thebevel upwards. Pullthe Thistech niquei s very painful ifthepH or plungerbac ktomakesuret heneedle osmolarity iswron g,orif them aterial is hasnot entered ablood vessel. irritant orcytotoxic. Tissuenec rosiscan occur. If osmolarity isin correct,tissue Thevolum eof¯uid that can be accom- ¯uids may diffuse into thesite. modated varieswiththe size ofanimal, the Whereth eeffectoftest substancesis site, loosenessof theskin andthephysico- unknowng ood pilot studies are essen- chemical nature of thesubstanc e. tial. Adversereac tionsm ust be fully Choosea site whereth ereis m ost resolvedbefore repeat dosing. looseskin toaccommodatethevolume Selectthesite withc are. Ifadverse andassociated skin movement(see effects may occurc lip thehair/ fur Table 4).Multiplesites c an be consid- around thesitetoenable early detection ered for administrationof greater ofproblemssothat theywill be easier volumesalthough, whenrepeate ddaily to resolve. administrationi sintended, four sites Ensurethe solutio nisst erileand that shouldnormallybe consideredth e pHan dosmolarity are correct.Reac- maximum.

LaboratoryAnimals (2001) 35 Administration of substances 35

Whenc arrying out repeat dosestud ies,th e cellc ulturesi fappropriate, thentest substancemay accumulate at asinglesite. oneanimal ®rst. Usesm aller volumesfor repeat dose Ingeneral, do not placeirritant sub- than for singledose studies. Massage stancesonthe skin, unlessc hecking theskin overlying thein jectionsit eto theirritanc ypotential isthereason for disperseany ¯uid. thetest .Insuc hcasesm onitor the Change sitesfor repeat dosestudies. animal particularly carefully and be prepared toremovet heanimal from the 3.14Topical— dermal studyifit becomesdist ressed. It may be possibleto quantify the Useofthetechnique erythemaand identify an earlier end- Thed ermal route isused to investigateboth pointby an intravenousin jectionof local andsystemic effectsfollowingdermal methyleneblue. absorptionand metabolism.It may be usedin Apply test substancesuniform lycover- testing potential therapeutic agentsfor skin ing nomorethan 10% of thebod y disease, orincheckingthepotential tocause surface (e.g. 5cm 65cmfor rats, skin irritation orsensitization/allergenicity. 12 cm614 cmfor rabbits and 7 cm610 cmfor guineapigs).Thearea Sum mary oftheprotocol of applicationfor highlytoxic sub- Almost without exceptionregulatory tox- stancesshouldbe muchless. Avoi d icology guidelinesnow only callfor the eyesand genital areas. conductofdermal toxicitytestsontheintact Unintended skinabrasionm ay occurwhen skin. clipping orshaving thehair. Thiscan Them ost commonlyusedspec iesare the increasepotential irritancyfor theanim al rabbit, rat, mouseand guineapig. Adequate and introduceexperimental errorby restraint of consciousani mals isrequired . increasing rate ofabsorption. Oil org rease Liquids may be applied diluted orundiluted. from clipperscan effect theresponsetothe Solid test materialsshouldbe pulverized and test substance. moistenedto apaste withsaline or an Onlyusean imals withclean, intactand appropriatesolventwhic hwilln ot `degrease’ ungrazed skin. Take great care when theskin.Theapplication site may orm ay not clippingor shaving theanim al, and be occluded orsemi-occluded dependingon make surecli ppersare clean. Prepare themost likely patternof exposure. thesi te 24 hpreviouslyto allow the Occlusionor semi-occlusionperm its useof animal timetorecover. greater dosevolum es,an dingeneral also increasesabsorption. At theen doftheexpo- For someteststhesk inis del iberately sureperiod thedressings shoul dbe carefully abraded before applying thetest substance. removed and thesite isusually washedclean Always question thenecess itytocon- withwarmwater andgently dried. duct this moresevere procedure and If thesite isnot occluded animalscan be make speci®c referral totheReg ulatory preventedfrom ingesting material, eitherby Authoritythat demandsit. If therei sno locating theapplic ationcl oseto thehead or alternativet henc heckwhetherapplica- by application ofan Elizabethan collar. tion and removal ofsellotape provides suf®cient abrasion (but note that this Potentialproblemswith re®nements can also causedisc omfort).Inany case it isnormally suf®cient to removethe Irritant substancesc an causeserious epidermis, e.g.by light scratchingwi th adverse effects. aneedlenotsuf®cient to causebleeding. Astep-wiseapproach isessen tial (HomeOf®ce 1994). First checkph y- Volumeslarg erthan about 500 ml tend to sico-chemical propertiesfor irritant runoff thebac kof smalleran imals, potential, thenassess this further in defeating theobjec tof theexperim ent.

LaboratoryAnimals (2001) 35 36 Joint Working Groupon Renement

Them aximumdose of atest material usedfor investigation ofpharmaceuticals that isrequired by regulatory authori- intended for long-term usein thehuman eye. ties for toxicological studies is2 g/kg. Administerthisin volumesoflessthan Sum mary oftheprotocol 500 ml. Very usefulg uidanceonth econductofeye Dressings can affectmovement. Overtight irritationstudies have beenprod uced by the dressingscan causeexc essiveabdom inal UKHomeOf®ce (1994)and therei sa pressurewhic hmay lead tointernal requirementin the U Ktofollowthese damage that isnot apparent. guidelines. Theprinc iplesare universally Ensuredressings are not too tight and applicable. do not restrict them ovementof the Substancescan be applied totheeyeby two animal orcauseexc essiveabdom inal methods: direct application intothec on- pressure.T hemaximumtim eofocclu- junctival sac, ord irectly onto thec ornea. sionshould be 24 h. Note :Thestandard Theconjunctival exposurem ethodhas been Magnusson and Kligman test requires commonly adopted becauseof easeof appli- 48 hocclusion(O rganization ofEco- cation.However, the current t hinkingisthat nomic Co-operation and Development conjunctival exposureis inappropri ate under 1992). many circumstances,espec ially whent he test material isapowderwhic hmay become Removal ofadhesive dressingsc an pullth e entrapped inthec onjunctival sac resultingin animals’ hair orskinwhichcan causepain. mechanical damage totheeye. Thec orneal Donot apply highly adhesivem aterials exposurem ethod morec losely mimicsacci- tothehair orski niftheyare tobe dental human exposureas occursfor example removed before theanim al iski lled. inch emical accidents. Determinethe least painful method of Astepwiseti ersystem approach(Home removing dressingsby practising on Of®ce 1994)shouldalways be followed yourself before applyingadressingt o wherethere is any chanceof irritationor theanim al. Consider whetherlight otherdam age.Donot proceedto using anaesthesia orsedat ionwould be bene- animals until initial physico-chemical char- ®cial. acterization, in vitro cytotoxicityorot her studies, and dermal toleranceassays have Theuse of collars inrats appears tobe particularly stressful. beenconduc ted. Aninit ial study inone animal usingoneeye onlyshouldalways be Avoid usingc ollars for rats by applying carried out. thesubst anceto asmall area of skin Various applicatorsfor cornealexposure closeto theh ead. have beend evelopedbut for routinepurposes All animals needtobe trained toaccept theeyelids are gently retractedand thetest a collar. formulationis applied directly tothecornea. Thetreated eyem ay be irrigatedwithwat er 20±30 safter application ofthetest sub- 3.15Topical— ocular stance. Animals must thenbe observed very closely withat least 24 hallowed for damage Useofthetechnique tobecomeapparent. Substancesare applied totheeye inorder to assesspotential irritanteffects. Application totheconjunctival mucousm embranes and Potentialproblemsand re®nements thecornealepith eliumi salso usedtoachieve Thistest has thepotential tocauseani mals highlocal concentrations ofdrugs for ocular seriousdistress. Substanceswhic hare severe therapy. Therabbit isuniversally usedfor eye skin irritantsmust neverbe testedintheeye. irritation studies,even though thereare Animals shouldbe closelymonitored for shortcomings and exceptionsin predic t- adverse effectsthroughoutthe procedure. If a ability. Dogs and primatesare sometimes severead versereac tion occursthe anim al

LaboratoryAnimals (2001) 35 Administration of substances 37 shouldbe killed and nofurther animals thel egal requirementfor thetest before shouldbe used . carrying out theproc edure.

Substancesmay causeopac ity, serious Volumesin excess of 50 ml may over- irritation,pain, in¯ammation orulc era- estimate human sensitivity according to tion. Themain area ofconcernis related to somescienti®c opinionand may simply effects onth ecornea, conjunctiva andiris over¯ow from theeye. althoughif theapplied material can pene- Anatomic andphysiological considera- trate deeperinto theeye otherstruc tures tionssuggest that themaximumprac - may alsobe affected. ticable volumewhichcan be instilled Donot apply knowncorrosive sub- into therabbit conjunctival sac is stances,those wi th ahighoxidation 30±50 ml (Note : 100 mlisrequired in potential, detergents, orknownirri- OECDguidelines),soquestion the tants, totheeye(Hom eOf®ce 1994). requirementfor larger volumes.A low Always checkthe physic o-chemical volumeeyetest can be consideredusing propertiesfor evidenceofirritancy.The 10 ml. pHlim itsare 2±11.5 but neutral pHis Animals dosedand thenheld to assess best (seeSec tion 2.3). recovery may suffer continuingdistress. Theeye can be anaesthetized the® rst Thenec essity for assessmentof recov- timethat thesubstanceisadm inis- ery should always be questionedand teredby applicationof atopical anaes- establishedwiththeregulatory author- thetic sothat ifany unexpected ity.Monitor animals very carefullyand irritationdoes oc curth eanimal will considergivi ngsystemic analgesics not feel it(Seabaugh e t al.1993). If the (Fielder et al. 1987). anaesthetic isin compatiblewithth e compoundor th ereare moreg eneral doubts, usea general anaesthetic for 3.16Footpad the®rst test. Thisroute has ahighdisturbanceindex Be aware ofthescale oflikely responses (Barclay et al.1988),and it isstrongly and be able torecognize andrespond recommended that it isnot usedun lesspro- rapidlyand appropriately totheadverse ven to be the only effective onefor achieving effects as set out inth eHomeOf®ce aspeci®c objective. 1994 guidelines. Footpads wereused as asiteof injectionfor Insoluble, hard substancesm ay cause antibody production, popliteal lymphnode mechanical damage whenapplied tothe assays, adjuvant arthritis, assessmentof conjunctiva. analgesic agentsand investigation of Donot placerough, hard, insoluble mechanismsofpainand in¯am mation, and substancesinanim als’ eyes. Crystals mycobacterial research,but alternative shouldbe micronized. routesfor virtually all theseappli cations are nowc onsidered satisfactory (Bennett et al. Irrigation may affecttheoutc omeof the 1992, Leenars et al.1997).For example,for test and,ifthis has not beentaken into antibody production, othersubc utaneous accounti ntheexperimental protocol, then sitesand careful selection ofadjuvant almost thetest may have tobe repeated using always allowsadequate titres tobe obtained moreanim als. and theuse of footpad injectionsfor this Theeffec tofirrigation onthe test (as purposeis c onsideredunnec essary. For opposedto theeffectont hean imal)is popliteal lymphnode assays, most investi- dependenton th echemical, thecon- gatorsn owc onsiderthat injectioninto any centration, theti melag betweenexpo- tissuewh ichdrainsinto thelym phnode wi ll surean dinitiationof theirrigation, and give good results. Alternativeassays for skin onthe volum eofirrigation. Always sensitization have beend eveloped,such as weighthesefac tors carefully vis aÁ vis skin painting,local lymphnod eassay and

LaboratoryAnimals (2001) 35 38 Joint Working Groupon Renement mouseear swellingtest. To induceadjuvant injected into thefootpad, andnot into arthritis, intradermal andsubcutaneous othertissues in the foot. routesare nowc onsidered effective enough toreplacethefoot pad. It isdif® cult to®ndapractical way of 3.17Uncommon routes avoiding thefootpad routeofinjection for Thereare anumber ofroutesthat have lim- assessmentofanalgesic agents and investi- ited usefor speci®c scienti®c purposes(e.g . gations of mechanismsof pain andin¯am - intra-anal; intrabursal; intrahepatic;intra- mation.Suc hexperimentsare shortterm and neural; intra-ocular; intra-oesophageal/intra- may involveinjec tion ofhyperalgesic or gastric;intrapenile;intrarenal;intraspinal; in¯am matory agents (suchas prostaglandins intrathecal; intravesicular; peri-, epi- and orcarageenan)into thefootpad whichsetsup subdural; retrovulvar; and rumen®stula).The an in¯ammatory reaction, orm imicsit, and general principlesof best practicedetailed in sosensitizes theanim al toasubsequent Section2 ofthis report apply tothese,as do painful stimulus,suc has pawpressure. Paw many oftheproblem sandre®n ements swellingand pawwith drawal timecan then described intherest ofthereport. be measured. TheWorkin gGroupwould lik etohear Inth ecase of Mycobacterium leprae , from anyonec arryingout thesetech niques infectionhas t obe inthe footpads because witha viewto publishingan addendumto thisis theonlysite whichpermitsgrowth of thisreport. theorganism.

4Special considerationsfor wild animals Sum mary oftheprotocolforMycobacterium lepra e Aninc reasingnumber ofpublications Mycobacterium leprae isinoc ulated sub- addresst heuse of wildanimals for research, cutaneouslyinto theh ind footpads. The both int helaboratory and®eld. Information bacteria are containedin a salinesuspension isavailable withregard totheirc apture and and amaximumof 10 mlisinjec ted. The transport, theet hicsof wildanimal usean d resultinginfection islim ited tothefoot pads theec ological effect of removing animals and can be easily quanti®ed. Nomacroscopic (Bekoff 1995, Putman 1995, Tribe &Spiel- lesionsd evelop.In the c ase ofnudemice, man 1996, Gaunt &Oving1999, Hawkins et infectiond evelopsto ahigherlevel and some al.2001).Information ont headm inistration footpad swellingmay occur. ofsubstancesto wild animals is, however, sparsean dmainly relates toanaesthetic Injectinginto both footpads interferes regimens.T hesam eprinciplesregarding the withlocomotion,therefore never inject substanceandtechniqueapply as whenusing moret hanone foot. laboratory animals. Inaddition, thefollowing Monitor animals closely. Any animal pointsare important. showingswelli ngofthefootpad accom- panied by weightloss, or decreased ¯uid Whenad ministering substancesto wild intake, orsubdued behaviourpattern s animals that are toremain in, orbe and/orimpairmentof normal mobility returnedto, thewild, theirc ontactwith shouldbe humanely killed. peopleshould be kept toaminimum.If, Animals m ust be givensoft bedding. Grid however,th eanimal isto be retainedin ¯oorsshould not be used.Food andwater captivityandrepeatedlydosed, thenit shouldbe placedadjacentto thebed to becomesless stressful if thean imal is avoid themh aving towalk far ifthefoot familiarized and accustomedtopeople. ispainful (Wolfensohn&Lloyd 1998). Food rewards can be very helpfulin Limit thefrequenc yand severityof familiarization andtraining. adverse effectsby limiting thed ose Familiaritywiththenatural behaviour volumeand ensuringthat them aterial is and habitat ofthespeciesis essen tial in

LaboratoryAnimals (2001) 35 Administration of substances 39

order todecide theleast stressfulway of thesubstan ceto theirnormal diet (see handlingthem.Wild mice, for example, Section3.10.1 )orsmearing it ontotheirfur oftenpanic and leap about trying to wherethey are likely tolickthemselves escapeif handled inopen areas inbrig ht whengroom ing. For predatory speciesit is light,whilstth osehandled underdim or often possibleto presentt hesubstanc ein redlightingand allowed tostay inshadow dead prey.For example,a solutionorsus- are muchc almer.T his® nding iscommon pensionof thesubstanc eina 5% (bovine tomany nocturnal species. spongiform encephalopathyfree) gelatin Anaesthesia orsedation of wildspecies solution can be injectedintotheabdom enof priorto administrationof substancesis adead, pre-chilled, chick.Thegelatin willset desirable tominimize thestressof hand- insideth echickwithno leakage and this can ling associatedwithsuc hprocedures. thenbe fed totheani mal. Whenan injection Aftersedationthe anim al shouldbe left hast obe usedthenth esubcutaneousroute is quietlyalonewith little stimulation until usually theleast painful and least stressful. thed rug has taken effect.Thisc an often T he most stressful procedureis intraperi- be achieved simply by coveringthe c age toneal administrationand thisshoul dbe orcontainerwitha blanket. Full recovery avoided. Intramuscular injectionis generally from sedation must be ensuredbefore quickand lessstressfu lbut may involve releaseoth erwiset hean imals’ chanceof persistentlocalized pain ornec rosis, which survival will be reduced. insom especiesm ay lead toself-mutilation Many speciesof mammal willwill ingly and inabilitytoescapea predator should the go intoadark spacesuchas abag orbox animal subsequently be released tothewild. and useshould be made ofthisbehaviour toreduceoverall stressof atechnique. Acknowledgment Theauthors wouldlik etothank Thean imals may thenbe injected MrD.Rutty whoattendedasaHomeOf® ce Observer. throughthe bag orwith inthe box ifit is equipped witharestraining mechanism. For example,wild rabbits may be heldina Note: During the preparation of this report itcameto the WorkingG roup’s attention that EFPIA(European blackcloth bag with just theirears Federationof PharmaceuticalIndustries Associations) protruding and them arginal ear veincan andECVAM (European Centrefor the Validationof thenbe injected. Wild ratswillrun into Alternative Methods) werealso produc ing`A G ood blackbagsortubes and can thenbe PracticeGuideto the Administration ofSubstances transportedintoventilated fumecham- andRemoval of Blood,includin gRoutes and bers tobe anaesthetized by inhalation. Volumes’, tobepublished inthe Journal ofApplied Squirrelsoften retreat into anestbox To xicology.Both reports areaim edatensuring good,if not best,prac tice. whenapproac hedand can be transferred from thisintoaventilated chamber. Somebirds entera stateoftonic immo- References bility(TI)whilebein ghandled, orduring ALZACorporation (1990)Productreview:Neuro- procedures,whic hmay facilitatethe scienceNow. Nature 347, 784 administrationof asubstance.TIisgen- AnimalWelfare Inform ationC entre(199 7) Info rm a- erally regardedas an acutefear response tionResourcesforAdjuvants andAntibodyPro- and isnot astate of`hypnosis’, soit duction.C omparisons andAlte rnative Technologies1990±1997 ,AWICResourc eSeries shouldnever be deliberatelyinducedto No.3. USDA,USA keepbirds stillas this willc auseavoid - BarclayRJ, HerbertWJ, Poole TB,eds(198 8) Th e able stress.Bird scan also recoververy Disturb anceIndex:A BehaviouralMethodof rapidly from TIsorestraintshouldbe Assessing theSeverityo fCommonLaboratory maintained continuouslyuntil theproc e- ProceduresonRodents .Potters Bar:U FAW,p 36 dure iscomplete. Bekoff M(1995)Marking,trapping andmanipulati ng animals:some methodologicaland ethical c onsi- The least stressful way todosewild ani- derations.In: Wildlife Mam mals asResearch malsisvia the oral route by simply adding Models: In theLaborato rya ndFie ld (Bayne KAL,

LaboratoryAnimals (2001) 35 40 Joint Working Groupon Renement

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