Adrian Smith, Norecopa, Norway

National Consensus Platform for the Replacement, Reduction and Refinement of Animal Experiments

adrian.smith@norecopa.no

http://norecopa.no/Brno

A practical approach

to 3Rs policy

AnimalModelsforHumanDiseases:BestPracticesforinvivoresearchBrno,6-7June2016

Industry

GovernmentResearch

Animalwelfare

www.ecopa.eu

European Consensus-Platform for Alternatives

ecopa supports the establishment of National Consensus Platforms (NCPs) where all 4 stakeholders are equally represented:

Norecopa is a member of ecopa

The Three RsThe result of a national survey of animal experimentation where the degree ofinhumanity was assessed.

• ReplacementMethods which replace or avoid the use of animalsAbsolute replacement (non-sentient animals) or relative replacement (e.g. cellcultures)

• ReductionMethodstomaximise bestquality andprecise information fromtheminimumnumber ofanimals - better experimental designandstatistical analysis

• RefinementMethodstoimprove experimental procedures,andother factors such ashousing andcare,toreduce suffering andimprovewelfare throughout the animals’lives

’Thegreatest scientific achievements havealways been the mosthumane’– awin-win situation foranimals andscientists

www.frame.org.uk/tag/russell-and-burch

Content

A few words on practical ethics and animal welfareThe 3Rs in practiceQuality assurance in animal studies

Planning (including literature searches)Conducting experimentsReporting animal experiments

Guidelines and other 3R resourcesCultures of Care and Challenge

Practical ethics behind the 3Rs

The paradox of “inverse veterinary medicine”SpeciesismWhat is the animal really experiencing?Weaknesses in animals as research models

History repeats itself when developing new models

GMO & GMM, nude mice, minipigs, isolators, IVC racks,cleaner fish, zebrafish

Two sets of “guidelines” emerge, that address very different questions:

• Wow that was cool, how do you do it?• Hmm, should we really be doing this?

The first group are more technical specifications than guidelines in the 3R sense…

The second set tend to be running fast in an attempt to catch up with the first set.

Reporting

Literaturesearch

Planning

ResearchIdentify and ensure the quality of (at least) the critical points in the

experiment:critical for animal welfare and scientific value

What is an alternative?

A method without the use of animals that gives the same answers as an animal experiment.

Are these alternatives?

• Cell cultures• Use of bacteria to test carcinogenicity• Chemical analysis of biologically active compounds

N.B. Animal experiments are usually needed to develop and validate alternatives.

Replacement alternatives

3D modelsAudiovisual aidsComputer simulationsMannekins, simulators, robotsQSAR (Quantitative Analysis of Structure/Activity Relationships)Cell and tissue cultures, organoids, organ perfusion

High Throughput Screening (HTS), organs-on-a-chipBiochemical & immunological methods (RIA, ELISA) Hybrid DNA technique, GMMTrials on “lower” organismsAcute experiments (terminal anaesthesia)Trials on dead animalsTrials on humans (microdosing and medical imaging)Synthesis of new evidence from experiments that have already been

performed

Next generation Multi-Organ-Chip

- Norecopa -

Marx et al., Altern Lab Anim. 2012 Oct;40(5):235-57

Organoids

e.g.Mini-liversDr Meritxell Huch,CambridgeUniversity

Adultmousestemcellsexpandintofullyfunctioning3Dlivertissue.

Cellsfromonemousecouldbeusedtotest1000drugcompoundstotreatliverdisease,andreduceanimalusebyupto50,000.

http://www.nc3rs.org.uk/news/mini-livers-show-promise-reduce-animal-use-science

• Charts,slides,manuals• Models,simulators,mannekins• Computerprograms&CD-ROM• Films,video• Virtualreality• Ethicallysourcedcadavers• Experiments onhumans,meatproducers,

slaughterhousematerial, invitro tissue&plants• Preservedmaterial(formalin,latex)

• Replacementwithatheoreticalsession• Animals intheirnaturalsettingorinbriefperiodsof

captivity• Animals inneedofclinicalveterinarycare• Researchanimalsthatwillbeusedanyway• Surplusbreedersfromlabanimalsuppliers

Alternativesineducationandtraining

virtualphysiology.com3dglasshorse.com

rescuecritters.com limbsandthings.com

The potential for 3R alternatives cannot be evaluated until the objective of the study is known.

This applies to all use of animals in research, testing, education and training

e.g. Possible objectives in education & training:

• Teaching and practising:- laboratory skills- general animal handling skills- preparation-specific animal skills

• imparting good ethical thinking• new knowledge and reinforcing existing• data handling skills• experimental design skills• communication skills (oral, written)• groupwork• staff-student interaction

Rats from IKEA

AJ Smith & K Smith, 2004

Guidelines for humane education: Alternatives to the use of animals in

teaching and trainingProceedings of the 4th World Congress on Alternatives and Animal

Use in the Life Sciences, New Orleans, August 2002.

http://www.atla.org.uk/wp-content/plugins/s2member-files/32_S1a_3_Plenary_specialcontribution.pdf

(log-inrequired,pages16-26)

2) Reduction alternatives

A good statistician is the lab animal’s best friend.

Combined with methods to reduce background “noise”.

http://norecopa.no/norecopa/vedlegg/Berdoy-handout.pdf

Sources of background “noise”:

• Age, sex, weight• Stress, subclinical disease• Room temperature, animal cage• Environmental “enrichment”• Temporal differences between treatments• Climatic factors• Position of cage in the room• Experimenter• Animal Technician (weekend workers)• and many more

FRAMETrainingSchools

Portugal,30March-1April2015Norway,February2016

www.frame.org.uk/training-schools

NC3Rswebsite

http://nc3rs.org.uk/experimental-design

Guidelines fortheDesignandStatisticalAnalysisofExperimentsUsingLaboratoryAnimalshttp://ilarjournal.oxfordjournals.org/content/43/4/244.full

NC3RsExperimentalDesignAssistant(EDA)http://nc3rs.org.uk/experimental-design-assistant-eda

TextBase

http://norecopa.no/textbasehttp://www.uk.sagepub.com/books/Book242188?siteId=sage-uk&prodTypes=any&q=9781853155130

3) Refinement alternatives

”Simple” techniques?

Are they feasible? e.g. intramuscular injections

Photo:NMBU

http://blogs.discovermagazine.com/notrocketscience/2011/01/12/flipper-bands-impair-penguin-survival-and-breeding-success/#.VLU6_8Y7_wo

Photo:colourbox.com

Photo:T.Poppe,NMBU

”Simple” identification methods?Do they affect the animal?

photo:NMBU

Givearewardinsteadofapunishment

Inacrossoverstudy,eightNewZealandWhiterabbitseachunderwentfourdifferenttreatmentsofactualorshameartattooing,withandwithoutpriorapplicationofatopicallocalanaesthetic(lidocaine/prilocaine).Changesinimmediatebehaviour,heartrate,arterialbloodpressure,serumcorticosteroneconcentrations,facialexpressionandhomepenbehaviourswereassessed.ChangesinfacialexpressionwereexaminedtodeveloptheRabbitGrimaceScaleinordertoassessacutepain.TattooingwithoutEMLAcreamresultedinsignificantlygreaterstrugglingbehaviourandvocalisation,greaterfacialexpressionscoresofpain,higherpeakheartrate,aswellashighersystolicandmeanarterialbloodpressurecomparedtoallothertreatments.PhysiologicalandbehaviouralchangesfollowingtattooingwithEMLAcreamweresimilartothoseinanimalsreceivingshamtattooswithorwithoutEMLAcream.Behaviouralchanges1hourpost-treatmentwereminimalwithnopainbehavioursidentifiableinanygroup.Serumcorticosteroneresponsesdidnotdifferbetweenshamandtattootreatments.ConclusionsEartattooingcausestransientandpotentiallyseverepaininrabbits,whichisalmostcompletelypreventedbypriorapplicationoflocalanaestheticcream.TheRabbitGrimaceScaledevelopedappearstobeareliableandaccuratewaytoassessacutepaininrabbits.

GrimaceScales

PLoSONE,2012,7(9):e44437.doi:10.1371/journal.pone.0044437

Laboratory Animals, 2000, 34, 430-433

Thelonely mouse

Malemice housed singly develop symptomswhich would becharacterised as

depression inhumans.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111065

photo:colourbox.com

”Contingent suffering”indirect suffering

fearboredomtransportstress

http://www.tecniplast.it/usermedia/us/3-5LTankwithFish-small.jpg

Zebrafish?

AlternativesinproductionoflabanimalsDirectiveArticle18:

MemberStatesshallfacilitate,whereappropriate,theestablishmentofprogrammesforthesharingoforgansandtissues ofanimalskilled.

AninternationalworkshopintheNetherlands”Bredbutnotused”.2012:579,338animalsusedinprocedures524,735animalsbredbutnotused

J-BPrins&CHendriksen,9thWorldCongressonAlternativesandAnimalUse,Prague,August2014

J-BPrins&CHendriksen,9thWorldCongressonAlternativesandAnimalUse,Prague,August2014

Causing factors: Survey 2011 by NVWA

y Max 5% illness, microbial status, …

y 38% unsuitable genotype

y 23.3% wrong sex

y 21.6% age (discrepancy between production and demand)

y 13.5% surplus breeding (not longer suitable for experimental purposes)

Quality assurance at all levels of the animal facility.For example:

• SOPs describing good techniques, carried out by competent operators• Checklist (“contract”) between researcher and the facility• The AAALAC Program Description template* as an overall performance

checklist• Institutional policies on animal care and use• Animal environment, housing and management• Veterinary care• Physical plant

• A Master Plan as a weekly checklist for the whole facility during the year

*https://www.aaalac.org/programdesc/index.cfm

www.aaalac.org

A simple but effective Master Plan

‘Ourlongexperienceandmoderncoffeemachinesareyourguaranteeofquality’

Reporting

Literaturesearch

Planning

Research

Report (at least) the critical points in the experiment:critical for animal welfare and scientific value – otherwise other people will

repeat your mistakes!

Reporting has historically been poor:

Jane Smith et al. (1997): 149 papers in 8 journals from 1990-1991:

Parameters not mentioned:

Number of animals: 30%

How the animals were killed: 45%

Sex 28% Room temperature 72%

Age 52% Relative humidity 89%

Weight 71% Photoperiod 72%

Source 53% Number of animals/cage 73%

Many journals now offer supplementary online space

(generally unlimited) where more information about

the methods and results can be posted.

And most people have access to a website where this

could be posted...

SCID-Humiceimmunizedwithapneumococcalvaccineproducespecifichumanantibodiesandshowincreased

resistancetoinfection.

photo: NMBUhttp://www.theodora.com/rodent_laboratory/blood_collection.html

Saphenous vein puncture for

blood samplingof the mouse,rat,hamster,

gerbil,guinea-pig,

ferret andmink

and it does not have to be in a high-impact journal.

Scientific

Ethically acceptableLegal

The animals

Animal Department Head

TechniciansResearchers

The animals

A useful additional (but largely unknown) tool…Carol M. Newton (1925-2014)

The three S’s

• Good Science• Good Sense*• Good Sensibilities*

*We can do this ourselves without scientific literature!

CarolMNewton,quoted inRowsell HC(1977):TheEthics of Biomedical Experimentation inTheFutureof Animals,Cells,Models,andSystemsinResearch,Development,Education,andTesting pp.267-281,NationalAcademy of Sciences,Washington,D.C.,ISBN0-309-02603-2.

NationalLibraryofMedicine

Criticalanthropomorphism:=empathy + objective,knowledge-basedconsiderationofwhatislikelytobesignificanttotheanimal

Smith AJ & Hawkins P (submitted) The Three S’s of Carol Newton

A Culture of Care

All about

• Attitude• Not just lip-service, but actually implemented• Commitment to continual improvement• Constant challenge of the way things are done• Everyone involved and having responsibility• Education in 3R alternatives from the day staff join• Finding the time to discuss the present situation and

possible 3R solutions• Transparency – involve all stakeholders

http://ilarjournal.oxfordjournals.org/content/48/1/3.full

https://www.nc3rs.org.uk/news/creating-culture-care

A Culture of Challenge

’because we’ve always done it that way...’

Choose the acceptable, not the accepted

Think ”3R-Alternatives” at all stages

• Breeding• Transport• Acclimation• Procedures, e.g. choice of

• dose• method of administration• methods of data collection (blood sampling, body temperature,

heart rate, blood pressure etc.)• Pilot studies

ConsultthetechniciansfromDay1:• theyknowthepossibilities(andlimitations)intheanimalfacility• theyoftenpossessalargerangeofpracticalskillsandaregoodatlateral

thinking• theyknowtheanimalsbest• theanimalsknowthembest

Focus on Alternatives (FoA)

A consortium of UK animal welfareorganisations

http://norecopa.no/media/6663/earlyplanningposter.pdf

and

http://norecopa.no/media/6672/

investigationposter.pdf

Reporting

Literaturesearch

Planning

ResearchCorrect literature searches are a vital part of

the work to advance the 3Rs

Are scientists good at looking for alternatives?

• Bibliographic databases are often not used adequately (poor overlapping)• Too few scientists are aware of the specialist databases on alternatives• Scientists rarely use ”3R” words when they write titles/abstracts/keywordsfor their papers• This is a major problem because we have no single ”Journal ofAlternatives”

AliceTillema,Radboud University:Howtoconstructaliteraturesearch

http://norecopa.no/how-to-construct-a-literature-search.pdf

“What’s the problem? We have the internet!”

The Internet doesn’t give us everything we need to know on alternatives

colourbox.com

The Surface Web

The Deep (Invisible) Web

TheSurfaceWebisusefulfor

• Searchingforaspecificdocumentwhichweknowexists• Lookingforastarting-pointforinformationonaspecifictopic• Finding”greyliterature”(e.g.unpublishedreports)

TheDeepWeb

ManythousandtimeslargerthantheSurfaceWeb,materialmaybe:

• Encrypted• Registration/subscription• Password-protecteddatabases• Notformattedfor,oraccessibleby,standardsearch

enginese.g.textinimageorvideofiles• Materialoncompanyintranets

colourbox.com

TheEURLECVAMSearchGuide

Canbeorderedfreeofchargefrom

bookshop.europa.eu

Contents

• DataRetrievalProcedures(basicprinciples)

• Check-listforsearchingforinformationonalternativemethods

• Tablescomparingthefeaturesof• Databases• Journals• Organisations

Contents

SevenGoldenStepstoSuccessfulSearching

1. Clearlydefineandbeawareofyourspecificinformationneed2. Identifythefundamentalcomponentsofyourscientific

approach3. Choosethemostappropriateinformationresources4. Compile relevantandnecessarysearchterms5. Startyoursearchwithasimplequeryina3Rsspecificcontext6. Limitsearchresultsfrommoreextensiveresources7. Broadenthesearchhorizon

Archiveyoursearches soyoucandocumentthemandavoidrepeatingthem

http://norecopa.no/search?q=bloodsampling%20mouse

Archivekeydocuments youretrieve,e.g.inMendeley

Searchstrategiesinanutshell

•Definethesearchaswellaspossible•Identifysynonymsand3Rterms•Remember thedifferences betweenBritish&AmericanEnglish•Useseveraldatabases(littleoverlapping)•Learnthedifferences betweenthesearchengines(readthe

instructions!)•GetusedtousingBooleanlogicandcheckwhichtermsaresupported

bythesearchengine•Learnhowtoexpand/narrowyoursearch•Lookforcorearticlesandkeyauthors•Use thepossibilitiesontheInternet togetintouchwiththebest

research labs

colourbox.com

Some search guides:

The EURL ECVAM search guide (2013):http://bookshop.europa.eu/en/the-eurl-ecvam-search-guide-pbLBN124391

CCAC Three Rs Search Guide:http://3rs.ccac.ca/en/searches-and-animal-index/guide

AltWeb: A step-by-step approach to an alternatives search:http://altweb.jhsph.edu/resources/searchalt/index.html

UC Davis guide to bibliographic databases for alternatives searching:http://lib.ucdavis.edu/dept/animalalternatives/databaseapproach.php

IMPI I3R working party report on Searching for 3Rs Information – Published Literature Sources (2002):http://www.impi.org.uk/i3r_v2_jul2002.pdf

Sources of 3R resources

• National 3R centres

• 3R congress proceedings

• Guidelines papers

• Databases

• Journals

• Discussion groups

• Training schools

National3RsCentres

www.nc3rs.org.uk

Animalwelfareorganisations

www.rspca.org.uk/sciencegroup/researchanimals

Centresgivinginformationonalternatives

UCCAAUniversity of CaliforniaCenter for Animal Alternatives

www.lib.ucdavis.edu/dept/animalalternatives

awic.nal.usda.gov

1996:2ndWorldCongressonAlternativesandAnimalUseintheLifeSciences,Utrecht

1997: Altweb (Alternatives to animals on the web)

http://altweb.jhsph.edu

Theworldcongressesonthe3Rsareimportant3R-driversanddisseminatorsofinformation:

wc9prague.org891abstracts,49countries,1000participants

(thenextoneisinSeptember2017inSeattle)

ec.europa.eu/animals-in-science

Expert Working Group report on severity classification

http://ec.europa.eu/environment/chemicals/lab_animals/pdf/report_ewg.pdf

Guidance on the severity classification ofprocedures involving fish

Report fromaWorking Groupconvened byNorecopa

DesignedtobeasupplementtotheEUWorkingGroupreportonthesamesubject,whichmostrelevantfor

traditional labanimals(http://ec.europa.eu/environment/chemicals/lab_animals/

pdf/report_ewg.pdf)

PHawkins,NDennison,GGoodman,SHetherington,SLlywelyn-Jones,KRyderandAJSmith

Laboratory Animals, 45: 219-224, 2011www.norecopa.no/categories

norecopa.no

Guidelines bleeding mice

Auto-complete function:

Synonym list:Bleeding, bloodsampling, blood sampling, venepuncture,blood collection, phlebotomy

Search engine

Redlinks:externalBlue link:internal

Searchfor‘bleedingmice’onGoogleandNorecopa.

‘Bleeding’notmentioned,butidentifiedbythesynonymlist

https://kmonadollaraday.fil es.w ordpr ess. com/2011/03/i nformation-sil os.j pg

Ourvision:Toaiddisseminationof3Rresourcesbetweenthedifferentscientificfields

Therearelotsofplatforms...

...butarethereenoughtrains?

Norecopa aimstobeafasttraintoglobal3Rresources

Photo: http://www.bbc.com/news/uk-engla nd-london-35882068http://www.london-gifts. co.uk

Thankstoourmainsponsors:

• Standing Committee on Business Affairs, Norwegian Parliament• Ministries of Agriculture and Fisheries• Novo Nordisk• Nordic Society Against Painful Experiments• Scottish Accreditation Board• Dag S. Stiansen Foundation• Laboratory Animals Ltd.• Norwegian Research Council

FundingoftheNORINAdatabase1991-2016:

NordicSocietyAgainstPainfulExperiments,DagS.Stiansen Foundation,TheNorwegianResearchCouncil,theNorwegianSchoolofVeterinaryScience,LaboratoryAnimalsLtd.,RSPCA,UFAW,AstraZeneca,SolvayPharmaceuticals,theSwedishFundforResearchwithoutAnimalExperiments,NorwegianFederationforAnimalProtection,Allkopi,TheHumaneSocietyoftheUnitedStates,St.AndrewAnimalFund,MicrosurgicalDevelopmentsFoundation,AAALACInternational,LASA,NEAVS,AmershamHealth