ab oratory

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lan imals

Working Party Report

Classification and reporting of severityexperienced by animals used in scientificprocedures: FELASA/ECLAM/ESLAVWorking Group report

David Smith1, David Anderson2, Anne-Dominique Degryse3,Carla Bol4, Ana Criado5, Alessia Ferrara6,Nuno Henrique Franco7, Istvan Gyertyan8, Jose M Orellana9,Grete Ostergaard10, Orsolya Varga11 and Hanna-Marja Voipio12

AbstractDirective 2010/63/EU introduced requirements for the classification of the severity of procedures to be appliedduring the project authorisation process to use animals in scientific procedures and also to report actualseverity experienced by each animal used in such procedures. These requirements offer opportunities duringthe design, conduct and reporting of procedures to consider the adverse effects of procedures and how thesecan be reduced to minimize the welfare consequences for the animals. Better recording and reporting ofadverse effects should also help in highlighting priorities for refinement of future similar procedures andbenchmarking good practice. Reporting of actual severity should help inform the public of the relative severityof different areas of scientific research and, over time, may show trends regarding refinement. Consistency ofassignment of severity categories across Member States is a key requirement, particularly if re-use is con-sidered, or the safeguard clause is to be invoked. The examples of severity classification given in Annex VIII arelimited in number, and have little descriptive power to aid assignment. Additionally, the examples given oftenrelate to the procedure and do not attempt to assess the outcome, such as adverse effects that may occur. Theaim of this report is to deliver guidance on the assignment of severity, both prospectively and at the end of aprocedure. A number of animal models, in current use, have been used to illustrate the severity assessmentprocess from inception of the project, through monitoring during the course of the procedure to the finalassessment of actual severity at the end of the procedure (Appendix 1).

KeywordsAnimals, severity, humane end-point, refinement, ethics & welfare, procedures

Date received: 31 October 2017; accepted: 1 November 2017

Introduction

Implementation of Directive 2010/63/EU has imposedadditional requirements related to the severity of pro-cedures carried out on animals for scientific purposes.Procedures need to be assigned a severity classificationprospectively and the actual severity experienced byeach animal during the course of a procedure has tobe determined and reported in the statistical informa-tion made publicly available annually (CommissionImplementing Decision 2012/707/EU, as amended by2014/11/EU).1,2

Good project planning is necessary to determine asuitable prospective severity classification and to

1FELASA, Federation for Laboratory Animal Science Associations,Eye, Suffolk, UK2LASA, PO Box 524, Hull, HU9 9HE, UK3Domaine de Mirabel, Puylaurens, France4Charles River Laboratories, ’s-Hertogenbosch, the Netherlands5Via Fleming 4, Verona, Italy6Aptuit, Verona, Italy7Instituto de Investigacao e Inovacao, Universidade do Porto,Portugal8Semmelweis University, Budapest, Hungary9Universidad de Alcala Campus, Universitario Alcala de Henares,Madrid, Spain10University of Copenhagen, Denmark11University of Debrecen, Hungary12University of Oulu, Finland

Corresponding author:David Smith, FELASA, Federation for Laboratory Animal ScienceAssociations, PO BOX 372, Eye, Suffolk, IP22 9BR, UK.Email: davidsmith1944@btinternet.com

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develop appropriate observational monitoring andassessment criteria and humane end-points, tailoredto the study.3,4 Sufficiently trained and competentstaff are an absolute requirement to assess animal wel-fare during the course of the study.

There needs to be an observational strategy and acommon recording system that captures all the necessarydata in a consistent format to facilitate continued appli-cation of refinement and enable an assessment of actualseverity to be made. This paper provides the rationale ofwhy such assessment is necessary and who is responsiblefor it. It then develops several real examples of animalprocedures of how to do so and how this then allowsevaluation of the actual severity score for each individualanimal that has gone through the procedures.

Annex VIII of the Directive has included some add-itional guidance on prospective severity classification tohelp achieve some common interpretation of theDirective’s intentions.

The examples give little information on how the severityclassification was derived and give a mix of simple singlestep procedures, such as short term restraint in ametaboliccage and more complex procedures which comprise mul-tiple steps such as organ transplantation requiring anaes-thesia, surgery and management of organ rejection.

Although the assignment criteria (set out in SectionII of Annex VIII) indicate that each study needs toconsider a range of factors before a classification ismade, many within the scientific community haveraised concerns that the examples provided in AnnexVIII contain insufficient information to satisfactorilyexplain the rationale for the severity classification andthat no examples are provided for some importantareas of research, such as, for example, pain and arth-ritis. Without additional explanation, there are likely tobe considerable differences in the assignation of sever-ity, which may ultimately give misleading informationon animal use, and, perhaps of greater concern, resultin inappropriate re-use of animals. A further potentialconcern related to animal models of pain is that it ispossible for the same model to be categorised across atleast two severity classifications, depending on therefinements in the procedure. In addition to the appli-cation of early end-points, the degree of amelioration ofpain, distress and suffering is a major factor.

During 2012, members of the FELASA/ECLAM/ESLAV Working Group contributed to discussions at ameeting arranged by the European Commission on sever-ity classification, and using material developed by thisgroup, assisted in the development of some additionalguidance and a few examples of severity classificationwhich were endorsed at a National Contact Point(NCP) meeting and can be found at the EC website.5

A main purpose of this joint FELASA/ECLAM/ESLAV report is to provide additional information

and guidance on prospective severity classificationand assessment of severity experienced by the animalsduring the course of a procedure (actual severity),through a number of further illustrative examplesfrom different fields of research drawing on existingsystems, providing examples of different severities andexpanding on the examples provided in Annex VIII. Anumber of ‘severe’ models have been deliberatelyincluded to illustrate areas of animal use in scientificwork not included in Annex VIII and to facilitate shar-ing and dissemination of good practice.

Models have been chosen which, at the time of prepar-ation of this report, were in use in fundamental and appliedresearch, togetherwith some examples used in safety evalu-ation. The examples include some information on howseverity can be reduced through application of refinementstrategies. Additional suggestions for refinement wereincorporated following review by the parent organisations.

Although the illustrative examples are representative ofcurrent practices, the principle remains that whenever theuse of any animal model is proposed, each component ofthe study should be reviewed and challengedwhere appro-priate to ensure that all 3R opportunities are applied.

With these additional requirements in 2010/63/EU, itis important for all involved in the use of animals,including those responsible for project evaluation, todevelop and agree a common understanding of andapproach to ‘severity classification’ in order to promotea ‘level playing field’ within the European research com-munity. This should thus ensure a consistent reportingof the severity experienced by the animals during theprocedures in the statistical returns on animal use.

The regulatory framework

The new Directive 2010/63/EU on the protection ofanimals used for scientific purposes was approved on22 September 2010 and took full effect in MemberStates on 1 January 2013.

As with Directive 86/609/EEC, the new Directiverequires that experiments are designed to cause theleast pain, suffering, distress or lasting harm.

All scientific procedures will be conducted under aproject authorisation approved in each Member State(MS) by the Competent Authority (CA). All thoseapplying for project authorisation will need to includean estimate of the likely severity of each procedure.These severity estimates will be considered by the CAduring the project evaluation process undertaken,before a decision on project authorisation is made.Having considered the information provided in theapplication, the CA will assign a severity classificationto each procedure (Article 38).

The actual severity experienced by each animalduring each individual procedure will be reported by

6 Laboratory Animals 52(1S)

each MS (Article 54(2)) annually, in the year in whichthe procedure is completed.

Furthermore, the actual severity experienced by ananimal in any previous procedures will be a key consid-eration in determining whether or not an animal maybe re-used in further procedures (Article 16). Animalsmay only be re-used provided that the severity of theprevious procedure was ‘mild’ or ‘moderate’; that theanimal’s general state of health and well-being has beenfully restored; that the further procedure is classified as‘mild’, ‘moderate or ‘non-recovery’ and is in accord-ance with veterinary advice, taking account the lifetimeexperience of the animal. The lifetime experience of theanimal includes all aspects of health, welfare and careand the impact of all scientific procedures. The combin-ation of all these effects may be considered as ‘cumula-tive’ severity. In exceptional circumstances, by way ofderogation by the CA and after a veterinary examin-ation, an animal which has previously experiencedsevere pain, distress or equivalent suffering may bere-used.

Article 3 defines a procedure as ‘any use, invasive ornon-invasive, of an animal for experimental or other sci-entific purposes, with known or unknown outcome, oreducational purposes, which may cause the animal alevel of pain, suffering, distress or lasting harm equivalentto, or higher than, that caused by the introduction of aneedle in accordance with good veterinary practice’.

This defines a lower ‘threshold’ for a scientific pro-cedure below which project authorisation will not benecessary. This definition gives an indication of thelevel of pain which could be considered as a ‘threshold’,but there is no information given on equivalent thresh-olds for suffering, distress or lasting harm.

Since the adoption of the Directive, EU guidance onseverity assessment was developed and endorsed in2012 and additional information to promote consistentreporting was included in a discussion paper from theNCP meeting in January 2016.6,7

Why do we need a severity classificationsystem?

The inclusion of a severity classification systemwithin thenew Directive provides an opportunity to focus continu-ously on refinement from inception to completion of aprocedure, improving the quality of science and animalwelfare, and, by the inclusion of the actual severity experi-enced by each animal during a procedure in the StatisticalReports, providing greater transparency and promoteimproved public confidence in the use of animals inresearch. Over time, these publications may provideinformation on trends in refinement.

A number of European countries, including Finland,Germany, Ireland, The Netherlands, Poland, Sweden,

Italy, Switzerland and the UK, and Australia, Canadaand New Zealand have, for a number of years, had inplace systems to categorise the severity of animal studies.

Many of the existing systems report prospectively,with the number of categories varying from 3 to 9.8

None of the systems, however, use the combination ofprospective, actual and cumulative suffering or the clas-sifications included in the new Directive. Assignment ofprospective classification and reporting of actual severityare necessary to enable comparison during retrospectivereview of a project, where such review is required.

The severity categories are defined in Annex VIII ofthe Directive as follows:

The severity of a procedure shall be determined bythe degree of pain, suffering, distress or lasting harmexpected to be experienced by an individual animalduring the course of the procedure.

Non-recovery:Procedures, which are performed entirely under gen-eral anaesthesia from which the animal shall notrecover consciousness shall be classified as non-recovery.Mild:Procedures on animals as a result of which the ani-mals are likely to experience short term mild pain,suffering or distress, as well as procedures with nosignificant impairment of the wellbeing or generalcondition of the animals shall be classified as mild.Moderate:Procedures on animals as a result of which the ani-mals are likely to experience short term moderatepain, suffering or distress, or long-lasting mildpain, suffering or distress as well as procedures thatare likely to cause moderate impairment of the well-being or general condition of the animals shall beclassified as moderate.Severe:Procedures on animals as a result of which the ani-mals are likely to experience severe pain, suffering ordistress, or long-lasting moderate pain, suffering ordistress as well as procedures, that are likely to causesevere impairment of the wellbeing or general condi-tion of the animals shall be classified as severe.

Note: There is the possibility with exceptional andscientifically justifiable reasons for Member States toadopt a provisional measure to permit the use of aprocedure involving severe, pain, suffering or distressthat is likely to be long-lasting and cannot be ame-liorated. Any such provisional measures must beconsidered and approved by an EU committee forsuch work to continue (Article 55).

Smith et al. 7

An estimate of severity expected to be experienced bythe animal has to be given for each scientific procedure.This requirement provides an opportunity during thedesign of the study to consider the application of the3Rs and to ensure that the severity is reduced as far aspossible within the scientific constraints of the study.9,10

This consideration of severity should thereforebenefit animals by reducing suffering, and may alsoimprove robustness of scientific design by giving oppor-tunities to consider the effects of the procedures on, forexample, physiology or behaviour where, for example,deteriorating health/welfare could affect outcomes, andways by which such changes can be minimised toimprove the quality and consistency of data.

The classification will furthermore help to defineclear upper limits on animal suffering, and thus canassist in the implementation of humane end-points.

The 3Rs should continue to be reviewed as the pro-ject develops both by those directly involved in the useof animals and by the Animal Welfare Body (AWB – asdetailed in Article 27 of the Directive).

When required, a retrospective assessment (RA) of aproject gives a further opportunity to review the welfarecosts/harms to the animals, to determine whether theobjectives have been met, and to re-consider the appro-priateness of the severity classification, prior to anyfuture study.

Who determines the severityclassification?

The application for a project authorisation by the useror the person responsible for the project requires that aproposed severity classification is included for each pro-cedure (Annex VI).

The CA which conducts the project evaluation(Article 38) shall include an ‘assessment and assignmentof the classification of the severity of procedures’. TheCA will consider expertise in relevant scientific areas,experimental design, laboratory animal science or wild-life veterinary practice and animal husbandry and care,as appropriate for the project proposal.

Prospective severity classification is assigned to theprocedures by the CA during project evaluation, andthis shall be based on the most severe effects likely to beexperienced by an individual animal after all refine-ments have been applied.

The AWB (Articles 26 & 27) is required to follow thedevelopment and outcome of projects and to advise onopportunities for the application of the 3Rs withinthese projects.

The actual severity of procedures will be reported byMS in the annual statistical returns. This reflects thehighest severity experienced by the animal as a conse-quence of the procedure. Such information will be

provided by the user or the person responsible for theproject, informed as necessary by input from scientists,care staff, veterinarians/suitably qualified expertsinvolved in the project.

The National Committees for the protection of ani-mals used in scientific procedures (Article 49) areexpected to promote and share best practices withinthe European Union. An important aspect of theirrole will be to promote consistency with regard toseverity assessment.

Terminology

Some clarification and standardisation in terminologyis necessary to ensure a common approach is takento the assessment and assignment of severityclassification.

The Technical Expert Working Group (TEWG) con-vened by the European Commission in 2003 to considervarious aspects of the composition of the new Directivemade several recommendations with regard to the ter-minology that should be used. These have not beendirectly transposed and the lack of further explanationin Annex VIII has contributed to further confusion onwhat aspects of the procedures (within a project) haveto be assessed for severity.

Directive 2010/63/EU – Article 3

‘procedure’ means any use, invasive or non-invasive,of an animal for experimental or other scientific pur-poses, with known or unknown outcome, or educa-tional purposes, which may cause the animal a levelof pain, suffering, distress or lasting harm equivalentto, or higher than, that caused by the introduction ofa needle according to good veterinary practice. Thisincludes any course of action intended, or liable, toresult in the birth or hatching of an animal or thecreation and maintenance of a genetically modifiedanimal line in any such condition, but excludes thekilling of animals solely for the use of their organs ortissues;‘project’ means a programme of work having adefined scientific objective and involving one ormore procedures;

The recommendation from the TEWGAuthorisation Sub-Group was to separate the defin-ition of a project from an ‘experiment’ and recom-mended that the term ‘procedure’ should be usedrather than ‘experiment’, to include both procedureswith known outcomes (e.g. procedures concerned withthe production of antibodies) and with unknown out-comes (e.g. a procedure conducted to test a hypothesis).

8 Laboratory Animals 52(1S)

This concept was included but the further recom-mended division was not.

Technique: A technical act on one or more animalsfor an experimental or other scientificpurpose and which may cause thatanimal or those animals pain, suffering,distress or lasting harm. Examples oftechnical acts would be gavage, injec-tion, laparotomy, withholding of food/water.

Procedure: A combination of one or more tech-nical acts carried out on an animal foran experimental or other scientific pur-pose and which may cause that animalpain, suffering, distress or lasting harm.

Project: A coherent programme of work aimedat meeting a defined scientific objectiveor objectives and involving a combin-ation of one or more procedures.

At a NCP meeting in October 2011, the concept of asingle and multiple-step procedure was preferred to theuse of ‘technique’, as this term is not included in theDirective.

The term ‘procedure’ should, therefore, be used todescribe the complete series of steps (techniques) thatneed to be applied to complete a particular experimen-tal or other scientific purpose. Some procedures mayinclude only a single step (technique) (e.g. withdrawalof blood from ‘normal’ animal to enable in vitro stu-dies), but the majority will include a number of steps(techniques) (e.g. a vaccine challenge study couldinvolve injection of vaccine, exposure to an infectiousagent, and sampling or biopsy of tissues).

Annex VIII in the Directive provides a number ofexamples of types of procedures in the different severitycategories, and includes a mix of single step proceduresand multiple-step procedures.

To determine the severity of a procedure, consider-ation will need to be given to the contribution to the over-all severity made by each step (technique) (and theconsequences of each step) within a procedure. For exam-ple, when injecting a substance, consideration needs to begiven on the impact of the injection itself, and also on anysubsequent effects of the substance being injected.

Prospective severity classification

The final classification of a procedure will be deter-mined by the most severe effects expected to be experi-enced by any individual animal – this then provides aprospective estimate of the highest level of suffering

likely to be encountered for any single animal withinany given procedure. This information may be helpfulin determining an appropriate monitoring strategy forthe animals and defining suitable humane end-points.

Actual severity reporting

In contrast to prospective classification, the actual(highest) severity experienced by each individual

animal during the course of a procedure will need tobe determined, using observations recorded during thecourse of the procedure, with the actual severity subse-quently reported in the annual statistical returns.

There will therefore likely be differences in severitybetween prospective severity classification for the pro-cedure and the actual severity reported for each of theanimals used in the procedure.

‘Below threshold’ for regulation

Directive 2010/63/EU defines a ‘procedure’ as ‘an inter-vention which may cause the animal a level of pain, suf-fering, distress or lasting harm equivalent to, or higherthan, that caused by the introduction of a needle accord-ing to good veterinary practice’.

Annex VIII gives some examples that fall below thisthreshold. These include minimal restraint of habitu-ated animals, application of external telemetry devicesand minor dietary manipulations, including variationsin composition and availability, provided these are notexpected to cause any adverse effects.

The Annex also notes that consideration needs to begiven when a frequency or combination of ‘belowthreshold’ interventions may result in a cumulativeeffect which leads to the classification of the procedureas ‘mild or higher’.

For example, scientific investigations into novel hus-bandry practices which involve significant changes tocleaning frequency and disruption to social groups arelikely to cause some suffering and distress and thereforewould require project authorisation.

Severity assessment of procedures andthe harm–benefit analysis of projects

The harm–benefit analysis that is required for projectevaluation (Article 38) will take into consideration thelikely impact of all animals used within the project, andtherefore needs to take account of all potential harmsto all animals.

In contrast, the severity classification of each proced-ure will give an indication of the limit of the suffering toa single animal used within the procedure.

It follows therefore that the information needed forproject evaluation needs more detail on the welfare

Smith et al. 9

harms to all animals than that provided by a simpleseverity classification applied to the procedures con-tained within the project.

For example, in a vaccination challenge study, someanimals (unvaccinated controls) may experience severeclinical disease, requiring an assignment of ‘severe’ tothe procedure.

However, and in contrast, it would be expected thatthe majority of the animals given an effective vaccine arelikely to experience no more than mild adverse effects.

This detailed understanding of the likely impact onall the animals to be used in a project is necessary topermit an informed harm–benefit analysis.10

In the example above, where the project consists ofdeveloping a novel vaccine against a disease that isassociated with high mortality, the harm–benefit ana-lysis is likely to be in favour of the project. Of course,this would be dependent on consideration of manyother factors, such as experience and implementationof the 3Rs in the procedures.

Planning of a procedure

The applicant should discuss the project proposal withthe veterinarian (or suitably qualified expert wheremore appropriate), care staff and/or AWB to considerthe procedures to be applied, the opportunities to applythe 3Rs, for example appropriate dosing and samplingstrategies, and to agree appropriate monitoring/assess-ment criteria, interventions to minimise suffering andwhere applicable humane end-points.

All those involved in severity assessment should havea sound understanding of animal behaviour and wel-fare and of the indicators of poor welfare, pain andsuffering in the species being used.4

This process ensures that all personnel involved inthe studies have an opportunity to contribute to thestudy design, and to ensure that all are aware of thepotential adverse effects, the animal monitoring whichwill be in place and the methods to be implemented tominimise suffering.

Effective teamwork among all those involved isnecessary to ensure consistent interpretation and mini-misation of suffering compatible with the scientificobjectives.

Training in severity assessment

Although the project leader will be responsible forreturning the data on actual severity to the CA, oftenthe assessment of actual severity will be undertaken bythose directly involved in carrying out procedures andobserving and caring for the animals.

Ensuring that all those involved have been appropri-ately trained and have a good knowledge of normal and

abnormal behaviour in the species/strain being used istherefore essential.

How should severity be assessed?

Prospective severity classification ofprocedures

Many factors have to be taken into consideration inorder to determine a suitable severity classification fora procedure.

Although assessment is largely subjective, as morescientific information becomes available, our under-standing of how to recognise pain, suffering and dis-tress improves, and it is therefore important to remainabreast of developments in this rapidly evolving field ofresearch.

Some examples of severity classifications of proceduresare included in Annex VIII of the Directive 2010/63/EUand in the endorsedEUSeverity Assessment Framework.

When determining an appropriate severity classifica-tion, it is necessary to consider the impact on theanimal of each step of the planned procedure:

What is being done to the animals?

What effect will this have on the animals?

How much suffering may it cause?

What interventions can be included to reduce the impact

on the animals?

(i) What is being done to the animals?

Consider all the steps involved in the procedure:

. Changes to normal environment, husbandry andcare practices

. Conditioning/training; handling and restraint

. Administrations/injections of substances – routes,volumes, frequency

. Sampling – what is being sampled, from where is thesample being taken, how much and how frequentlyare the samples being taken

. Surgical and other invasive interventions

. Use of anaesthesia – local, general, regional and/oranalgesia

. Duration of study

. In the case of work in the wild – method and fre-quency of capture, accidental capture of non-targetspecies, temporary housing, etc.

(ii) What effect will these interventions have on theanimals?

Changes to the environment, husbandry and carepractices may initially impact animal welfare and

10 Laboratory Animals 52(1S)

cause changes in behaviour (e.g. increased aggres-sion), but habituation of the animals will reduce poten-tial distress.11 The same is true for handling andrestraint procedures. Behavioural conditioning ofthe animals, such as adaptation to handling or struc-tured positive reinforcement training may preparethe animals better for procedures and mitigate theseeffects,

Administration of substances and sampling proced-ures may have a negative impact on welfare, in theshort or long-term dependent on the routes, volumesand the effects of the administered substances.

Surgical interventions are likely to cause some pain,even with good peri-operative care (including the use ofanalgesics).

(iii) How much suffering will these interventions cause?

Consideration needs to be given to all the individualelements, and how these will interact.

The nature, intensity and duration of each interven-tion will impact on the overall severity.

The frequency of interventions and recovery timebetween interventions also need to be considered.

Duration of the study is an important factor to con-sider and the period over which the animal may experi-ence pain, suffering or distress. For example, in a safetyevaluation/toxicology study, depending on the dose, anacute study may cause major discomfort as a conse-quence of drug administration but this would generallybe of short duration. In contrast, an animal may beexposed to contaminated material (e.g. scrapie/BSE)as a juvenile with no initial adverse effects and, due tothe very long incubation period, will remain in goodhealth until the onset of clinical disease.

In chronic toxicology studies, animals may experi-ence minor/moderate adverse effects over many monthsas a combination of daily dosing and the effects of thetest substance.

For work in the wild, a careful scrutiny of the projectauthorisation is extremely relevant, as severity classifi-cation of only animals used and as described underproject authorisation is to be reported.7

When, in agreement with Article 10, exemptions aregiven to use wild animals captured from nature, it isthen very important to check whether capture from the

wild is considered as part of the scientific procedure andthus that the severity reporting of that action needs tobe included. When capture and transport are not part ofthe project (e.g. 100 fish captured and transported to alab where they will be allocated to several projects overa period of time and two fish die during capture processdue to injury in nets) the severity is not to be reported, as

neither the capture nor the transport are a specific com-

ponent of the scientific objective.

However, if ‘taking animals from the wild’ is one of the

scientific objectives (e.g. effect of capture and transport on

behaviour of animals) the severity observed must be takeninto account in the reporting of the actual severity.

It goes without saying that the appropriate welfareduring capture and transport under the Directive mustbe ensured: the capture may only be carried out bycompetent person(s), using methods which do notcause avoidable pain, suffering, distress or lastingharm; animals must be transported under appropriateconditions using appropriate methods of containment;any animal found to be injured or in poor health shallbe examined by a veterinarian or other competentperson and actions shall be taken to minimise suffering.Special considerations shall be given and appropriatemeasures taken for the acclimatisation, quarantine,housing, husbandry, and care of animals taken fromthe wild and, as appropriate, equally provisions forsetting them free at the end of procedures.

(iv) What interventions can be included to reduce theimpact on the animals?

How can suffering be minimised? How are the prin-ciples of the 3Rs embedded in the procedure/project?

. Plan to minimise disruption to accommodation, hus-bandry and care practices.

. Develop processes for acclimatisation, implementtraining programmes as appropriate.

. Consider dosing and sampling procedures to minim-ise impact on animals. For example, mini-pumpadministration may have reduced adverse welfareimpact compared with multiple daily dosing, whichmay require stressful restraint and acclimatisation tohandling. However, this needs to be balanced withthe need for surgery and the relative size of theimplant which may impact on locomotion and/orbehaviour.

. Surgical interventions must be accompanied byeffective peri-operative care, and appropriate pre-emptive and post-operative analgesia.

. During the planning of procedures, consider thedevelopment of welfare monitoring/scoring systemsand identifying likely clinical effects and determiningearly end-points (consistent with scientificobjectives).

Each element of a procedure should be challenged toensure that the 3Rs have been properly addressed.These issues should be considered initially by theProject Applicant, and should be informed by discus-sions with the veterinarian and animal care staff, andthe AWB before the details of the procedure are fina-lised and the severity assigned in the application.

Smith et al. 11

Prospective discussions with all relevant personnelwill ensure that the most refined procedure is developed.

Discussions should continue throughout the proced-ure to ensure that all opportunities are taken to furtherdevelop and adopt refinements as these become avail-able, for example through new publications.

Retrospective assessment (RA) andassessment of ‘actual’ severity

As indicated earlier, there are requirements in theDirective for the assessment of actual severity experi-enced by each animal and, for certain projects, arequirement that an RA shall be performed (Article 39).

Assessment of actual severity is necessary for anumber of reasons:

. to provide information for the annual statisticalreturns on animal use;

. to enable consideration of requests for re-use ofanimals;

. to contribute to the RA of projects (where required).

RA, in addition to the consideration of actual sever-ity experienced by the animals, also requires consider-ation of whether or not the objectives of the Projecthave been achieved, and whether further opportunitiesfor implementation of the 3Rs have been identified. Allprojects using non-human primates and all those invol-ving ‘severe’ procedures must undergo RA.

RA may be required for other projects – these aredetermined by CA during project evaluation and theapplicant informed where and when RA is required.

In order to be able to determine ‘actual’ severitythere is a need to develop recording and assessmentsystems, tailored to each project which will capture allthe necessary information in a format which will facili-tate subsequent assessment and categorisation of theactual severity.

Development of a system for the monitor-ing and assessment of welfare

There are many publications that offer guidance on theassessment of welfare in animals undergoing scientificprocedures (see references below). The Working Grouprefers readers in particular to three articles on the cre-ation and use of follow-up and evaluation sheets,namely articles by Morton, Buchanan-Smith and thereport by the BVAAWF/FRAME/RSPCA/UFAWJoint Working Group on Refinement.12–14 These art-icles not only provide background on this topic, theydiscuss the benefits of using such sheets for the experi-mental animals, the animal care staff, and the science ofa project. Other useful references to consider are the

Guidelines for the Assessment and Management ofPain in Rodents and Rabbits, the Rabbit GrimaceScale – a new method for pain assessment in rabbitsand the 1994 FELASA publication giving guidelines onpain and distress that offers general guidance on clinicalsigns in rodents and lagomorphs, equating to a severitycategory.15–17

An ideal assessment system would include simple,objective measurements which could be applied consist-ently and used to detect the onset, and monitor thedevelopment of pain, suffering and distress in animalsundergoing scientific procedures. Unfortunately, such asystem is not available, nor is likely ever to be so, due tothe wide variation in behaviours and behaviouralresponses among different species, strains and individ-ual animals and the specifics of procedures.

The use of tailored assessment systems specific to theproject, using trained and experienced personnel cancontribute to significant refinements in animal models.Thus, it is important to list possible and/or observedbehavioural or clinical responses, which moreovershould be assessed and quantified or scored whereverpossible, and may allow for identification of humaneend-points. For example, in some infection studiesbody-temperature monitoring has been successfullyused to identify early suitable end-points in advanceof clinical evidence of disease, while still allowing thescientific objectives of the study to be met.18

The following points should be considered in thedevelopment of a monitoring and assessment system:

. Targeted at an individual animal and not at a groupof experimental animals, although this may be chal-lenging where large groups are involved (e.g. in somefish studies).

. Level of severity that is experienced by each animalneeds to be reported using the categories of mild,moderate, or severe.

. Consideration should be given to the administrativeburden in the design of the recording system

. System should, where possible, use objective meas-ures to assess the level of pain, suffering or distressexperienced by the animal during the procedure.

. System should require a definition and description ofhumane end-points.

. The monitoring must capture (a) any welfare-relatedissues, both expected and unexpected, that occursduring the course of the project, (b) any refinementactions that were taken during the course of a project.

. Assessment criteria should be included to facilitatethe severity classification. Many of the published sys-tems advocate some form of numerical scoringsystem, and rank clinical signs with severity alloca-tion. Evidently, expertise and professional judgmentwill better allow for objective scoring.

12 Laboratory Animals 52(1S)

. Standardised recording is essential. Although it isacknowledged that all scoring systems will containsubjective criteria to some degree, the informationrecorded should be specific for the model andspecies used.

Development of a suitable recordingsystem

. Develop an animal welfare assessment sheet tailoredto the research project through discussions withresearchers, care staff and veterinarian (or suitablyqualified expert where more appropriate).

. Where applicable, score the signs of discomfort on aconvenient scale from normal (score¼ 0) to the high-est level of severity. Use objective scoring wherepossible.

. Identify intervention criteria – for example statesigns which require veterinary check/intervention

. Define the limit of acceptable or permissible severity(e.g. a maximum score of clinical signs/behavioursfor the procedures that should not be exceeded.This score can then be used as the score for adoptinga humane end-point.)

. Include consideration of the assessment of cumula-tive suffering and criteria for re-use where applicable

. An electronic format may facilitate data entry,allowing the details to be modified at any time inorder to permit the recording of unexpected eventsand any new events when they occur during the pro-ject and also allows easy sharing of information withall those involved (technicians, researchers, veterin-arians, AWB, etc.).

. The assessment sheet should be simple and easy-to-use for experienced and inexperienced or noviceobservers, based on and using agreed terminology(e.g. FELASA Glossary of clinical signs).19

The assessment sheet should be structured insuch a way that the results of different momentsof observing the experimental animals, such as onhandling, close up, or from a distance, can berecorded.

. The assessment sheet should be structured such thatit can be easily modified for changing situations,type of project, and animal species.

. The assessment sheet should, when required, allowthe recording of the time when each procedure, tech-nique was performed, or refinement introducedduring the project.

. The assessment sheet should be useful for theentire duration of a project. Records of such assess-ments will be useful for subsequent review of theproject and tailoring improvements for futurestudies.

Assessment of actual severity

An animal’s overall or cumulative suffering can be esti-mated from the nature and number of adverse and unex-pected effects that appeared during the course of aprocedure. The level of severity experienced by eachanimal during the course of a procedure is influencedby several factors, each of which should be incorporated.

The non-exhaustive list below gives an indication ofthe factors which can influence the amount and level ofsuffering that an experimental animal may experienceduring a scientific procedure, and which need to betaken into account when determining cumulative suf-fering of an experimental animal:

. the duration of the project/procedure;

. the duration of any adverse effect

. the number of procedures that were carried out onthe animal;

. the frequency of performing the procedures;

. information on whether the animal used in this pro-ject is being re-used

. an assessment of the animal’s clinical condition andphysical wellbeing at the end of the procedure, whichshould include determination of those factors thatinfluence body weight and body condition;

. an assessment of the impact on the animal’s behav-iour or psychological wellbeing, for example, theincidence of abnormal, stereotypic or aggressivebehaviours.

The following factors could also be given consider-ation in the assessment of actual severity:

. how the animal was conditioned (e.g. adaptation,training);

. the number of (surgical) interventions;

. the routes, volumes and frequencies of compoundand drug administration;

. the physical and chemical characteristics of theadministered compound or solution, for example,whether the repeated injections or the injections ofacidic or basic substances induced local irritationand necrosis;

. the routes, frequency and volume removed duringblood samplings;

. the method and frequency of restraint;

. changes in social structure/separation and singlehousing of social animals.

Although each element has the potential to impacton severity, the actual severity experienced will largelybe determined by the effectiveness of the actions takento reduce the negative impacts of the procedure – forexample, the use of analgesics will reduce post-operative pain.

Smith et al. 13

Determination of actual severity requires a review ofthe application and consequences of the applied proced-ures and the effectiveness of actions taken to minimisesuffering. The actual severity can only be determinedfollowing a review of all the effects on the animalthroughout the procedure – this necessitates the main-tenance and consideration of focussed clinical records.

The actual severity to be reported for each individualanimal should be based on the highest level of severityexperienced during the course of the procedure and notbased on the severity at the end of the procedure.

Assigning severity to an animal found deadduring study

Despite the best efforts to monitor animals closely, it ispossible that an animal might be found dead as a con-sequence of either the experimental procedure or otherunrelated causes. Some guidance on assignment ofseverity in such cases is given in the Commission work-ing document on a severity assessment framework andin the 2016 Discussion paper.6,7 These state that: ‘Forthe purposes of statistical reporting, actual severityshould primarily relate to the severity of the experimentalprocedures and not unrelated incidents such as diseaseoutbreaks or cage flooding’ and that ‘the actual severityof an animal found dead should be reported as ‘‘severe’’unless an informed decision can be made that the animaldid not experience severe suffering prior to death. If it isunlikely that the death was preceded by severe suffering,the actual severity classification should reflect the knownexperience prior to death. Factors such as frequency ofmonitoring, use of analgesia, etc. will need to be given dueconsideration.’

All deaths of animals should be carefully reviewedamong those involved (e.g. scientist/care staff/veterin-ary surgeon) as soon as possible to ensure that all rele-vant information is available to determine whether ornot the death was procedure related and to determinean appropriate level for reporting purposes. Whateverthe cause, measures should be taken to avoidrecurrence.

When the actual severity experienced exceeds thatpredicted prospectively for the procedure, there maybe a need to notify authorities and/or revise projectauthorisations.

Further guidance on the assignation of actual sever-ity where animals are found dead can be found in theEU discussion paper of January 2016, which providesan illustrative decision tree to assist determination inassigning the severity of death for the purposes of stat-istical reporting (reproduced below).7

1. Is the death unrelated or related to the procedure theanimal was undergoing?

1.1. Unrelated

Examples of unrelated deaths:

. deficiencies in equipment or environmental controlssuch as cage flooding, heating/ventilationmalfunction;

. inappropriate husbandry or care practices such asfailure to provide adequate diet (e.g. inappropriatelybalanced) or diet contaminated (e.g. poor storage);

. aggression between animals in a group housing;

. unrelated disease and infections;

. Ageing animals: deaths in animals on long-term stu-dies should be evaluated to clearly differentiate

deaths as a result of the procedure from those as aconsequence of the natural ageing process. Deaths insuch studies should not be automatically classed assevere, and the clinical history and condition of theanimal at the time of the last observation should begiven due consideration;

. In the case of GA breeding of an established line,when the genetic alteration is not considered to

cause any mortalities on the basis of the welfareassessment performed on the established line, there-fore, it is unlikely that deaths during the breedingprogramme are due to the genetic alteration.

The actual severity for the animal should reflect the

highest level of severity experienced during the

course of the procedure by the animal (excluding

the level of severity related to the death).

1.2. Related: proceed to question 2.2. Can an informed decision be made about the events

leading to the death? Factors such as frequency ofmonitoring, use of analgesia, etc. will need to begiven due consideration.

2.1. Yes, for example:� animal failing to fully recover consciousness in

post-operative period, but under appropriateanalgesic regime throughout;

� no clinical abnormalities recorded throughoutthe procedure, nor anticipated, but found deada few hours after a clinical examination.

The actual reported severity should reflect the sever-

ity as the result of the assumed events leading to

death.

2.2. No

The actual severity should be reported as ‘severe’.

14 Laboratory Animals 52(1S)

Re-use of animals and cumulativesuffering

Re-use of animals in further procedures is permissibleunder the Directive 2010/63/EU, but is dependent on anumber of factors including the actual severity of theprevious procedure, a demonstration that the animal’sgeneral state of health and well-being has been fullyrestored and that re-use is in accordance with veterin-ary advice, taking into account the lifetime experienceof the animal.20

Assessing the severity that an individual scientificprocedure will cause to an animal can be difficultwhen animals undergo several multi-step proceduresover prolonged periods, especially when the nature ofthe procedures means that the animals may also besubjected to alterations in normal housing and carepractices (e.g. periods of single housing).

However, such an assessment is necessary to allowre-use, and this needs to take account of the animal’slifetime experience. This introduces a further area forconsideration as now not only does the direct pain,suffering or distress caused by the various steps in theprocedure need to be taken into account, but also someconsideration is needed of any contingent suffering dueto the animal’s husbandry and care environmentthroughout its lifetime.

Lifetime or cumulative suffering can be consideredas the combination of direct suffering (the applicationof scientific procedures), any clinical conditions fromwhich the animal has suffered (which may or may notbe due to the procedure being carried out, e.g. inter-current disease or surgical wound) and contingent suf-fering (housing, husbandry, transport etc.); the dur-ation of these events must be taken into account.

The key issues which need to be taken into accountwhen considering lifetime experience are:

. the duration of exposure to the pain suffering dis-tress or lasting harm – longer duration is more likelyto cause higher severity;

. the nature and intensity of the effects on the animals;

. the interval between procedures – the shorter theinterval (usually) the less opportunity the animalhas to return to normal;

. the nature of interventions and actions that will betaken to relieve the suffering;

. consideration of any contingent suffering.

Illustrative examples of severityclassification and reporting - Appendix 1

The attachedworked examples (Appendix 1)were currentduring the evolution of this report and were real examplesof how severity classification and reporting can be

approached. They highlight the welfare and scientificissues to be considered, suggesting improvements thatcan be made through critical review of a study design,and provide illustrative realistic severity classifications.The examples also include illustrations of recording sys-tems that can be used during the course of studies tomonitor and assess actual severity and contribute elem-ents to retrospective assessment of a project.

It is acknowledged that there may be further 3Ropportunities that have evolved since these were devel-oped, and therefore these illustrations are not intendedto be used unaltered by research workers. Each projectwill be different in particular with regard to scientificobjectives that can influence significantly the overallseverity of a procedure. The intention however is toexplain and promote this stepwise approach to severityassessment. If applied as intended, the desired out-comes of improved science and welfare and consistentassessment and reporting of actual severity should beachieved.

The WG has chosen animal models commonly usedin the scientific community and has for each of themaddressed the previously stated four questions askednamely: What is being done to the animals? Whateffect will this have on the animals? How much sufferingmay it cause? What interventions can be included toreduce the impact on the animals?

Models included in this report

1. Control of infection: assessment of protection ofvaccine candidates in a murine model of tuberculosisand screening of novel drug candidates

2. Neuropathic pain: spinal nerve ligation in the rat3. Stroke: efficacy of a novel therapeutic agent on intra-

luminal thread middle cerebral artery occlusion(MCAO) in the marmoset

4. Cardiovascular evaluation of novel therapeutics: tel-emetered dog model

5. Atrial fibrillation: evaluation of novel antiarrhyth-mic substances in the rabbit

6. Ecotoxicology: determination of bioaccumulationusing the fish flow through test

7. Regulatory toxicology: assessment of acute oral toxi-city in the rat

8. Pharmacokinetics: determination of the pharmaco-kinetics after a single administration of a test sub-stance in the dog

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with

respect to the research, authorship, and/or publication of thisarticle.

Smith et al. 15

Funding

The author(s) disclosed receipt of the following financial sup-

port for the publication of this article: Publication of our workwas supported by a bursary from Laboratory Animals Limited.

Acknowledgements

The authors would like to thank Laszio Dezsi for technical

assistance with the spinal nerve ligation model and Pascalevan Loo for technical assistance with the pharmacokineticmodel.

References

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December 2013 correcting Annex II to implementing

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Council on the protection of animals used for scientific pur-poses (2014/11/EU). Off J Eur Union 2014; L10: 18.

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ment/chemicals/lab_animals/pdf/examples.pdf (2013,accessed 14 May 2017).

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mental technique, Special ed. Wheathampstead, UK:

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ments across Europe: FELASA Working Group on

Ethical Evaluation of Animal Experiments. Lab Anim

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Harmonising the definition of refinement. Anim Welf

2005; 14: 379–384.14. Hawkins P, Morton DB, Burman O, et al. A guide to

defining and implementing protocols for the welfare

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in rodents and rabbits, www.aclam.org/Content/files/

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Resume

La directive 2010/63/EU a introduit des exigences de classification de la gravite des procedures a appliquer aucours du processus d’autorisation de projet visant a utiliser des animaux dans les procedures scientifiques et

16 Laboratory Animals 52(1S)

egalement a rendre compte de la gravite reelle vecue par chaque animal utilise dans de telles procedures.Ces exigences offrent des possibilites d’examiner les effets nefastes des procedures sur les animaux et lafacon dont ils peuvent etre reduits avant le commencement du travail. Un meilleur systeme d’enregistrementet de signalement des effets indesirables devrait aussi aider a mettre en evidence les priorites d’ameliorationde procedures semblables a l’avenir et l’analyse comparative des bonnes pratiques. Le signalement de lagravite devrait contribuer a informer le public de la gravite relative des differents domaines de la recherchescientifique, et pourrait au fil du temps indiquer les tendances en matiere de raffinement. La coherence del’affectation des categories de gravite dans les Etats membres est une condition essentielle, surtout si lareutilisation est envisagee, ou si la clause de sauvegarde doit etre invoquee. Les exemples de classification dela gravite fournis a l’annexe VIII sont limites en nombre, et ont peu de pouvoir descriptif pour aider l’affect-ation. De plus, les exemples donnes sont souvent lies a la procedure et ne tentent pas d’evaluer les resultats,tels que les effets indesirables qui peuvent se produire. L’objectif de ce rapport est de fournir des conseils surl’affectation de la gravite, tant de maniere prospective qu’a la fin d’une procedure. Un certain nombre demodeles animaux actuellement utilises ont servi a illustrer le processus d’evaluation de la gravite a partirde la conception du projet, par le biais d’une surveillance au cours de la procedure jusqu’a l’evaluation finalede la gravite reelle a la fin de la procedure (Appendix 1).

Abstract

Die Richtlinie 2010/63/EU regelt die Anforderungen fur die Einstufung des Schweregrads bei derProjektgenehmigung zur Verwendung von Tieren in wissenschaftlichen Verfahren sowie fur die Meldungdes tatsachlichen Schweregrads jedes des in diesen Verfahren eingesetzten Tieres. Diese Anforderungenbieten die Moglichkeit, die negativen Auswirkungen von Verfahren auf Tiere zu berucksichtigen und zu prufen,wie diese vor Arbeitsbeginn reduziert werden konnen. Bessere Erfassung und Berichterstattung uber nach-teilige Wirkungen sollten auch dazu beitragen, Prioritaten fur die Verbesserung ahnlicher Verfahren und dasBenchmarking bewahrter Verfahren hervorzuheben. Die Berichterstattung uber den tatsachlichenSchweregrad sollte dazu beitragen, die Offentlichkeit uber den relativen Schweregrad der verschiedenenBereiche der wissenschaftlichen Forschung zu informieren, und konnte im Laufe der Zeit Trends in Bezugauf Verbesserung aufzeigen. Eine einheitliche Zuordnung der Schweregrade durch die Mitgliedstaaten ist einezentrale Anforderung, insbesondere wenn die Wiederverwendung in Betracht gezogen oder die Schutzklauselangewendet werden soll. Die in Anhang VIII aufgefuhrten Beispiele fur die Schweregradklassifizierung sindzahlenmaßig begrenzt und beschreiben die Vornahme der Zuordnung unzureichend. Außerdem beziehen sichdie genannten Beispiele oft auf das Verfahren, ohne dass versucht wird, das Ergebnis zu bewerten, wie z. B.moglicherweise auftretende nachteilige Wirkungen. Ziel dieses Berichts ist es, sowohl vorausschauende alsauch verfahrensabschließende Leitlinien fur die Schweregradzuweisung zu liefern. Es wurde eine Reihe vonTiermodellen verwendet, die derzeit im Einsatz sind, um den Prozess der Schweregradbewertung vom Beginndes Projekts uber die Uberwachung wahrend des Verfahrens bis hin zur abschließenden Bewertung dertatsachlichen Schwere zum Schluss des Verfahrens darzustellen (Appendix 1).

Resumen

La directiva 2010/63/UE introdujo requisitos para la clasificacion de la crudeza de los procedimientos a seraplicados durante el proceso de autorizacion de proyectos para utilizar animales en procedimientoscientıficos y tambien para informar sobre la crudeza real ejercida sobre cada animal utilizado en dichosprocedimientos. Estos requisitos ofrecen oportunidades para considerar los efectos adversos en los animalesutilizados en los procedimientos y como pueden reducirse antes de comenzar con el proyecto. Unos registrosy unos informes de los efectos secundarios tambien deberıan ayudar a poner de manifiesto las prioridadespara conseguir un refinamiento de otros procedimientos futuros parecidos y un referente de buenas practi-cas. La creacion de informes sobre la crudeza real deberıa ayudar a informar al publico sobre la relativacrudeza de varias areas de la investigacion cientıfica y, con el tiempo, se podrıan conseguir cambios en elrefinamiento. La consistencia en la asignacion de categorıas de crudeza en todos los Estados Miembro es unrequisito clave, particularmente si se considera una reutilizacion o, de lo contrario, debera invocarse laclausula de salvaguarda. Los ejemplos de la clasificacion de crudeza indicados en el Anexo VIII tienen unnumero limitado y tienen poco poder descriptivo para facilitar la asignacion. Asimismo, los ejemplos

Smith et al. 17

facilitados a menudo guardan relacion con el procedimiento y no tratan de evaluar los resultados, como losposibles efectos adversos. El objetivo de este informe es ofrecer unas directrices sobre la clasificacion decrudeza, tanto con anterioridad al procedimiento como posteriormente. Se ha utilizado una serie de modelosanimales, en activo actualmente, para ilustrar el proceso de evaluacion de crudeza desde la creacion delproyecto, durante el transcurso del procedimiento y en la evaluacion final de la crudeza real al final delproyecto (Appendix 1).

Appendix 1: Illustrative examples ofseverity classification and reporting

Control of infection – murine models of TB

General context. In 2010, 8.8 million cases ofactive pulmonary disease were identified, with around1.2–1.5 million people dying of this disease in thatyear, making it the second largest cause of infection-related deaths worldwide, after HIV/AIDS.1 Althoughthere are some effective drug regimens availablefor treating human TB, there are presently somehard-to-tackle challenges in the fight against this infec-tion, that include the rise in co-infection with HIV,the emergence of multidrug-resistant strains ofMycobacterium tuberculosis, compliance problemswith current long-term drug regimens and the needfor new vaccines to replace BCG, which efficacy hasbeen reported to vary between 0 and 80%.1–4 Thismakes the use of murine models of TB in pre-clinicalas important as ever.5,6

Experimental infection of mice with M. tuberculosishas been used to model human TB since the early worksof Robert Koch, and these models have since then beenof pivotal importance for the understanding of host-pathogen interaction and for testing therapeutic andpreventive approaches to this disease.5,7–10

There are marked differences in susceptibility to TBinfection between mouse strains. However, and contraryto what happens in most humans, no mouse strain iscapable of controlling disease to a truly latent state, andall animals eventually succumb to the infection as a resultof progressive disease, if left untreated.11 In all strainsexperimental infection is quickly followed by an accentu-ated and continuous growth of bacterial numbers in thelungs. The more resistant strains (e.g. the C57BL/6) arecapable of mounting a specific immune response after thisprimary response, being thus able to control the disease toa chronic stage from 3–4 weeks post-infection. Duringthis stage bacillary numbers in the lungs remain highbut relatively stable for several months and up to morethan a year (although lung pathology ensues) and animalsare seemingly asymptomatic. Eventually, disease recru-desces, progressing in severity until death,1,12 if notaverted by humane end-points.13 As for the more suscep-tible strains, these either fail to inhibit bacillary growth in

the lungs after primary infection or cannot maintain it,resulting in rapidly progressive and overtly symptomaticdisease, which culminates in early death.14,15 Aside mousestrain, other important parameters affect resistance toinfection, such as the inoculum size (in CFUs) and thechosen route of infection.11,16,17 Depending on these vari-ous parameters, median survival times of M. tuberculosis-infected mice may vary between less than 20 days to morethan 300 days.11,18

Two different procedures are described below, onefor vaccine testing and the other for screening of noveldrug candidates.

Illustrative procedure (1) – assessment ofprotection of vaccine candidates in a murinemodel of TB infection

Study design. The aim is to find vaccine candidates thatcan decrease a lung CFU count to at least 1.5 log lowerthan that conferred by BCG. In a pilot study it has beenestablished that a standard deviation of 1.2 log puts theeffect size around 1.65. For an alpha¼ 0.01 and a 90%power, a minimum of 11 animals is required to detectthis mean difference between BCG and another antigen’sprotection. A group size of 12 animals will be used toaccount for unexpected deaths. Ten groups of 12 femaleBALB/c mice each will be used to test vaccine candidatesfor M. tuberculosis infection. Each of the eight test com-pound groups will be immunised by intramuscular injec-tion (on three occasions at two week intervals) with avaccine candidate; one control group will be injectedwith vehicle only and another control group with BCG(‘gold-standard’ control). Thirty days after the lastimmunisation, all mice will be aerosol infected with alow dose of M. tuberculosis. Three mice from eachgroup will be sacrificed one, two and three monthspost infection to assess bacillary burden in lungs andspleen. The remaining mice will be monitored using anadapted clinical scoring system used for infection stu-dies,19 helping to determine humane end-points. Micewill be group housed in solid floored cages with litter andnesting material and cardboard tubes. Animals will beprovided food and water ad libitum. All animals will beeuthanised by anaesthetic overdose with pentobarbitalsodium.

18 Laboratory Animals 52(1S)

Consideration of specific refinements and humane end-points

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might makeit worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology and interven-tions to minimise severity End-points

Intramuscular immunisa-tion

Injection of vaccine candi-date antigens with adju-vant into both hind legs.

Injection can be painfuland/or cause mechan-ical trauma and poten-tial nerve damage.Immune reaction maylead to painful swellingPrevious trauma by thisantigen inoculationtechnique may exacer-bate pain experienced insubsequentimmunisations.

Well-trained personnelFollowing of recom-

mended guidelines foradministration of sub-stances.

Animals closely monitoredafter each antigeninoculation.

Animals showing perman-ent lameness and/orother signs of non-tran-sient distress frominjection-related inju-ries will be euthanised.

Aerosol infection30 days after last immun-

isation, Balb/c mice willbe placed in groups inan exposure chamber ofan aerosol generationdevice. The device iscalibrated to deliver aspecific dose of viablebacilli into the lungs ofeach animal from asuspension ofmycobacteria.

Possible minor distressfrom handling and con-tainment in aerosolchamber for a 20 mincycle. Primary infectionis usually characterisedas asymptomatic. Micehave however beenreported to manifesttransient fever duringthis stage Three to fourweeks after infection,animals are expected tocontrol infection to achronic state. Duringchronic infection, miceare seeminglyasymptomatic.

Animals will be monitoreddaily.

Relatively low inoculatesizes (�2 log10 CFU) willbe used, to avoid exa-cerbated immune reac-tion and excessivebacillary burden duringchronic stage.

No markedly adverseeffects are expectedduring primary infec-tion. All animals areexpected to controlinfection to a chronicstage.

No adverse effects areexpected.

Assessment of vaccineprotection

Groups of three mice fromeach group will beeuthanised, by anaes-thetic overdose, todetermine bacillarynumbers in lungs andspleen, after one, twoand three months of theexperimental infection.

All time-points for datacollection are expectedto coincide with asymp-tomatic or mildly symp-tomatic stages ofdisease.

Close monitoring of ani-mals during this stageby use of clinical scoresheets

The time-points for theseobservations areexpected to occur priorto any overt clinicalsigns.

If for any reason animalsreach a predefined clin-ical score, they will beeuthanised.

Determination of survivaltime

Three remaining micefrom each group will bemonitored for signs ofdisease recrudescence.

Disease recrudescence ischaracterised by thetransition from a non-clinical chronic stage toan overtly symptomaticstage, of progressive

Close monitoring of ani-mals and daily weighingof animals to determinethe turning point fromsub-clinical chronic dis-ease to overt disease,

Humane end-point deter-mined by clinical score.

Regardless of clinicalscoring, all animalslosing more than 15% ofbody weight (compared

(continued)

Smith et al. 19

Initial prospective assessment. While bacillaryburden and immunological parameters are to be usedas indicators of vaccine protection (see interim evalu-ation), measurement of survival provides importantdata and is commonly used in this sort of study.Objective criteria, as assessed by clinical scoring, willhowever be used to implement humane end-points toprevent animals of reaching advanced stages of disease.Nevertheless there is the risk of at least some ani-mals unpredictably reaching significant levels of suffer-ing and distress as a result of experimentalinfection (particularly vehicle controls), as well asfrom accidental nerve damage or exacerbated immuneresponse, hence swelling from antigen inoculation (cf. 3i.m. injections in the thigh of mice).20,21 A prospectiveseverity category of MODERATE is thereforeappropriate

Could the severity category be SEVERE?

Not if the proposed refinement measures are prop-erly applied. The use of the scoring sheets in particularmay prove valuable for identifying early signs of active

disease and thus allow identifying early humane end-points, preventing animals from significant suffering.

Could the severity category be MILD?

Depending on the time point of euthanasia, animalsmay not yet show signs of onset of disease thereforeclassification would be MILD.

For survival studies, in cases where the vaccine con-trol has been effective and refinement opportunitiesmaximised, there is the possibility that those animalsmay only exhibit MILD clinical signs. However, as inmice, even BCG vaccination only reduces bacillaryburden (see introduction) and thus eventually all losecontrol of the infection a severity classification ofMILD is unlikely.

Clinical observation/scoring system. Animals werecarefully monitored from experimental infectiononward. An example of the combined clinical observa-tion and scoring system used to help monitor the clin-ical condition of animals throughout the procedure isincluded at the end of this example.

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might makeit worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology and interven-tions to minimise severity End-points

The end-point will beassessed by clinicalscore. Post-mortemanalysis will be con-ducted to provide fur-ther data.

severity. Signs asso-ciated with active dis-ease include respiratorydistress, hunched pos-ture, lack of grooming;inability to eat or drink,fever and cachexia.

If not prevented by humaneend-points, severity ofsymptoms progressesuntil reaching hypoki-netic irresponsive(‘moribund’) state, cul-minating in death.

which will call for use ofa humane end-point.

with peak body weight)will be euthanised.

20 Laboratory Animals 52(1S)

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Smith et al. 21

Apart from one animal that had to be euthanisedand removed from the study for showing a pronouncedlimping gait in one hind leg after the second immunisa-tion, all animals recovered from the immunisationscheme with no unexpected complications. No compli-cations were associated with aerosol infection.

All mice euthanised at the predefined time-points(three per group, three time-points) showed no overtsigns of disease.

Of the eight vaccinated groups, five of the vaccinatedgroups showed lower bacillary burden in all organsanalysed, a delayed onset of disease and a significantlylonger survival time than non-vaccinated groups, twovaccinated groups showed levels of protection similarto the BCG group and in the one remaining grouppathology resembled that of controls, which were thefirst to show signs of recrudescent disease. Despite dif-ferences in time-of-onset, rate of progression and sur-vival, all animals in the survival study eventuallyshowed signs of recrudescent disease and were timelyeuthanised according to the predefined clinical score.No unexpected animal loss (e.g. spontaneous death)was registered.

89 animals (euthanised at the predefined time-pointsexcept the one limping) were considered to have experi-enced MILD severity.

31 animals (30 used in survival studies and oneeuthanised because of limping were considered tohave experienced MODERATE severity

Illustrative procedure (2) – screening ofnovel drug candidates in a murine model ofTB infection

Study design. The aim is to find drugs that can matchcurrent effective antibiotics, and allow all animals tosurvive up to six months without relapse, post-infec-tion. For this period, we have observed a survival of20% in untreated controls. To detect such a survival

Example clinical observation/scoring system

Vocalisations No vocalisation 0

Vocalisation when provoked,during handling

1

Vocalisation unprovoked, painrelated

2

Activity Interaction with peers, spe-cies-typical movementsand behaviour, curiousresponsive, normal pro-voked patterns of behav-iour (e.g. escape reaction,on approach)

0

Interruptions in activity, sub-dued behaviour patterns,even when provoked,reduced food and waterintake

1

Unresponsive, lethargic, nofood and water intake

2

Coat condition Shiny coat, close fitting 0

(continued)

Continued

Shiny coat, partialpiloerection

1

Stared, lustreless coat,marked piloerection

2

Dehydration Normal skin turgor 0

Skin turgor reduced (remain-ing skin fold)

1

Discharges(oculo-nasal)

No discharges 0

Oculo-nasal discharge,serous secretion

1

Oculo-nasal discharge, copi-ous, and suppurative

2

Grooming Normal grooming 0

Reduced grooming, smearedanal region

1

No grooming, smeared andincrusted anal region,facial impaction

2

Locomotion Spontaneous locomotionwithout being provoked, orwhen sleeping, after open-ing of the cage andprovocation

0

Locomotion after being pro-voked, staggering, atactic,laboured gait

1

No locomotion 2

Posture Normal 0

Hunched intermittently 1

Hunched permanently, par-tially in lateral position(mice in lateral position

2

Respiration Normal 0

Tachypnea or abnormalbreathing pattern

1

Tachypnea, laboured breath-ing, abnormal breathingpattern, occasionaldropouts

2

Other

A total score of 10 signals the humane end-point, at which theanimals will be euthanised.

22 Laboratory Animals 52(1S)

difference, with a 90% power and an alpha¼ 0.05, onlysix animals are needed (Fisher’s exact test). However, toaccommodate unexpected losses, eight animals will beused per group. 13 groups of n¼ 8 TB-susceptible C3H/HeJ female mice will be used to compare the efficacy offour novel drug candidates against M. tuberculosisinfection with existing licensed products. All mice willbe infected via the intratracheal route (3 logs of CFU in100�l PBS, a dose deemed appropriate during prelim-inary testing) by an incision in the trachea. Four weekspost infection, four groups of mice will be administereda low dose of their assigned test compound daily fivedays a week by oral gavage, for two weeks. Fourgroups will be given high doses of the test compound.One group of control mice will be administered vehicle

only, and four other groups will be given either one oftwo drugs currently used as gold-standards (GS-A, GS-B; positive controls), in two doses for each drug.Survival will be recorded for all groups of mice. A clin-ical score will be used to define humane end-points.Mice will be housed in solid floored cages with litterand nesting material and cardboard tubes. Animals willbe provided food and water ad libitum. All survivinganimals will be euthanised by anaesthetic overdose withpentobarbital sodium at the end of the experiment (14days after dosing).

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doingto the animals?

What will the animalsexperience? How muchsuffering might it cause?What might makeit worse? How will suffering be reduced to a minimum

Adverse effectsMethodology and interven-tions to minimise severity End-points

Intratracheal instillationMice will be infected byan incision in the tra-chea under generalanaesthesia induced byintravenous injection ofa combination of keta-mine/medetomidine.

Adverse effects of generalanaesthesia

Post-surgical complica-tions, pain in woundsite; reopening ofwound, infection

The wound typically healsin 2–3 days.

Appropriate anaesthesiaand analgesia, attendingto known adverseeffects during induce-ment and recovery

Aseptic technique,heating pad duringanaesthesia.

Non-pharmacologicalcontrol of pain anddistress

Daily observationWell-trained personnel

Post-surgical complica-tions as per end-pointsdefined

Drug administrationOral administration ofcandidate drugs by dailygavage, for two weeks,starting one week post-infection.

Oral gavage is a difficultprocedure that maycause fluid aspiration bythe lungs, perforation ofoesophagus or gastricwall, irritation, swellingand ulceration of theoesophagus fromrepeated dosing.

Inappetence and weightloss from gavage-induced stress or sideeffects of the drug

A refinement for oralgavage will beattempted in a pilot-study, by pre-coating agavage needle withsucrose, a method thathas been reported topacify mice and inducethem to swallow, redu-cing procedure-relatedstress.

Well-trained personnel

Any animals showing signsof gavage-related inju-ries will be euthanised.

Disease Progression andassessment of survivaltimeMice will be monitored

Signs associated withactive disease includerespiratory distress,hunched posture, lack of

All animals will be closelymonitored and assigneda clinical score, throughthe use of a clinical

Humane end-points deter-mined by clinical score

Euthanasia when bodyweight loss greater than

(continued)

Smith et al. 23

Initial prospective assessment. The use of intratra-cheal instillation, when compared with other routes ofinfection, raises additional welfare issues since itrequires general anaesthesia and, aside recovery-asso-ciated distress, complications may arise before fullwound cicatrisation.23 However, it allows for an accur-ate, standardised and successful inoculation. An opti-mised surgical procedure will aim to avoid post-surgicalcomplications and reduce variability.

While a measure of survival is required, death will bereplaced by humane end-points based on objective andeasily measurable clinical parameters. Nevertheless, asC3H mice cannot establish a long-lasting control of M.tuberculosis infection, there is the possibility of at leastsome animals reaching advanced stages of disease at thetime of treatment onset (particularly the vehicle con-trols), or as a result of low experimental treatmentefficacy.

A prospective severity classification of SEVERE istherefore appropriate.

Could the severity classification be MODERATE?Where disease progress is interrupted by drug treat-

ment in gold-standard controls, and possibly also intest-compound-treated groups, these animals, mayretrospectively be found to not have exceeded theMODERATE category.

Could the severity classification be MILD?No – surgical intervention under general anaesthesia

is by definition considered MODERATE.

Clinical observation/scoring system. Animals arevery carefully monitored; analgesia and local support-ive therapy are provided as necessary.

The combined clinical observation and scoringsystem used to help monitor the clinical condition ofthe animals throughout the procedure is the same asthat used in the preceding example.

Example of a clinical score sheet of the animal inGroup Test Compound 3 LD found dead on Day 5(gavage error)

Continued

What does this studyinvolve doingto the animals?

What will the animalsexperience? How muchsuffering might it cause?What might makeit worse? How will suffering be reduced to a minimum

Adverse effectsMethodology and interven-tions to minimise severity End-points

for signs of diseaserecrudescence. Theend-point will beassessed by clinicalscore. Post-mortemanalysis will be con-ducted to provide fur-ther data.

grooming, inability toeat or drink, fever andcachexia. If unrelievedprogresses to a hypoki-netic irresponsive(‘moribund’) state andculminating in death.

score sheet.For survival studies, a pre-

defined clinical scorewill be used to deter-mine the humane end-point.

15% (compared withpeak body weight)

Any animals showing signsof severe distress, asdetermined by bodycondition score, will beeuthanised.

Project number: Procedure number: . . .. . .. . .. . .. . .

Animal number: . . .. . .. . .. . .. . .. . ..Experiment number: . . .. . .. . .. . .. . ...Date of start of procedure: . . . /. . . /. . .

Clinical signs D1 D2 D3 D4 D5 D6 D7

Activity (0–2) 0 0 1 2

Coat condition (0–2) 0 0 1 2

Dehydration (0–2) 0 0 0 2

Discharges (0–2) 0 0 0 0

Grooming (0–2) 0 0 1 1

(continued)

24 Laboratory Animals 52(1S)

Continued

Locomotion (0–3) 0 0 0 0

Posture (0–2) 0 1 0 0

Respiration (0–2) 0 0 0 0

Vocalisations (0–1) 0 0 0 0

Other Found dead

Time of day 10.00 am 10.00 am 10.00 am 10.00 am 10.00 am

Initials of observer

Smith et al. 25

One animal died under anaesthesia. All other ani-mals survived and recovered from intratracheal instil-lation without any complications from this proceduralstep. Gold-standard controls showed no signs of dis-ease at the end of the treatment. The proposed pilotstudy showed animals to be more compliant with oralgavaging when sugar-coated gavage needles were used.Nevertheless, three animals were found dead at the endof the drug treatment (one from gold-standard con-trols, two from Test Compound 3 LD), with post-mortem analysis showing gavage-related injuries asthe most likely cause.

Two groups of mice treated with test compound1(low and high dose) showed results comparable tothe vehicle controls, along with one group with lowdose of test compound 3. All of these animals reachedsymptomatic stages of disease, but spontaneous deathwas prevented by close monitoring for humane end-points. Test compounds 2 and 4 prevented diseasedevelopment in both low and high- dose groups, andtest compound 3 only in high-dose treated animals.

1 animal died under anaesthesia during initialimmunisation (from negative control group): NON-

RECOVERY

71 (31 low and high-dose gold-standard drug con-trolsþ 32 low and high dose test-drug mice and 8 high-dose test-drug mice): although disease developmentwas prevented and no clinical signs developed, due tothe surgery it is MODERATE

29 animals (7 negative controlsþ 16 low and highdose test-drug mice and 6 low-dose test drug mice):MODERATE

3 gavage related incidents (1 animal from gold-stan-dard group, 2 from the same low-dose treated group):SEVERE

References

1. World Health Organization. Global tuberculosis control.

Geneva, Switzerland: WHO, 2011.2. Palomino JC, Ramos DF and da Silva PA. New anti-

tuberculosis drugs: strategies, sources and new molecules.

Curr Med Chem 2009; 16: 1898–1904.3. Kaufmann SHE, Hussey G and Lambert PH. New vac-

cines for tuberculosis. Lancet 2010; 375: 2110–2119.4. Koul A, Arnoult E, Lounis N, et al. The challenge of new

drug discovery for tuberculosis. Nature 2011; 469:

483–490.5. Apt A and Kramnik I. Man and mouse TB: contradic-

tions and solutions. Tuberculosis 2009; 89: 195–198.6. Rylance J, Pai M, Lienhardt C, et al. Priorities for tuber-

culosis research: a systematic review. Lancet Infect Dis

2010; 10: 886–892.7. Orme IM and Collins FM. Mouse model of tuberculosis.

In: Bloom BR (ed.) Tuberculosis: pathogenesis, protec-

tion, and control. Washington, DC: American Society

for Microbiology, 1994, pp.113–134.

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nd

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nt

of

act

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ima

ls

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NT

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st

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mp

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nd

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D

Te

st

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mp

ou

nd

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D

Te

st

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mp

ou

nd

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D

Te

st

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mp

ou

nd

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Te

st

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Te

st

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mp

ou

nd

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mp

ou

nd

4L

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26 Laboratory Animals 52(1S)

8. Gupta UD and Katoch VM. Animal models of tubercu-

losis. Tuberculosis 2005; 85: 277–293.

9. Flynn JL. Lessons from experimental Mycobacterium

tuberculosis infections. Mic Infect 2006; 8: 1179–1188.

10. Dharmadhikari AS and Nardell EA. What animal

models teach humans about tuberculosis. Am J Respir

Cell Mol Biol 2008; 39: 503–508.11. Medina E and North RJ. Resistance ranking of some

common inbred mouse strains to Mycobacterium tubercu-

losis and relationship to major histocompatibility com-

plex haplotype and Nramp1 genotype. Immunology

1998; 93: 270–274.12. Rhoades ER, Frank AA and Orme IM. Progression of

chronic pulmonary tuberculosis in mice aerogenically

infected with virulent Mycobacterium tuberculosis. Tuber

Lung Dis 1997; 78: 57–66.

13. Franco NH, Correia-Neves M and Olsson IA. How

‘‘humane’’ is your endpoint? Refining the science-driven

approach for termination of animal studies of chronic

infection. PLoS Pathog 2012; 8: e1002399.14. North RJ and Jung YI. Immunity to tuberculosis. Annu

Rev Immunol 2004; 22: 599–623.15. Cooper AM. Cell-mediated immune responses in tuber-

culosis. Ann Rev Immunol 2009; 27: 393–422.

16. North RJ. Mycobacterium tuberculosis is strikingly more

virulent for mice when given via the respiratory than via

the intravenous route. J Infect Dis 1995; 172: 1550–1553.17. Chackerian AA and Behar SM. Susceptibility to

Mycobacterium tuberculosis: lessons from inbred strains

of mice. Tuberculosis 2003; 83: 279–285.18. Nikonenko BV, Averbakh MM, Apt AS, et al.

Comparative analysis of mycobacterial infections in sus-

ceptible I/St and resistant A/Sn inbred mice. Tuber Lung

Dis 2000; 80: 15–25.19. Munder A and Tummler B. Assessing pseudomonas viru-

lence using mammalian models: acute infection model.

In: (eds) Pseudomonas methods and protocols. The

Netherlands: Springer, 2014, pp.773–791.

20. Morton DB, Jennings M, Buckwell A, et al. Refining

procedures for the administration of substances. Lab

Anim 2001; 35: 1–41.21. Baumans V and Pekow C. Common nonsurgical tech-

niques and procedures. In: Handbook of Laboratory

Animal Science, Volume I. 3rd ed. London: CRC Press,

2010, pp.401–445.

Neuropathic pain – spinal nerve ligation

General context

Neuropathic pain (NP) refers to a group of pain syn-dromes (e.g. spontaneous burning pain, allodynia,hyperalgesia, aftersensation, paraesthesias, etc.) thatresult from an initial nerve injury that causes anincreased responsiveness and pathological signal trans-mission in the pain pathways of the nervous system.

The estimated prevalence of NP is about 1–1.5% inthe population. The largest group of patients arethose with diabetic peripheral neuropathy (representingabout 45–70% of diabetic patients) while post thera-peutic neuralgia is the second most common cause ofNP. AIDS and cancer/chemotherapy may also predis-pose to NP. Traumatic nerve injury may lead to a painsyndrome termed causalgia or complex regional painsyndrome II (CRPS II) which is considered as themost severe NP conditions. NP is resistant to conven-tional pain medications, and as such represents a majortherapeutic challenge. In spite of recent improvementsin therapy by the introduction of certain novel drugs,there is still a huge unmet medical need for new medi-cations with higher efficacy, more rapid onset of actionand better side effect profile.

A typical and hardly tolerable NP symptom is allo-dynia, when a mechanical or thermal stimulus (e.g. skincontact by clothes) which does not normally provokepain becomes painful. Drugs with anti-allodynic effectsmay substantially alleviate the suffering of neuropathicpatients.

Experimental nerve injury in animals can be inducedby several methods which mimic different NP aetiolo-gies.1 Animals with streptozotocin-induced diabetesalso develop painful peripheral neuropathy, whiletoxic polyneuropathy can be modelled by cytotoxicdrug treatment, e.g. vincristine or cisplatin. Variousmodels based on surgical injury to a major nervetrunk (partial ligation or transection) have also beendeveloped which are more or less direct translationsof human CRPS II. The model under analysis usesspinal nerve lesion to induce mechanical allodynia fortesting the effects of analgesic compounds.2 A unilateralligation of the fifth lumbar (L5) spinal nerve is per-formed, and then animals are allowed to recover for 2weeks. The advantage of the spinal nerve lesion modelover other nerve injury models such as the chronic con-striction or the partial ligation of the sciatic nerve isthat the degree of injury is more uniform and thereforethe development of allodynia is more consistent.Moreover, the motor deficit and the foot deformitiesafter the selective L5 lesion are less severe. Themechano-nociceptive threshold of rats are determinedby dynamic plantar aesthesiometer and/or von Freyfilaments before and after surgery.3 Thermal allodyniacan also be measured with radiant heat paw stimulator.Test compounds are usually administered under arepeated dose regimen starting after the recoveryperiod when allodynia has already developed.

Illustrative procedure

Study design. In this example, 30 male Sprague-Dawley rats will undergo unilateral spinal nerve

Smith et al. 27

ligation. After the operation they will be allowed torecover for 2 weeks, during which the condition ofthe wound, the affected limb and the general healthstatus including body weight measurements and obser-vation of home cage behaviour will be monitored daily.On the 14th postoperative day mechano-nociceptivethresholds will be measured to confirm the developmentof mechanical allodynia. Only animals having a min-imum of 20% decrease of the pre-operation thresholdmeasured by the dynamic plantar aesthesiometer and avon Frey threshold <5.4 g are included in the treatmentgroups. Allodynic rats are randomised to form threetreatment groups to be treated intraperitoneally (i.p.)

with test compound A at a low dose, at a high dose anda vehicle control, respectively. An 8-day-long repeateddose regimen is utilised with daily treatments and theanti-allodynic effect is determined on Day 1, Day 4 andDay 8 of treatment at 30 and 60min after the injection.At the end of the study animals are euthanised. In thisstudy, no analgesia will be provided as this would inter-fere with the study results.

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might makeit worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology and interven-tions to minimise severity End-points

Baseline measurement ofmechano -nociceptivethreshold

Repeated tactile stimula-tion to the paw up to thesensory threshold or thecut off value

Using von Frey hairs anddynamic plantaraesthesiometer

Use of cut off values ofstimulus strength

Rats with low baselinethreshold are excluded

Surgery: L5 spinal nerveligation

Possible complicationsduring surgery:

Potential cardio-respira-tory depression/arrest

Tissue damage due tosurgery

HaemorrhageDamage to adjacent nerve

trunks

Choosing appropriateanaesthesia; carefuldosing

Heating, oxygen monitor-ing

Expert surgical skillsRefinement to original

published model inorder to reduce tissuedamage by an alterna-tive surgical explorationtechnique which sparesthe transverse processof the vertebra

Cardio-respiratory dys-function, failure torecover from anaesthe-sia

(very low probabilityevents, in case of GoodSurgical Practice onlytheoretical possibility)

Recovery for two weeks Possible complicationsduring post-operativerecovery:

Respiratory distress(during post-operativerecovery)

Wound infectionPain and discomfort

Adequate post-operativecare upon awakening.

Well-trained personnelAseptic surgical tech-

niquesUse of antibiotics when

neededObserving appropriate

wound healingGeneral observation of the

animalBody-weight

measurement

Poor general conditionsIn case of wound infec-

tions, animals will bekilled as treatmentwould interfere withscientific outcomes.

(very low probabilityevents)

(continued)

28 Laboratory Animals 52(1S)

Initial prospective assessment. Interventionsinvolved in this model individually do not exceed mod-erate severity. If the surgery is carried out with properskills and, consequently, no complications occur, thispart of the procedure is within the moderate category.Following surgery, careful monitoring allied to clearend-points will ensure no animals exceed moderateseverity.

A prospective severity classification of MODERATEis therefore appropriate.

Could the severity classification be MILD?No. As the prolonged pain resulting from these

interventions renders the model moderate, and

assessment of the pain itself is the objective of thestudy, it is not possible to conduct this procedurewithin a MILD classification.

Clinical observation/scoring system. Animals werecarefully monitored from surgery until the end of theprocedure. Two nociceptive assays were applied tomeasure latency of hind feet withdrawal: (1) von Freyhairs of different stiffness were used to determine theone that evoked a hind paw withdrawal; (2) dynamicplantar aesthesiometer. During the assays no additionalagent was used.

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might makeit worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology and interven-tions to minimise severity End-points

Effect of surgery:Pain in the affected leg,

sparing limb fromweight bearing.

Motor deficit in theaffected paw

Soft bedding in home cage;careful handling of theanimal

Ensure plenty availablespace to allow animalsto rest without contactfrom other animals.

Animals showing majormotor deficit in theaffected limb or signs ofautotomy, beyond nailchewing on the affectedpaw are euthanised.

(extremely low probabilityevents if surgery isaccordingly performed)

Determination of mechan-ical allodynia

[Thermal allodynia canalso be measured]

Pain stimulus to theaffected limb

Using techniques thatdetermine mechano-nociceptive threshold onunrestrained animals(e.g. von Frey hairs,dynamic plantaraesthesiometer) insteadof applying supra-maxi-mal stimuli

[Radiant heat is applied fordetermination of ther-mal nociceptivethreshold]

Animals failing to developthe pre-determineddegree of allodynia aftertwo weeks of recoveryare excluded andeuthanised.

Administration of testcompound or vehicle(i.p.)

Repeated daily dosing for 8days

Transient discomfortassociated with admin-istration route

Possible side effect of testdrugs

Competent personneltrained to properly injectthe compound accordingto Good Practice.

Daily clinical observationof rats

Dose selection based onprevious analgesia andbehavioural side-effecttesting (e.g. locomotoractivity)

Euthanasia at the end ofthe procedure

Doses of the test com-pound are not expectedto cause adverseeffects, but animals willbe killed if severe clin-ical signs are noted.

Smith et al. 29

An example of an observation sheet and a samplescore sheet to help monitor the clinical condition ofanimals throughout the procedure are included at theend of this example.

Results and assessment of actual severity. All ani-mals, except one in the vehicle treated group, recoveredfrom surgery with no unexpected complications, due tothe intensive peri-operative support provided.

Nociceptive assays indicated that mild or moderatepain was experienced.

Vehicle group1/10 animals did not recover from surgery. NON-

RECOVERY1/10 animals showed signs of automutilation and

was euthanised. MODERATE severity.1/10 animals reached a humane end-point and was

euthanised. MODERATE severity.7/10 animals showed a poor performance in the

nociceptive assays and the behavioural tests comparedto treated animals. However they did not show anyother clinical effects and maintained body weight.Clinical score was similar to treated animals after thesurgery. These animals developed moderate neurologi-cal-locomotor deficit, and showed a gradual reductionin clinical score over time, possible resulting from their

ability to compensate and adapt to long term neuro-logic deficits. MODERATE severity.

Treatment groups10/10 animals treated at lower doses showed mild

improvement in motor function, together with animprovement in clinical scoring. The agent had anti-allodynic effect, compared to vehicle. No specific side-effect was reported. MODERATE severity.

10/10 animals treated at higher doses showed signifi-cant improvement in motor function, together with animprovement in clinical scoring. The agent had clearanti-allodynic effect, compared to vehicle. No specificside-effect was reported. MODERATE severity.

Although animals in the treated groups experiencedless pain, due to the surgery and prolonged allodyniathe severity category for all animals was considered tobe moderate.

Example clinical observation/scoringsystem

Severity is assessed using a cumulative score from acombination of general clinical observations (body-weight, appearance, behaviour, cage environment,food and water intake, body function) together with aprocedure-specific evaluation.

General clinical signs Score

Appearance5–10% weight loss 1

11–15 % weight loss 2

16–20% weight loss 3

20%þ weight loss HEP

Coat slightly unkempt 1

Slight piloerection 2

Marked piloerection 3

Body functionRapid, slow or deep breathing – slight 1

Rapid, slow or deep breathing – moderate 2

Rapid, slow or deep breathing – marked 3

Food and water intakeNot drinking up to 10% of body weight per 24 h 1

Not drinking at all 3

Reduced food intake 1

Anorexia 3

BehaviourSlightly decreased mobility 1

Markedly decreased mobility 2

Significant mobility problems 3

Immobility >24 h HEP

(continued)

30 Laboratory Animals 52(1S)

Score 0–5 plus surgery¼MODERATE

Either 2 scores of 3 in any of the categories or a totalscore of 12 and above¼HEP

Note: that as surgical complications are generallynoted in the immediate post-op recovery period, closemonitoring and expert, empathetic judgement areessential during the first 24 h to ensure that adverseeffects are identified and actions taken to addressthese. Animals are humanely killed if their sufferingexceeds of the moderate category.

1 – Review frequency of monitoring.

4 – Provide appropriate supplementary care, e.g.mash and additional fluids

Dehydration/diarrhoea: Ringer Lactate or regularserum

Abdominal dilation (ascites): draining for pressurereduction

Weight loss: soft food

5 – Review progress with vet

Either 2 scores of 3 in any of the categories or a totalscore of 12 and above¼HEP

References

1. Jaggi AS, Jain V and Singh N. Animal models of neuro-

pathic pain. Fundam Clin Pharmacol 2011; 25: 1–28.

2. Kim SH and Chung JM. An experimental model for per-

ipheral neuropathy produced by segmental spinal nerve

ligation in the rat. Pain 1992; 50: 355–363.3. Chaplan SR, Bach FW, Pogrel JW, et al. Quantitative

assessment of tactile allodynia in the rat paw.

J Neurosci Methods 1994; 53: 55–63.

Stroke – efficacy of a novel therapeuticagent on intraluminal thread MCAO modelin the marmoset

General context

Stroke is defined as loss or alteration of normal bodyfunction that results from an insufficient supply ofblood to part of the brain. Despite better understandingof the pathophysiology of vascular brain injury, aneffective treatment for stroke remains an importantunmet medical need, and research is on-going to findappropriate preventive and therapeutic measures.

Three different types of stroke can be seen in humanpatients: ischaemic, intra-cerebral haemorrhage andsubarachnoid haemorrhage, but most of the animalmodels currently available are based on the ischaemictype. Stroke models, by their very nature, represent achallenge from the perspective of animal welfare. Goodinteractions and communication between all individ-uals involved in the scientific procedures, (veterinar-ians, investigators, animal technologists and carestaff), are critical to ensure that there is adequate bal-ance between achieving a valid model in this researcharea and minimising animal suffering.

Stroke is routinely induced in rodents and non-human primates by temporarily or permanently occlud-ing the middle cerebral artery (MCAO model).This‘MCAO’ model aims to reproduce experimentally thefocal cerebral ischemia that occurs in stroke, and it hasbeen extensively used to study the mechanisms ofinjury, to identify potential targets and to test putativeneuroprotective agents.

Continued

General clinical signs Score

Tense and nervous on handling 2

Markedly distressed on handling, e.g. shaking, vocalising, aggressive 3

Procedure-specific indicators Score

Wound healingWound edges are smoothly closed, no sign of inflammation 0

Wound edges are slightly swollen and erythematous 1

Wound is clearly infected/partially opened 3

No improvement of wound infection to topical and systemictreatment/wound is completely opened

HEP

Status of the operated limbSlightly reduced weight bearing 1

Marked gait impairment (lameness) 2

Severe gait difficulty/paw is often held in a constant elevated position 3

Major motor deficit >24 h postop/signs of autotomy HEP

TOTAL

HEP: humane end-point

Smith et al. 31

In a standard study design, the animals are trainedto perform certain behavioural tests prior to theMCAO procedure. During the therapeutic timewindow, established according to the mechanism ofdrug action and objective of the study, animals aregiven the test compound. The outcome analysisshould include information on infarct size, mortalityrate, frequency of complications (e.g. subarachnoidhaemorrhage), together with functional and neuro-logical evaluation to monitor progress. Serial magneticresonance imaging (MRI) has proven to be a powerfultool to gain information on variation of infarct sizeover time, but can also provide additional informationon blood flow or metabolic state. Histological, bio-chemical and molecular end-points can also beincluded.

There are various behavioural tests that may beapplied to stroke models. The simplest tests includeneurological scoring systems, which assess globalneurological status, and limb placing tests, used tomeasure motor reflexes. These are generally used toassess animals in the acute post-stroke phase. In long-term studies, more complex tests may be used to assesssensory and motor functions (e.g. bilateral sticky labeltest, beam walking, rotarod or staircase) and cognitivefunctions such as memory (e.g. passive avoidance tests,or evaluations of learning strategies).

It is good practice to perform a group of behaviouraltests, including at least one for each phase (acute andlong-term), so as to gather comprehensive informationon the impact on sensory, motor and cognitive func-tions. These tests have to be carefully chosen to captureany effects of the putative therapeutic strategies.Detailed descriptions of each of these behaviouraltests, including training schedules, are not includedhere, but for a comprehensive review and discussionof their use see Schaar et al.1

Many recent recommendations for preclinical inves-tigations designed to develop stroke therapies recom-mend the use of higher order species such as non-human primates in addition to rodent models.2–5 Thecommon marmoset (Callithrix jacchus) may be con-sidered the species of choice to study the pathophysi-ology and the treatment of cerebral ischemia.Compared with rodents, this primate is closer tohumans in term of cerebrovascular system, brain

metabolism, grey-to-white matter ratio and has aricher behavioural repertoire. In addition, comparedto old world monkeys, they are easier to handle,which is advantageous for behavioural testing and post-operative care management. The location and anatomyof the MCA in the marmoset have historically restrictedthe approaches used in rats and thus a more invasivesurgical model was developed that included the turningof a large bone flap to access the brain and the MCA.Nevertheless, more recently the intraluminal threadapproach has also been described in the marmoset.6

The intravascular approach presents a number ofadvantages compared to previously used methods,and should be considered a clear refinement. In particu-lar the absence of craniotomy and the comparativenon-invasiveness results in fewer adverse effects (e.g.severe disability/mortality) encountered in the post-operative period.

As in rodents, the first 48 h post-surgery are critical.The animals will have difficulties caring for themselves,and typical impairments can include left arm hemipar-esis, abnormal grasp reflex, left-sided neglect, nystag-mus and rotation of eye.7 Generally, after 3–7 days theanimals are capable of self-care and can return to theirhome cage. Gradually (around 2 weeks post-surgery),they will recover the majority of motor abilities and willbe able to freely jump and climb around their cages.

Illustrative procedure

Study design. In this example, on efficacy of a newcompound, six male and six female laboratory-bredmarmosets (Callithrix jacchus) will undergo 3 h-transi-ent MCAO using the intraluminal filament techniqueunder general anaesthesia. Before surgery, marmosetswill be trained and tested on a number of neurologicaltests, which assessed general neurological function,motor ability, and spatial awareness. Immediatelyafter tMCAO, marmosets will receive a bolus ofsaline (n¼ 6) or test compound A (n¼ 6), and osmoticmini-pumps will be implanted subcutaneously, provid-ing 48-h saline or drug infusion. Sensory-motor deficitswill be assessed weekly up to 45 days after MCAO, andMRI scans performed under general anaesthesia, at 1 h,D8 and D45. Animals will be killed 46 days afterMCAO.

32 Laboratory Animals 52(1S)

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might it makeit worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology andinterventions End-points

Behavioural tests trainingfor 1 week precedingMCAO: tactile stimula-tion, hill-and-valleystaircase, six-tubechoice, adhesiveremoval

These are behaviouralobservations, performvoluntarily by the ani-mals and will not causepain, distress or lastingharm

All tests are performed inthe modified home cage

Typically, a maximum timeto perform therequested task is set,and a final score isgiven. Animals notreaching a baselineperformance will beexcluded from the study

Under general anaesthe-sia, transient (3 h)occlusion of the MCAusing an intraluminalthread advanced via thecommon carotid artery.

Pain and discomfort asso-ciated with surgery

Potential for unexpectedsurgical complications,e.g. subarachnoidhaemorrhage, ipsilat-eral retinal injury,intraluminal thrombusformation, brainoedema, hypothalamusinvolvement with con-sequent hyperthermiaor temporal musclenecrosis. These canpresent clinically in anumber of differentways, for example:sudden collapse, par-alysis, severe head tilts,seizures

Potential effects of anaes-thesia on physiologicalvariables, e.g. hypo-thermia, hypotension,hypoxia

Poor nutritional intakeresulting from reducedconsciousness level,impaired masticationand poor motility, gen-erally in the first 48 hpost-MCAO

Use of appropriateanaesthetics, withappropriate analgesics(i.e. effective yet withminimal neuroprotectiveproperties)

Well-trained surgeonusing appropriate asep-tic surgical technique(with regular reviews ofsuccess rates)

Maintenance of homeosta-sis during anaesthesia

Use of standardisedmonofilaments and sur-gical technique toreduce variability andcomplications derivedfrom extensive lesions

Intensive post-operativecare for first 3–5 days,including external heatsources.

Regular body weightchecks; daily observa-tion and wound care

Providing easy accessiblefood and water duringthe recovery period, oradditional food (mash,liquid) and assistancewith feeding if neces-sary; rehydrate (e.g. viasaline injection) ifnecessary

Animals will be humanelykilled if any of the fol-lowing occur –

� Significant technicalproblems during sur-gery.

� Failing to fully recoverfrom anaesthesia

� Signs of unexpectedsurgical complications

� If animal’s bodyweightloss exceeds 20% pre-surgical weight, despiteadditional feeding and/or rehydration, or if theyremain immobile forover 24 h

Intravenous bolus ofsaline/test compound,

Pain and discomfort asso-ciated with surgical

Animals will be closelyobserved for adverse

Animals will be humanelykilled if any severe side

(continued)

Smith et al. 33

Initial prospective assessment. This model is con-sidered SEVERE because of the surgical procedureinvolved and the deleterious effects of the MCA occlu-sion on the welfare of the animal, especially during thefirst week. Nevertheless, intensive post-operative care inthe first 48 h up to 7 days, and close monitoring of thesubsequent phase can greatly contribute to reducenegative impact on animal welfare. From the experi-mental point of view, attention to refinement andstandardisation of the individual steps in the procedurecan lead to reduced incidence of complications andvariability, and consequently better quality of thedata obtained and therefore a reduction of thenumber of animals required.

A clinical score sheet should be agreed upon by theresearcher, veterinarian and animal technologists to setup criteria for monitoring and euthanasia; it will needto include the neurological score, together with otherclinical criteria such as body weight, ability to care forthemselves or reaction to stimulus.

Could the severity classification be MODERATE?

Experience/training of personnel involved, veterin-ary supervision, and intensive care in the early postMCAO period together with agreed end points can sig-nificantly reduce the incidence of negative effectsexperienced by the animals.

Clinical observations/scoring system. Animals arevery carefully monitored in the post-operative period.Analgesia and local supportive therapy are provided.

An example of a combined neurological/clinicalscoring system which is used to help monitor the clin-ical condition of the animals throughout the procedureis included at the end.

Results and assessment of severity. All animalsrecovered from surgery with no unexpected complica-tions. Clinical scoring in all animals was similar in thefirst 48 h after MCAO, and all of them received inten-sive peri-operative support. No significant ipsilateraldeficit was observed after induction of ischemia in nei-ther control nor treated animals. A partial recovering

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might it makeit worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology andinterventions End-points

followed by subcutane-ous implantation of amini-pump under gen-eral anaesthesia (aspart of the MCAO surgi-cal procedure, i.e. noadditional anaesthesiarequested)

procedure (mini-pumpimplantation)

No adverse effect expectedat the dose levelsadministered

effects of testsubstances

effects due to the noveltherapeutic agents arenoted

Behavioural tests per-formed daily during thefirst week post-MCAO,and weekly for 6 weeks:tactile stimulation, hill-and-valley staircase,six-tube choice, adhe-sive removal

Animals may find the tasksstressful if their motorabilities arecompromised

Monitor for behaviouralindicators of anxiety ordistress

Animals will be continu-ously observed byexperienced staff

Typically, a maximum time(cut-off) to perform therequested task is set,and a final score is given

Longitudinal imaging (MRI)under anaesthesia at1 h, 8 and 45 days afterMCAO

Repeated anaesthesiaEffect of anaesthesia, and

physiological variables(hypothermia, arterialblood pressure, arterialblood gases) on strokeoutcome

Use of appropriate anaes-thetics and analgesics(i.e. effective yet withminimal neuroprotectiveproperties)

Maintenance of homeosta-sis during anaesthesia

Animals failing to fullyrecover from anaesthe-sia will be euthanised

34 Laboratory Animals 52(1S)

of contralateral neurological deficit was observed in allanimals.

– All six vehicle-treated animals developed moder-ate contralateral neurological deficit, togetherwith a poor performance in the behavioural testscompared to treated animals. Clinical score wasnevertheless improving over time possible result-ing from their ability to compensate and adapt tolong term neurologic deficitsAssessment: SEVERE

– 2/6 treated animals developed moderate contralat-eral neurological deficit, together with a poor per-formance in the behavioural tests compared totreated animals. Clinical score was neverthelessimproving over time, possible resulting fromtheir ability to compensate and adapt to longterm neurologic deficitsAssessment: SEVERE

– 4/6 treated animals showed a significant improve-ment in neurological scoring after 48 h post-MCAO, together with an improvement in clinicalscoring.Assessment: MODERATE

In conclusion, 8 animals were considered asSEVERE and 4 animals were considered asMODERATE

Example clinical observation/scoringsystem

Severity assessment is performed by a combination ofgeneral clinical observations (bodyweight, appearance,behaviour) together with a procedure-specific neuro-logic evaluation. The neurological score used was amodified version of that described by Bihel et al.where absence (score 0), moderate presence (score 1)or presence (score 2) of a number of abnormal move-ments/postures are evaluated separately for each bodyside (i.e. ipsilateral and contralateral to MCAOlesion).6

In addition, a separate neurological scoring will becarried out. The limbs will be assessed independently,with the maximum hemilateral score being 10. In con-trol animals, a high scoring (up to10) is expected in thecontralateral side of the body during the first week afterMCAO, gradually recovering in the subsequent weeks.No significant ipsilateral deficit is expected (as thiswould mean the ischemic area is too large).

Each limb will be assessed and scored separately;severe contralateral neurological deficit together withpoor clinical conditions are expected during the firstweek post-MCAO while ipsilateral neurologicaldamage should be very limited/absent.

The neurological score for each limb will be added tothe clinical observation scoring to obtain a cumulativescore.

Score

Appearance5–10% weight loss 1

11–15 % weight loss 2

16–20% weight loss 3

20%þ weight loss HEP

Lack of grooming 1

(continued)

Continued

Score

Slight piloerection 2

Marked piloerection 3

BehaviourSlightly abnormal gait 1

Markedly abnormal gait 2

Significant mobility problems 3

Immobility >24 h HEP

Tense and nervous on handling 2

Markedly distressed on handling,e.g. shaking, vocalising, aggressive

3

HEP: humane end-point

Neurological score

Score AbsenceSlightpresence

Markedpresence

Forelimbs limbs slip-ping or danglingunder the perch, atrest or duringmovement

0 1 2

Hind limbs slipping ordangling under theperch, at rest orduring movement

0 1 2

Hand crossing the chest 0 1 2

Head tilting (before andafter stimulation)

0 1 2

Lack of reaction tovisual stimulus

0 1 2

Smith et al. 35

Week 1 post MCAOIt is well known that immediately after MCAO, a

high cumulative scoring is to be expected with a pro-gressive improvement over the first week post- surgery.During this critical period, animals will receive inten-sive care and will be frequently monitored under thesupervision of a veterinary surgeon. In particular, thefirst 48–72 h are key to identified potential surgicalcomplications and ensure that adverse effects are iden-tified and actions taken to address them in a timelymanner.� 22 – HEPtpb 1pc

Weeks 2–6 post MCAO1–10 – monitor regularly, evaluate together with the

clinical score10–20 – monitor frequently, provide care if not able

to care for itself, evaluate clinical scoring and team toreview all experimental data available (i.e. MRI, behav-ioural tests) to rule out unexpected complications, suchas brain haemorrhage, oedema, etc.� 20 – HEP

Example of an Individual observation sheet(Days 0–4)

References

1. Schaar KL, Brenneman MM and Savitz SI. Functional

assessments in the rodent stroke model. Exp Transl

Stroke Med 2010; 2: 13.2. Freret T, Bouet V, Toutain J, et al. Intraluminal thread

model of focal stroke in the non-human primate. J Cereb

Blood Flow Met 2008; 28: 786–796.

3. Freret T and Bouet V. Improvements of the stroke model

guidelines: animal body weight and long-term functional

concerns. Exp Transl Stroke Med 2009; 2: 28–31.4. Stroke Therapy Academic Industry Roundtable

(STAIR). Recommendations for standards regarding pre-

clinical neuroprotective and restorative drug develop-

ment. Stroke 1999; 30: 2752–2758.

5. Virley D, Hadingham SJ, Roberts JC, et al. A new pri-

mate model of focal stroke: endothelin-1-induced middle

cerebral artery occlusion and reperfusion in the common

marmoset. J Cereb Blood Flow Met 2003; 24: 24–41.6. Bihel E, Pro-Sistiaga P, Letourneur A, et al. Permanent

or transient chronic ischemic stroke in the non-human

primate: behavioural, neuroimaging, histological, and

immunohistochemical investigations. J Cereb Blood

Flow Met 2010; 30: 273–285.7. Marshall JW and Ridley RM. Assessment of cognitive

and motor deficits in a marmoset model of stroke.

ILAR J 2003; 44: 153–160.

Cardiovascular evaluation of noveltherapeutics – telemetered dog model

General context

Any medical drug to be marketed will need to proveefficacy but also safety. Therefore, even compoundsintended for use in very different areas

Day 0 1 2 3 4

Appearance

Body weight (g) (score) 340 (1) 305 (3) 320 (2) 323 (2) 335 (1)

Coat unkempt/piloerection 1 1 1 1 1

Behaviours

Gait 3 3 3 3 3

Response to handling 0 0 2 0 0

Total clinical score 4 7 8 5 5

Total neurological score 15 14 14 10 10

Lesion volume (MRI assessment)* 7%

Other observations Recovereduneventfullyfrom surgery

Moving aroundincubator (with severecontrolateral deficit)

Attempt toreach food withipsilateral hand

*‘Lesion volume’ (assessed using MRI) is included for the investigator to fill in at the end of the study. This data can then be correlatedwith clinical and behavioural observations to enable further refinement of monitoring, animal care and procedures.

36 Laboratory Animals 52(1S)

(e.g. neuropharmacology) will have to be evaluated fortheir potential cardiovascular effects.

Cardiovascular effects of compounds can be tested ina variety of ways: in invasive terminal procedures onanesthetised animals, in animals which are momentarilyrestrained and externally equipped with monitoringdevices or in freely moving animals previouslyimplanted with monitoring devices (telemetry systems).Over the past decades, telemetry systems have beenincreasingly applied in drug research and developmentfor measurement of physiological and bioelectrical vari-ables (e.g. blood pressure, heart rate, ECG).1 Theabsence of tethering, handling and restraint duringmeasurement provides a unique opportunity to studylaboratory animals without additional stress or physio-logical disturbance (anaesthesia) during a longer periodof time. In between measurements, animals can more-over be group-housed.

Telemetry can also enable reduction in animal num-bers. Firstly, because telemetry systems are stable formonths (and possibly even years), animals can be usedas their own controls, reducing data variability andconsequently the number of animals needed per treat-ment group. Secondly, telemetry provides an ability tocontinuously record a number of variables so that thereis a significant increase in the amount of data that canbe obtained from a given number of animals, comparedto the use of conventional methods. Thirdly, in theabsence of potential stressors, such as restraint or exter-nalised catheters, the quality of data obtained isimproved. Telemetry systems can also provide indica-tors of animal wellbeing to help implement earlier,more humane end-points.2 Telemetry thus is widelyregarded as benefiting science while minimisingimpact on animal welfare. However, the requirementfor appropriate surgical training should not be under-estimated, as this has a direct impact upon animal wel-fare. A sound basic and advance training inexperimental surgery and good working knowledge ofthe devices are absolutely essential before progressinginto implant insertion

Illustrative procedureStudy design. In this example, three pre-selected malebeagle dogs (suitable temperament) will be used.Animals will act as their own control.

After overnight fasting, animals will be anesthetised.Telemetry devices allowing for continuous measurementof body temperature and cardiovascular parameters(ECG, blood pressure) will be surgically implanted. Theimplantation of these telemetric devices is a surgical pro-cedure not invading body cavities, as the emitter is notimplanted intraperitoneally, but in an inter-muscularpouch. The catheter is inserted in the femoral arteryand further advanced into the aorta. ECG electrodesare tunnelled subcutaneously. The discomfort provokedis linked to the implantation and the surgical woundscreated, and the animals’ need to be anaesthetised.

During the 3 week recovery period, animals will betrained to be socially isolated for 2 h (e.g. telemetricrecording).

After recovery, the procedure calls for continueduse: animals will be used weekly (one day with vehicle,and another with compound) to evaluate cardiovascu-lar effects of the novel therapeutic agent at two differentdose levels. The first treatment will consist of vehicleadministration, followed by administration of the novelpharmaceutical agent. In-between recordings, animalswill be socially housed. A minimal wash-out period willbe allowed between test sessions (as determined by PKstudies).

The study is intended to assess the potential cardio-vascular effects of novel agents at proposed thera-peutics doses.

At the end of the experiment, animals will be sociallyhoused, while awaiting possible re-use.

Consideration of specific refinements and humaneend-points.

Initial prospective assessment. The procedure is clas-sified as MODERATE as it requires anaesthesia andsurgical intervention.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might make it worseAdverse effects

How will suffering be reduced to a minimum

Methodology and interven-tions to minimise severity End-points

Pre-surgical prepar-ation of the animal

Single housing prior tosurgery

Food withdrawal overnightprior to surgery

NA

Surgical implantation oftelemetry devices

Possible pain and discom-fort

Appropriate perioperativecare: appropriate

Decision to humanely kill theanimal during surgery if

(continued)

Smith et al. 37

The administration of the compound is expected tohave no clinical effect on the animal. However, admin-istration of the compound is MILD, as it involves injec-tion according to Good Veterinary Practice.

Could the severity classification be MILD?No: the procedure involves anaesthesia and surgery;

thus classification is MODERATE.In the case of re-use of previously implanted animals

that have been used in another project, the simpleadministration and evaluation through the use of tel-emetry of test compounds at therapeutic dose levels,and as no restraint is involved (freely moving) the pro-spective severity is to be classified as MILD

Could the severity classification be SEVERE?Yes, if the compound tested at doses administeredwould

have toxic effects (cardiovascular or other). This would def-initely mean that the flow chart of testing is not accurate, asa model of telemetry is definitely not intended to evaluate

toxic doses, but rather clinical/therapeutic doses of com-pounds. The study design would need to be reviewed.

Clinical observation/scoring system. Animals arecarefully monitored at every step of the procedure. Aspecific anaesthesia sheet, as well as a clinical observa-tion sheet, are developed, and included at the end ofthis example. During techniques, any clinical observa-tion is recorded daily. During the wash-out periods,animals are observed and weighed at least weekly.

Results and assessment of actual severity. Threedogs underwent the procedure. All recovered from sur-gery without any complication, no cardiovascular side-effects were observed in any of the animals at any of thedoses tested.

The three animals were considered to have experi-enced MODERATE severity.

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might make it worseAdverse effects

How will suffering be reduced to a minimum

Methodology and interven-tions to minimise severity End-points

Potential adverse effectsof anaesthesia and sur-gery (hypothermia,dehydration, distress onrecovery

anaesthesia and anal-gesia, etc.

Non-pharmacological con-trol of pain and distress:aseptic technique, heat-ing,

well-trained personnel,optimised surgical

procedure

unacceptable complicationsarise and persist

Period of recovery (3weeks), includingtraining

Possible pain, infection,discomfort

Post-operative complica-tions

Single housing duringpost-operative period(24 h) and training (2 h aday)

Adequate post-operativecare (analgesics, anti-biotics) as needed

Daily observation andwound care

Any animal with clinical signs ofinfection will be examined,treated and temporarilyexcluded from the study

Administration of testcompound (i.v., s.c.,i.p., p.o.) before rec-ording parameters

Possibly transient discom-fort following adminis-tration of compound

Potential compound-related unexpected sideeffects

Animals are individuallyhoused for short-termperiods (determined bythe pharmacokinetics ofthe drug).

Blood pressure, ECG andbody temperature arecontinuously monitoredin freely moving animal,housed in pen equippedwith telemetry receiver.

Between studies animalsare group housed andobserved daily,

Body weight is recordedweekly.

In case of prolonged side effectsafter test compound treat-ment: possibly treat and allowanimal longer wash-outperiod and clinical check-upbefore new dosing and deci-sion on possible euthanasia.

End of battery life: animal iseither proposed for re-use asblood donor, or if competentauthorities authorise,explanted and depending onvet examination proposed forrehoming or proposed for re-use as blood donor.

38 Laboratory Animals 52(1S)

At the end of the procedure the animals were againsocially housed. The level of severity wasMODERATE, due to the surgical implantation ani-mals underwent, but the general state of health hasbeen fully restored. No cumulative severity wasobserved due to administration of the novel therapeuticagent under evaluation. Therefore it is considered that

animals may be re-used in future MILD, MODERATEor Non-recovery procedures pending an appropriatewash-out period and after positive veterinary advice.

Example clinical observation/scoring sheets

Food intake

Water Intake

Urineprod.

Faecesprod.

Woundaspect

Other Analgesic Antibiotics Wound care Other drug

D-1h:T°:

D 0h:T°:

D 1h:T°:

D 2h:T°:

D 3h:T°:

D 4h:T°:

D 5h:T°:

…

D17h:T°:

D18h:T°:

D19h:T°:

D20h:T°:

*: Administer analgEsics and antibiotics according to procedureIn case of abnormalities detected, contact the veterinarian

DateBW/ kg

TimeTemp.

°C

Post-op care*Observations and other actions Visa

Batch n° of telemetry device

Post-op follow-up sheet Micro-chip: Dog n°:

Dog Name:Researcher:

Batch: Date surgery:

gk:thgiewydoB:etad lavirrAdI erudecorP

Date Time Fasting IsolationAdmin. of compound

(dose, route)food

distributionblood

SamplingVisa

Score behaviour as 0, +, ++, +++Observe Salivation, Myosis, Mydriasis, Defaecation, Miction, Posture of animal, Sedation, Locomotor activity, Rigidity, Vocalisation, etc.Describe any unexpected abnormalities and contact the VeterinarianAlso note time quantity and containers of blood samples obtained

gk:thgiewydoB:etad lavirrA:dI erudecorP

Start/end of telemetric recordings

Behavioural observations Other ac�ons

Batch n° of telemetry device

Study follow-up sheet Micro-chip: Dog n°:

Dog Name:Researcher:

Batch: Date surgery:

Smith et al. 39

References

1. Morton DB, Hawkins P, Bevan R, et al. Refinements in

telemetry procedures: seventh report of the BVA(AWF)/

FRAME/RSPCA/UFAW Joint Working Party on

Refinement, Part A. Lab Anim 2003; 37: 261–299.

2. Hawkins P, Morton DB, Bevan R, et al. Husbandry

refinements for rats, mice, dogs and non-human pri-

mates used in telemetry procedures: seventh report of

the BVA(AWF)/FRAME/RSPCA/UFAW Joint

Working Party on Refinement, Part B. Lab Anim 2004;

38: 1–10.

Evaluation of the efficacy of novelantiarrhythmic substances – rabbit modelof atrial fibrillation using implantedmini-pump for administration ofsubstances

General context

Paroxysmal atrial fibrillation (AF) is a condition inwhich an irregular heart rhythm occurs periodically.The heart returns to its normal sinus rhythm on itsown in a few minutes, hours or days. People whohave this type of AF may have episodes every day,or only a few times a year. When these episodesbegin and end is usually unpredictable, which canbe very unsettling. About 1 in 4 people with parox-ysmal AF eventually develop the permanent form ofthe condition.

The causes of paroxysmal AF aren’t alwaysknown. Although AF can occur in patients withoutevident heart disease, organic heart diseases, such ascongestive heart failure and mitral valve disease, areinvolved in about half the cases of paroxysmal AFand 80% of persistent or permanent AF. People withparoxysmal AF appear to be at just as high a risk ofdeveloping blood clots as those with chronic AF.However, the benefits of blood-thinning medicationshave not been shown to be as effective in people withparoxysmal AF as those with chronic AF. AF is acommon arrhythmia and a potent risk factor forcardio-embolic stroke. There is evidence for manyion current changes in studies of the ionic propertiesfrom atrial tissue of patients with AF, and this is thereason that ion channel blockers require to beinvestigated.1,2

The development of numerous animal models of AFwith clinically relevant disease paradigms has been amajor advance over the past years. Models have been

developed in rats, dogs, goats, sheep and more recentlyin genetically altered mice.3 The severity and invasive-ness of the model will depend on the underlying causesstudied.

Screening based on electrophysiological, ionic, andmolecular mechanisms in in vitro models allow identifi-cation of potential efficacy of compounds, which arethen evaluated in in vivo models. The ideal animalmodel for drug testing should be simple, stable, inex-pensive, clinically relevant, allow for reliable sustainedAF production (for testing drug-induced termination)and eventually allow for easy conversion to sinusrhythm (for multiple dose studies). The example pro-vided here concerns a surgical model which has provengood reproducibility in producing AF, together withminor clinical discomfort to the animal.

Illustrative procedure

Study design. In this example, 10 large adult maleNew Zealand White (NZW) rabbits (n¼ 5 per dosegroup (saline control and test substance)) will beused. Based on power analysis from previous data (pre-liminary studies) this was determined to be the minimalnumber of animals required per sample size.

In order to allow the test compound to be present asa preventive treatment in a steady state level, a subcuta-neous mini-pump will be implanted which allows deliv-ery of the compound for 21 days. This allows not onlyfor continuous diffusion of the drug in the animal’sbody (thus preventing peaks of drug distribution) butalso minimises disturbance of the animal for drugadministration (which can momentarily provoke sup-plementary changes in heart frequency and lead tospontaneous AF).

Under the same anaesthetic procedure, bipolar elec-trodes will be implanted on the left atrium and con-nected to an electrical stimulator. The electricalstimulator will be contained in a jacket. Animals willbe habituated to the jacket before surgery.

A post -operative period of 7 days will allow theanimals to fully recover from the surgery. Thereafter,implanted electrodes will be stimulated daily for 14 con-secutive days. This allows a graded stimulus, graduallyincreasing heart rate and provoking paroxysmal AF.This will involve occasional handling and immobilisa-tion of the animal in order to modify stimulationparameters.

14 days later, the animals will be terminally anaes-thetised. Once hemodynamic parameters are stable, AFwill be provoked by a fixed time period of electricalstimulation and hemodynamic parameters and bio-logical markers will be measured.

40 Laboratory Animals 52(1S)

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might make itworse? How will suffering be reduced to a minimum?

Adverse effectsMethodology and interven-tions to minimise severity End-points

Single housing (as adultmales and surgicallyequipped)

Habituation of the animalsto the jacket& weightedbackpack

May cause some distressMinimal stress to jacket

Provision of environmentalenrichment

Calm and empathetichandling

If animal fails to acclima-tise then will not beincluded in study

Surgical implantation ofs.c. mini-pump contain-ing novel therapeuticagent and placement ofbipolar electrodes onheart wall via thoracot-omy

Administration of thera-peutic agent throughmini-pump

Complications during sur-gery and anaesthesia:Hypothermia, cardiacarrhythmias, respiratoryarrest

Pain and discomfort asso-ciated with surgery

Therapeutic substance notexpected to cause sig-nificant adverse effects(based on previousdata).

Appropriate perioperativecare: aseptic technique,Suitable anaesthetic proto-col, artificial ventilation,heating pad, antiarrhythmicdrugs during surgery, i.v.perfusion of saline

Non- pharmacological controlof pain and distress: Well-trained surgeon, optimisedsurgical procedure toreduce variability.

Analgesia will be providedroutinely, and continued asindicated.

Use of very experiencedpersonnel.

Decision to humanely killthe animal during sur-gery if unacceptablecomplications persist,for example, uncon-trolled arrhythmias,ventricular fibrillation

Period of recovery (7 days)and thereafter continu-ous stimulation of theimplanted electrodesfor 14 consecutive days

Complications during post-operative recovery:

Pain and discomfort,hypothermia or hyper-thermia, respiratorydistress, cardiacarrhythmias, woundinfection

Mini-pump presence anddrug diffusion can pos-sibly cause discomfortdue to localisation ofmini-pump and/or diffu-sion of drug

Heart frequency is grad-ually increased. AF maysuddenly occur if animalis stressed

Adequate post-operative care(heating, food, etc.)

Appropriate housing and ajacket will prevent theanimal from injuring itselfor interfering with theelectrodes/wound site (e.g.large and high cage).

Daily massage to preventadhesions of the mini-pump.

Maximum dose levels to bedetermined from previousprocedures.

Appropriate perioperativecare:

Analgesia and/or anti-inflam-matory drugs suitable forspecies for at least 2 dayspost-op, depending on bodytemperature and clinicalsigns

Topical antibiotics/antiseptics

Animals will be humanelykilled if weight lossexceeds 20% of initialbody weight in less than48 h.

Decision to euthanise ifunacceptable complica-tions persist duringpost-operative period(clinical signs of dis-tress; failure to returnto normal by 48 h post-op

Animals with clinical signsof infection will beexamined and treated

(continued)

Smith et al. 41

Initial prospective assessment. The study is intendedto assess the efficacy of novel therapeutic agents in pre-venting/minimising occurrence of AF following elec-trical stimulation of the left atrium.

The s.c. implantation of a mini-pump is a very fastprocedure; only takes a few minutes for an experiencedperson. The thoracic surgery is an invasive procedure.There is a need to anaesthetise the animal and the sur-gery may certainly provoke some pain and discomfort.The cardiac stimulation itself does not seem to provokeany pain, no visible discomfort: animals do not seem toreact to increased heart frequency when this is done in aslow and progressive manner.

A prospective severity of MODERATE is deemedappropriate.

Could the severity classification be MILD?No; the thoracic surgery will provoke pain and dis-

comfort, which however is minimised by pain allevi-ation and best practice surgical techniques.

This procedure involves major surgery, even theimplantation of the mini-pump alone would be a pro-cedure which must be carried out under anaesthesia.

Could the severity classification be SEVERE?

If for whatever reasons no adequate analgesia orpost-operative analgesia would be given this procedurecould potentially provoke severe pain.

If cardiac stimulation would not be gradual, it mightprovoke spontaneous AF, which may lead to spontan-eous death of the animal and thus should be classifiedas SEVERE. This is rare, and should with appropriateprogressive stimulation only occur very rarely

Clinical observation/scoring system. Animals arevery carefully monitored at every step of the procedureusing a specific anaesthesia log and a clinical observa-tion sheet. In the post-operative period, analgesia andlocal supportive therapy are provided as necessary andrecorded on the individual clinical observation sheet.Sheets are filled out daily and include recordings ofbodyweight, body temperature, clinical signs, standardpost-operative treatments, any other treatments.Stimulations and reactions to it are also recorded.Examples of observation sheets are included at theend of this example

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might make itworse? How will suffering be reduced to a minimum?

Adverse effectsMethodology and interven-tions to minimise severity End-points

on skin wounds. Post-operative monitoring withdaily clinical scoring sheet(body temperature, bodyweight, food intake, woundcare, etc.)

Animals are habituated toweight of backpack.

Clinical daily follow up ofanimals

Housing and handling undernon-stressful conditions

Terminal anaesthesia (withprovoked AF) 14 dayslater

AF may occur spontan-eously,

Presents as sudden death;very rare, with no clin-ical signs noted

Appropriate anaesthesia andanalgesia

Animals will be humanelykilled by anaestheticoverdose if fibrillationoccurs spontaneouslybefore the start of theprovoked fibrillation (astest compound clearlynot efficacious), other-wise at end of 3 hmeasurement duringterminal procedure

42 Laboratory Animals 52(1S)

Results and assessment of actual severity. Ten ani-mals underwent the procedure. All recovered from sur-gery without complications. One animal in the salinecontrol group was found dead shortly after the firstelectrical stimulation. All other animals underwent theterminal anaesthesia during which efficacy of the thera-peutic agent was evaluated versus vehicle. Severityassessment for the five saline-treated animals:

- 1 out of 5 rabbits was found dead at D11 (firstelectrical stimulation): SEVERE

- 4 out of 5 completed the terminal anaesthesia pro-cedure: MODERATE

- 5 compound treated animals were used. All 5 com-pleted the terminal anaesthesia procedure:MODERATE

Example of post-operative follow-up sheet

References

1. Benson MD, Ju Li Q, Kieckhafer K, et al. SUMO modi-

fication regulates inactivation of the voltage-gated potas-

sium channel Kv1.5. PNAS 2007; 104: 1805–1810.2. Burashnikov A and Antzelevitch C. Role of late channel

current block in the management of atrial fibrillation.

Cardiovasc Drugs Ther 2013; 27: 79–89.

3. Nattel S, Shiroshita-Takeshita A and Brunde BJ.

Mechanisms of atrial fibrillation: lessons from animal

models. Prog Cardiovasc Dis 2005; 48: 9–28.

Determination of ecotoxicity of a testsubstance – bioconcentration flow-throughfish test

General context

The bioconcentration test aims at determining thebioaccumulating potential of a chemical in tissues offish, when triggered by (a.o.) its octanol-water parti-tioning coefficient. This is a legal requirement for allchemicals when exposure of the aquatic environmentis possible. A study can be conducted with waterborneexposure; this will provide a bioconcentration factor

(BCF, ratio of concentration in fish divided by concen-tration in water). The study may also be conducted withdietary exposure (e.g. when the substance is very poorlysoluble in water); this will provide the biomagnificationfactor (BMF, ratio between concentration in fishdivided by concentration in food).

Other drugJ-1 h:

T°:J 0 h:

T°:J 1 h:

T°:J 2 h:

T°:J 3 h:

T°:J 4 h:

T°:J 5 h:

T°:J 6 h:

T°:J 7 h:

T°:J 8 h:

T°:J 9 h:

T°:J10 h:

T°:J11 h:

T°:J12 h:

T°:J13 h:

T°:J14 h:

T°:

Date BW: kgTime

Temp.Post-op care*

VisasnoitcarehtodnasnoitavresbOscitoibitnAciseglanA

Post-op follow-up sheet Tatoo:: Rabbit n° Researcher: Batch: Date implantation stimulator

gk:thgiewydoB:etadlavirrA

Smith et al. 43

The BCF or BMF is used in the assessment of thepersistence, bioaccumulation and toxicity (PBT) andvery persistent and very bioaccumulative (vPvB) cri-teria, and is used for the environmental risk assessmentfor the aquatic food chain (i.e. water – fish – top preda-tors, such as birds and mammals). If a substance isbioaccumulative it will accumulate in living organismsonce it enters the environment, which may lead toadverse effects higher in the food chain. A substancefor which all criteria for PBT and/or vPvB are fulfilledwill be identified as a PBT/vPvB substance. Substancesidentified as being PBT or vPvB are considered candi-dates for substitution, which means that they should bereplaced by substances that are not PBT/vPvB ifpossible.

A PBT/vPvB assessment is required for all sub-stances (i.e. industrial chemicals, agrochemicals, bio-cides and human and veterinary drugs). Though thereare some QSARs available, these are not always suit-able, depending on the molecular structure or physico-chemical characteristics of the chemical. Furthermore,QSARs will only estimate the BCF of the unchangedchemical, while a study in fish allows for the determin-ation of degradation products and elimination timeupon transfer to uncontaminated water.

Fish species used in the test may involve: carp(Cyprinus carpio), zebra-fish (Danio rerio), fatheadminnow (Pimephales promelas), bluegill (Lepomismacrochirus) or rainbow trout (Oncorhynchus mykiss).

Illustrative procedure

Study design. The study is performed based on theOECD guidelines for testing of chemicals, GuidelineNo. 305: Bioaccumulation in Fish: Aqueous andDietary Exposure, October 2012.1 The procedure isdesigned to meet the test methods prescribed by

Commission Regulation (EC) No 440/2008 of 30 May2008, Part C: Methods for the determination of eco-toxicity, Publication No. L142, C.13‘Bioconcentration: Flow-through Fish Test’.2 The fishare exposed during an uptake phase of 28 days and adepuration phase of 56 days. The duration of bothphases may be changed based on the course of uptakeand/or depuration. During the uptake phase a group offish is exposed to the test substance at one or morechosen concentrations. The numbers of fish per testconcentration are selected such that a minimum offour fish are available at each sampling point. Theyare then transferred to a medium free of the test sub-stance for the depuration phase. The fish are kept intanks with a continuous supply of water, to which thetest substance is added during the uptake phase. Thewater volume should be replaced at least five times perday. Non-toxic concentrations of test substances areused. Radiolabelling of the test substance may beused for analytical purposes. Water samples are takenprior to and during uptake and depuration phase. Fishare selected for tissue sampling following euthanasia atsequential time points during the uptake phase (e.g. 1,3, 7, 14, 21 and 28 days) and the depuration phase(30, 35, 42, 56 and 84 days); and for lipid extractionat 0 or 28 days of exposure. Weighing is per-formed before test and post-mortem; sampling of fishduring test and post-mortem. Four or more fish areneeded per sampling point and per test concentration.A total number of 130 rainbow trout were used andexposed to two different concentrations of a test sub-stance: 32 fishes will be used for the control group(1 replicate) and 49 fishes for each test concentration(2 replicates).

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might itcause? What mightit make it worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology andinterventions End-points

Fish to be used for test arecaptured, weighed andmeasured

Mild agitation due to cap-ture/handling)

Good practice, appropriateequipment/technique

Application of humaneend-points, e.g.haemorrhages/sug-gillation, skinlesions, gill defects,abnormal position

(continued)

44 Laboratory Animals 52(1S)

Initial prospective assessment. Animals are onlyexpected to experience MILD discomfort based onexposure to non-toxic concentrations of test substance,and the use of good practice for handling andeuthanasia.

A prospective severity classification of MILD istherefore appropriate.

Could the severity be MODERATE?Normally not, as during the bioaccumulation phase

only slight or no adverse effects, and thus only MILDdiscomfort are expected based on use of non-toxic doseconcentrations, good practice handling and euthanasiamethods. If contrary to expectations clinical signs indi-cating moderate severity are observed, an informeddecision is made on the follow-up of the experiment,e.g. euthanasia of the animal based on humane end-points, intensified monitoring or need for change ofprospective severity assessment of similar experimentsand notification to CA.

Clinical observation/scoring system. Animals aremonitored for mortality/viability and any adverseeffects daily. Any clinical score sheet and observationprocedure should be agreed upon by the researcher,veterinarian and animal technologists to set up criteriafor monitoring and timely euthanasia (humane end-points).3–5 Temporary use of light to improve observa-tions is possible but should be limited. Fish areobserved for adverse effects and mortality after 2–4 h,and for mortality or possibly more adverse effects forpossible implementation of humane end-points at theend of the day. Then following mornings, the fish arechecked for any mortality, and for mortality and any(more) adverse effects on the observation moments, andat the end of the day. An example of the clinical obser-vation and scoring system used to help monitor theclinical condition of animals throughout the procedureis included at the end of the example. Unless typicalclinical signs or abnormalities (e.g. typical swimming

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might itcause? What mightit make it worse? How will suffering be reduced to a minimum?

Adverse effectsMethodology andinterventions End-points

swimming for 2 daysor more, convulsions

Fish are held in test solu-tions in a flow-throughsystem with completelydissolved test substanceconcentrations

No or mild clinical signs NA Application of humaneend-points, e.g.haemorrhages/sug-gillation, skinlesions, gill defects,abnormal positionswimming for 2 daysor more, convulsions

Euthanasia by anaestheticoverdose or blow to thehead:

Selected fish at samplingtime are caught by net,rinsed, blotted dry andinstantly killed by striketo the head followed bycervical incision;

Remaining fish areimmersed at end of theexposure in euthanisingsolution, e.g. tricainemethanesulfonate (TMS,MS-222), 2-phenoxyethanol

Mild agitation due to cap-ture/handling immedi-ately followed byeuthanasia or anaes-thesia-euthanasia,respectively)

Good practice, appropriateequipment/technique

Good monitoring/control ofeuthanasia procedure(quality, concentration,effects)

NA

Smith et al. 45

behaviour or damaged fin/tail) are present in one indi-vidual or very small number of fish, any follow-up orre-identification of an individual fish is not or hardlypossible. Whenever it is possible to identify an individ-ual affected fish with subsequent observations, this maybe helpful in monitoring the clinical condition, appro-priate for possible implementation of humane end-points. If not, counting of the number of fish with thetypical clinical signs should be used to assess the con-dition of the group of fish per tank.

Results and assessment of actual severity. Duringthe 84 days, 124 fish did not show any clinicalsigns and were considered to have experiencedMILD severity. In test concentration 2, discolour-ation was observed in one fish on three consecutivedays, followed by complete recovery. This was likelyto be the same fish on each day. Severity was con-sidered MILD. In the control group, test concentra-tion 1 and 2, one fish was found dead without priorclinical signs. Severity was considered MILD. In both

the control group and test concentration 1, one fishwas circle swimming. After consultation of the desig-nated veterinarian, these fish were kept in study andclosely monitored. Clinical signs disappeared in thefish after two days. Severity in both fish was con-sidered MODERATE. Finally, in test concentration1, one fish was observed without a tail and humanelyeuthanised. Severity: SEVERE.

Assessment: MILD for 127 fish, MODERATE for 2and SEVERE for 1 fish.

Actions: As clinical signs exceeded prospective sever-ity classification, and after veterinary consultation,

closer monitoring was performed and the CA wasnotified.

Example clinical observation/scoringsystem

A clinical score sheet can be used for daily observationson mortality and adverse effects. A clinical score sheetmay include several signs regarding swimming behav-iour, pigmentation, appearance, reactive behaviour orconvulsions. Their presence or absence is documenteddaily or more frequently is necessary. Any abnormaland unexpected behaviour should be reported to theresearcher and/or designated veterinarian or suitablyqualified fish expert. If severe adverse effects areobserved, it should be decided to monitor more fre-quently and/or to implement humane end-points.Considering the absence of identification and groupdensity, follow-up on any observations on individualanimals may be impaired or non-realistic.

Example of a clinical score sheet is given below.

Severity assessment is performed by a combinationof these observations (together with a procedure forevaluation). In case of combinations of observationsas described in the table, the highest score present isused for assessment. If an observation is present formore than one day in a row, the scores may be adjusted(þ1, þ2 or þ3) in consultation with the researcher,animal technologists and/or designated veterinarian orfish expert. Close monitoring is helpful in the assess-ment of actual severity on MILD, MODERATE orSEVERE, especially in cases of sudden death with orwithout previous clinical signs or in helping taking

Day of observationTarget concentration

Control Test concentration 1 Test concentration 2

0 no abnormalities no abnormalities no abnormalities

1 1 fish is dead no abnormalities no abnormalities

etc.

7 no abnormalities no abnormalities 1 fish is dead

etc.

14 no abnormalities 1 fish is circle swimming 1 fish is discoloured

etc.

16 no abnormalities 1 fish is dead 1 fish is discoloured

etc.

37 1 fish is circle swimming no abnormalities no abnormalities

etc.

72 no abnormalities 1 fish without tail (euthanised) no abnormalities

etc.

46 Laboratory Animals 52(1S)

informative decisions on timely implementation ofhumane end-points.

Suggested severity assessment and actions based onclinical observations:

References

1. Organisation for Economic Co-operation and

Development (OECD). Test no. 305: bioaccumulation

in fish: aqueous and dietary exposure. In: OECD guide-

lines for the testing of chemicals, section 3: degradation and

accumulation. Paris: OECD Publishing, 2012.2. European Commission. Commission Regulation (EC)

No 440/2008 of 30 May 2008, Part C: Methods for the

determination of ecotoxicity: ‘‘Bioconcentration: Flow-

through Fish Test’’. Off J Eur Union 2008; L142: C.13.

3. Organisation for Economic Co-operation and

Development (OECD). Guidance document on aquatic

toxicity testing of difficult substances and mixtures.

OECD series on testing and assessment number 23.

Paris: OECD Publishing, 2000.

4. Organisation for Economic Co-operation and

Development (OECD). Guidance document on the recog-

nition, assessment, and use of clinical signs as humane end-

points for experimental animals used in safety evaluation.

Paris: OECD Publishing, 2000.5. Humane Endpoints in Laboratory

Animal Experimentation. What are humane endpoints?,

www.humane-endpoints.info/en (2017, accessed 14 May

2017).

Description Scorea Action Severity assessmentb

Normal swimming, behaviour, appearance 1 NA MILD

Swimming behaviour

Bottom swimming 1 Check similarity controls MILD

Surface swimming 1 Check similarity controls MILD

Surface air gasping 2 NA MILD

Vertical swimming 3 Cumulative discomfort MODERATE

Swimming in side or supine position 3 NA MODERATE

Circle swimming (normal to supine position) 3 NA MODERATE

Pigmentation

Darker colouring 2 NA MILD

Appearance

Tail contraction 3 NA MODERATE

Haemorrhages/suggillation 4–5 (Consider) HEPc SEVERE

Visible damage to skin and/or fins 4–5 (Consider) HEP SEVERE

Reactive behaviour

Slow compared with control 2 NA MILD

Immobile 2 NA MILD

Hyperactive swimming after ticking against vessel 2 NA MILD

Hyperactive swimming 3 NA MODERATE

Other

Sudden death without previous signs 2 NA MILD

Sudden death with previous signs 2–5 NA MILD, MODERATE or SEVERE

Irreversible discomfort like convulsions 4–5 (Consider) HEP SEVERE

aIf an observation is also present on the second or third day in a row, the score is added þ1, 4 days in a row þ2 and 5 days in a row þ3.This may facilitate severity assessment and implementation of HEP.bIf a combination of observations is present, the severity assessment will be adjusted to the highest severity score of the observationspresent.cHEP¼ humane end-points.

Smith et al. 47

Assessment of acute oral toxicity with atest substance in rats

General context

The Organisation for Economic Co-operation andDevelopment (OECD) test guidelines are a collectionof methods used to assess the hazards of chemicals andof chemical preparations such as pesticides. Thesemethods cover tests for physical and chemical proper-ties, effects on human health and wildlife, and accumu-lation and degradation in the environment. The OECDtest guidelines are recognised worldwide as the stand-ard reference tool for chemical testing and are period-ically reviewed in the light of scientific progress orchanging assessment practices. Acute oral toxicitydata are used to satisfy hazard classification and label-ling requirements, for risk assessment for human healthand the environment, and when estimating the toxicityof mixtures. The objective of an acute oral toxicitystudy is to assess the (near-)lethal toxicity of the testsubstance following a single dose. The oral route isselected as this may be a possible route of humanexposure.

The conventional acute oral toxicity test (formerlyOECD Test Guideline 401, the classic ‘LD50’ test),1

requiring more than 20 animals is the most heavilycriticised test in terms of animal welfare and this con-cern was the driving force behind its deletion and thedevelopment of three alternative tests for acute oraltoxicity (OECD test guidelines 420, 423 and 425),2–4

respectively, the fixed dose method (420), the acutetoxic class method (423), and the up-and-down proced-ure (425). All guideline tests involve giving the test sub-stance in graduated doses to groups of animals, whichare observed with respect to effects and deaths (orhumane end-points). The result is given as a calculatedLD50 (the dose that kills 50% of the animals) or rangeestimates of LD50. The fixed dose method does notrequire the death of animals as an end-point and useson average 5–7 animals with 1 death. The acute toxicclass and up-and-down procedure use on average 6–9animals, with 0–3 deaths.5

Illustrative procedure

Study design. The acute toxic class method is a step-wise procedure with the use of three animals of themost susceptible sex (if unknown, female) per step.All available information on the test substance (e.g.identity and chemical structure, physico-chemical prop-erties, other in vivo or in vitro toxicity tests, toxico-logical data on the structurally related substances,anticipated use(s) of the substance) should be con-sidered prior to conducting the study.

The test substance should be formulated using a suit-able vehicle and concentrations should be adjusted toallow for constant dosage volumes. Animals should befasted prior to dosing to avoid interference with the testsubstance by food present in the stomach; the guidelinerequires ‘overnight’ fasting. Dosing volumes are chosenaccording to good practice and vehicles according tointernal/external recipients databases.

The dose level to be used as the starting dose isselected from one of four fixed levels (5, 50, 300 and2000mg/kg). There is an option to use an additionaldose level of 5000mg/kg, but only when justified by aspecific regulatory need. The starting dose level shouldbe that which is most likely to produce mortality insome of the dosed animals. When available informationsuggests that mortality is unlikely at the highest startingdose level (2000mg/kg body weight), then a so-called‘limit test’ should be conducted (6 rats, 3 rats per step).When there is no information on a substance to betested, for animal welfare reasons it is recommendedto use the starting dose of 300mg/kg body weight.Depending on the mortality and/or the moribundstatus of the animals, further groups of animals aredosed with higher or lower fixed doses, depending onthe presence of mortality, until the study objective isachieved; that is, the classification of the test substancebased on the identification of the dose(s) causing mor-tality, except when there are no effects at the highestfixed dose.

The time interval between treatment groups will bebased on onset, duration and severity of the toxic signs.Treatment of next group will be delayed until no fur-ther mortality is expected in previously dosed group(s).On average 2–4 steps may be necessary to enable ajudgement with respect to classifying the test substanceto one of a series of toxicity classes defined by fixedLD50 cut-off values.

In total, six rats will be used in this study. According tothe stepwise procedure, in step one, three rats will bedosed by oral gavage at 300mg/kg as a starting dose.Animals will receive a single oral dosing and then beobserved for 14 days. Observations include clinicalsigns, body weight measurements and macroscopicabnormalities during necropsy at the end of the observa-tion period, or earlier if found dead or humanely killed.

48 Laboratory Animals 52(1S)

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might itcause? What mightmake it worse? How will suffering be reduced to a minimum

Adverse effectsMethodology and interven-tions to minimise severity End-points

Food deprivation over-night prior to dosingand until 3–4 h afterdosing of test sub-stance. Water will beavailable.

No pain, suffering or lastingharm as food is deprivedonce per rat, and BW is notconsidered to get lowerthan 80% of the mean ofthe same sex/strain/age.As fasting is performedduring the active phase andmay take up to 24 h, thefood deprivation is con-sidered Mild as it maycause some distress basedon behaviour andphysiology.

Fasting not longer thanphysiologically advisable(rat 6 h) and scientificnecessary to empty GI tractor improve GI absorption ofsubstance is preferred overovernight fasting accordingto guideline but would needprior acceptance by com-petent authorities. Next(2nd) group will be givenfood immediately and notbe fasted/started, if (pro-longed/suspected) mortal-ity in earlier (1st) group ispresent.

NA

Single dosing of testsubstance by oralgavage

Transient discomfort follow-ing gavage is consideredMild. Good dosing tech-niques/skills essential butgavage accident may occurrarely.

Well-trained personnel.Gavage tube chosen toprovide minimum discom-fort (appropriate size, flex-ible plastic if allowed byinertness of test sub-stance, relatively wideinner diameter). Goodpractice administrationvolumes based on weight/species: e.g. maximumdose volume of 10 ml/kgBW. Maximum of threesingle dosing actions within24 h if higher volumes(based on concentration offormulation) are necessary.

Humane end-points incase ofgavageaccident,e.g. rup-turedoesophagus(rare).

Immediate and delayedtoxic effect of sub-stance after dosingand during a 14-dayobservation periodwith group housing

Depending on the test sub-stance. Local or systemicMild, Moderate or Severeclinical signs or mortalityduring the standard obser-vation period of 14 daysafter dosing may bepresent.

Monitoring of mortality/via-bility (at least 2�/day).Monitoring of clinical signs(at least 3�/dosing day andonce daily thereafter) andbody weights (at leastweekly). Interval betweensubsequent groups at least24 h. Next (2nd) group at thenext dose level will not befasted/started, if (pro-longed/suspected) mortal-ity in earlier (1st) group is

Humane end-pointsdeterminedby totalclinicalscore. An-imals show-ing severeclinicalsigns or inmoribundconditionwill be

(continued)

Smith et al. 49

Initial prospective assessment. SEVERE: Substancetoxicity may result in up to SEVERE severity, as start-ing dose levels are most likely to cause mortality in oneor more animals. Frequent observations after dosing onday 1 (directly after dosing, 2 and 4 h later) and morn-ing and evening checks are necessary to apply effectiveearly end-points minimising severity.

In general, a severity classification of SEVERE istherefore appropriate.

Could the severity classification be MODERATE?

Yes, if test substance information suggests mortalityto be unlikely at the highest starting dose level(2000mg/kg BW) and mortality or any relevant(delayed) clinical signs or mortality remain absent in6 rats (3 animals per step), the severity may be con-sidered MODERATE or even MILD.

Clinical observation. According to the guideline(OECD TG 423), observations should be made in indi-vidual rats after dosing at least once during the first30min, then periodically during the first 24 h, with spe-cial attention given the first 4 h, and daily thereafter for14 days.3 Duration of observation should be deter-mined by toxic reactions, time of onset and length ofrecovery period and may be extended when considerednecessary. Body weights are measured before dosingand at least weekly thereafter. Body weight changesshould be recorded and used for welfare assessmentand/or early humane end-points. Any relevant macro-scopic abnormalities during necropsy may be useful toconfirm the correctness of chosen humane end-points.Additional observations may be necessary if signs oftoxicity continue. Observations should include changes

in skin and fur, eyes and mucous membranes, and alsorespiratory, circulatory, autonomic and central nervoussystems, and somatomotor activity and behaviour pat-tern. Attention should be directed to observations oftremors, convulsions, salivation, diarrhoea, lethargy,sleep and coma. The principles and criteria summarisedin the OECD Humane Endpoints Guidance Documentshould be taken into consideration.6 Animals found ina moribund condition and animals showing severe painor enduring signs of severe distress should be humanelykilled.

A clinical score sheet or program (FELASAWorking Group Report, 2015) is useful for monitoringanimal welfare, identifying humane end-points as wellas reporting according to the OCDE guidelines.6,7 Anexample of a clinical scoring system which is used tohelp monitor the clinical condition of the animalsthroughout the procedure is included at the end ofthis example.

Results and assessment of actual severity. All trea-ted rats in step 1 at starting dose of 300mg/kg showedclinical signs. Clinical signs comprised lethargy,hunched posture, uncoordinated movements, piloerec-tion, ptosis, rales and/or salivation on Days 1 and 2where one rat showed hunched posture also on days 6and 7. No rats were found dead or needed to be killedhumanely, thus a second step was necessary accordingto the guidelines. In step 2, with 3 rats dosed at2000mg/kg, one female rat was found dead on Day 2,showing slight body weight loss and clinical signs likelethargy, hunched posture, piloerection, ptosis and sali-vation between Days 1 and 2. No macroscopic

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might itcause? What mightmake it worse? How will suffering be reduced to a minimum

Adverse effectsMethodology and interven-tions to minimise severity End-points

present. Be alert fordelayed mortality/toxicity.Frequency/period ofobservation may beextended based upon(expected) clinical signs tominimise risk of lethalityusing humane end-points.

euthanised(this doesnot com-promise thetest result,as such ani-mals areconsideredsimilar toanimals thatdied on thetest).

50 Laboratory Animals 52(1S)

abnormalities were found during necropsy of thisanimal and other rats at the end of the study. No mor-tality was observed in other rats. Body weight gain ofsurviving rats was comparable to untreated animals ofsame strain and age. For this purpose, historical dataon untreated or on same vehicle treated animals ofsame strain and age may be used. Assessment:

SEVERE for the rat found dead, MODERATE forthe other rats.

Scoring system and examples of scoringsheets

Severity assessment is performed by a combination ofclinical observations (changes in bodyweight, skin, fur,eyes and mucous membranes, occurrence of secretionsand secretions and respiratory, circulatory, autonomicand central nervous systems, somatomotor activity andbehaviour pattern.) based on a clinical scoring systemused in toxicology, and necropsy findings. Time ofonset, grade and duration of observed signs are rec-orded. Signs are graded for severity and the maximumgrade is predefined at 3 or 4. Grades are coded as slight

(grade 1), moderate (grade 2), severe (grade 3) and verysevere (grade 4). For certain signs, only its presence(grade 1) or absence (grade 0) is scored.

Example of a clinical score sheet is given below. Abroad but not complete range of typical clinical signs,are presented on the left (the range of scores is given perclinical sign between brackets, e.g. 0–3 ranging fromabsent to maximally present). This example refers toan individual rat dosed at 300mg/kg BW in the firststep with three rats.

Another example of the same study, now an individ-ual rat dosed at 2000mg/kg BW in the second step withthree other rats. This rat dies on Day 2 after scheduledobservation. Only abnormalities in the clinical scoringsheet over the observation period are shown.

Clinical signs D1-0 h D1-2 h D1-4 h D2 D3 D4 D5 D6D7 etc.up to 15

Bodyweight (g) 150 – – 145 150 152 155 158

Skin and fur: stains (0–1) 0 0 0 0 0 0 0 0 0

Skin and fur: pale (0–3) 0 0 0 0 0 0 0 0 0

Involuntary clonic movements (0–3) 0 0 0 0 0 0 0 0 0

Involuntary tonic movements (0–3) 0 0 0 0 0 0 0 0 0

Tremors (0–3) 0 0 0 0 0 0 0 0 0

Salivation (0–3) 0 1 0 0 0 0 0 0 0

Lacrimation (0–1) 0 1 0 0 0 0 0 0 0

Diarrhoea (0–1) 0 0 0 0 0 0 0 0 0

Vocalisations (0–1) 0 0 0 0 0 0 0 0 0

Stereotypy: excessive grooming (0–1) 0 0 0 0 0 0 0 0 0

Stereotypy: repetitive circling (0–1) 0 0 0 0 0 0 0 0 0

Bizarre behaviour: self-mutilation (0–1) 0 0 0 0 0 0 0 0 0

CNS: lethargy (0–3) 0 2 2 0 0 0 0 0 0

CNS: convulsions (0–3) 0 0 0 0 0 0 0 0 0

Posture: hunched posture (0–1) 0 1 1 0 0 0 0 1 1

Posture: rearing (0–1) 0 0 0 0 0 0 0 0 0

Posture: ventro-lateral recumbency (0–1) 0 0 0 0 0 0 0 0 0

Gait abnormalities: uncoordinated movements (0–3) 0 1 1 0 0 0 0 0 0

Piloerection (0–1) 0 1 1 0 0 0 0 0 0

Ptosis (palpebral closure) (0–3) 0 1 1 0 0 0 0 0 0

Respiratory abnormalities: laboured respiration (0–3) 0 0 0 0 0 0 0 0 0

Respiratory abnormalities: Rales (0–3) 0 1 1 0 0 0 0 0 0

Smith et al. 51

References

1. Organisation for Economic Co-operation and

Development (OECD). Test no. 401: acute oral toxicity.

In: OECD guidelines for the testing of chemicals, section 4:

health effects. Paris: OECD Publishing, 1987.

2. Organisation for Economic Co-operation and

Development (OECD). Test no. 423: acute oral toxicity

– fixed dose procedure. In: OECD guidelines for the test-

ing of chemicals, section 4: health effects. Paris: OECD

Publishing, 2001.

3. Organisation for Economic Co-operation and

Development (OECD). Test no. 423: acute oral toxicity

– acute toxic class method. In: OECD guidelines for the

testing of chemicals, section 4: health effects. Paris: OECD

Publishing, 2002.

4. Organisation for Economic Co-operation and

Development (OECD). Test no. 425: acute oral toxicity

– up-and-down procedure. In: OECD guidelines for the

testing of chemicals, section 4: health effects. Paris: OECD

Publishing, 2008.5. Organisation for Economic Co-operation and

Development (OECD). Guidance document on acute

oral toxicity testing. In: OECD guidelines for the testing

of chemicals, section 4: health effects. Paris: OECD

Publishing, 2000.6. Organisation for Economic Co-operation and

Development (OECD). Guidance document on the recog-

nition, assessment and use of clinical signs as humane end-

points for experimental animals used in safety evaluation.

Environmental Health and Safety Monograph Series on

Testing and Assessment No. 19. Paris: OECD Publishing,

2000.7. Fentener van Vlissingen JM, Borrens M, Girod A, et al.

The reporting of clinical signs in laboratory animals:

FELASA Working Group Report. Lab Anim 2015; 49:

267–283.

Pharmacokinetic study after singleadministration of a test substance inthe dog

General context

The aim of the study is to study the pharmacokineticsof a compound after single administration.

The difference in systemic exposure following oral (cap-sule/tablet/gavage/diet), intravenous, dermal or sub-cutaneous (bioequivalence) administration is assessed.For substances intended to be ingested via the oralroute in humans, the oral route is compared to theintravenous route, which is the reference route giving100% bioavailability. The applicable testing guidelinesfor the compound relate to any human exposure, pro-spective use or legislation.1–10 The compound may con-cern pharmaceuticals for human or veterinary use,feeding ingredients or (agro-)chemicals.

Illustrative procedure

Study design. A common study design is to use thesame animals for two periods. In this example, threedogs were used. In the first period, plasma levels ofthe compound following an intravenous dose are mea-sured, then after an appropriate estimated wash-out, inthe second period, plasma levels of the compound fol-lowing oral dosing of the same animals are measured.Third or more periods with appropriate wash-outs maybe used to compare different oral test substance formu-lations (e.g. different solutions, tablets, capsules). Forbioequivalence studies where a test compound is com-pared to a known compound, a randomised blockdesign may be used to randomise the order of exposureto different routes and thus prevent any possible timeeffects from previous dosing. For bioavailability stu-dies, where an unknown test compound is tested forsufficient bioavailability in order to be used for furtherstudies, this may not be typically required. Urine and/or faeces may be collected from animals kept in metab-olism cages for the collection. After the study, it maystill be scientifically plausible to re-use the animal, andit may be allocated to the ‘stock animals’. Fitness forpossible re-use will be based on age, lifetime and pro-cedure-specific welfare assessment, restoration of gen-eral health and well-being in accordance with veterinaryadvice and classification of further procedures. A totalof 4 periods, first period of intravenous dosing followedby three periods of different capsule dosing, each with a1 week wash-out period in between the dosing periods,were used in the study. Observations comprised clinicalsigns and body weight measurement before each dosingperiod.

Clinical signs D1-0 h D1-2 h D1-4 h D2 D3 D4 D5 D6 D7 etc. up to 15

CNS: lethargy (0–3) 0 1 1 1 – – – – –

Posture: hunched posture (0–1) 1 1 1 1 – – – – –

Piloerection (0–1) 0 1 1 1 – – – – –

Ptosis (palpebral closure (0–3) 0 2 1 1 – – – – –

Salivation (0–3) 0 0 0 1 – – – – –

52 Laboratory Animals 52(1S)

Consideration of specific refinements and humaneend-points.

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might make itworse? How will suffering be reduced to a minimum?

Adverse effectsMethodology andinterventions End-points

Food deprivation in oraldosing study up to 20–24 h before dosing(‘overnight fasting’) toimprove GI absorption,to prevent vomiting offood and/or possibleclinical biochemistryblood sampling.

Or: diet may be offered forsame GI absorptionreasons in parts pre-orpost-dosing)

In general, not regarded as aprocedure causing dis-comfort in adult dogs rou-tinely fed once daily (ifnormally fed twice daily, itwould be regarded as aprocedure). If exceeding24 h based on post-dosingfasting or in juveniles, itmay cause discomfort.Depending on duration offood deprivation and effectof test substance likevomiting or local GI effects:ranging from no effect tohungry feeling, nausea,indigestion, pain, vomiting,food aspiration, diarrhoeadue to GI effects.

Preferably no longerthan physiologicallyadvisable (dog 12 h)and scientific neces-sary to empty GI tractto improve GIabsorption of sub-stances, but also toprevent vomitingreflex or possiblelocal test substanceeffects

Changing in time-related workingprocedures.

NA

Water restriction (usuallynot necessary but ifindicated, of limitedduration like 2 h pre-and 2 h post-dosing,exact amount of watermay be dosed by oralgavage after dosing oftest substance)

No discomfort or dehydrationexpected, transient MILDdiscomfort if oral gavagedosing of water is used.

Water restriction notlonger than physio-logically advisableand scientificallynecessary toimprove/harmoniseGI absorption of sub-stances.

Appropriate equipment/technique e.g.appropriately sizeddisposable flexiblecatheter for oraldosing of water.

NA

Single dosing by oral(capsule/tablet/gavage),subcutaneous, intra-muscular intravenous(via superficial veins),dermal, sublingual,rectal, vaginal, intratra-cheal or intranasalroute.

Some study design mayinvolve testing ofmetabolites next to test

Transient MILD discomfortdepending on dosing route,restraining, possibleanaesthesia (with intratra-cheal or intranasal route)and local/systemic testsubstance and vehicleeffects.

Though non-toxic doses areused, unexpected effectsmay cause up toMODERATE effects.

Limitation of adminis-tration volumes.11

Appropriate vehicleselection (optimalsubstance exposure,biological inertness,no/limited biologicaleffects and toxicity).

Appropriate dosingequipment e.g.appropriately sizeddisposable flexible

Humane end-pointsdetermined bytotal clinicalscore.

Animals showingsevere clinicalsigns or in mori-bund conditionwill be euthanised

(continued)

Smith et al. 53

Continued

What does this studyinvolve doing tothe animals?

What will the animalsexperience? How muchsuffering might it cause?What might make itworse? How will suffering be reduced to a minimum?

Adverse effectsMethodology andinterventions End-points

substance and/or refer-ence products (bioequi-valence) and thusinvolve repeated dosingin the animal with con-sideration of appropri-ate wash-out periods.

MILD to MODERATE discom-fort due to repeated dosingor use of anaesthesiaduring intra-trachealdosing.

catheter instead ofsteel gavage needle).

Appropriate anaesthe-sia. Mixing with pal-atable food if thisdoes not hamperuptake.

Repeated blood sampling(e.g. jugular vein) aftersingle dosing for toxico-kinetics (oral dosing:e.g. pre-dose, 8� in first24 h, then 5 time pointsup to day 5; IV dosing:e.g. pre-dose, 10–12� infirst 24 h, then 2 timepoints until day 3, or upto 4–5 time points up today 4–5)

(Re)Introduction of needlecausing no or MILD dis-comfort depending on pos-sible habituation effect toprocedure or possible localinflammatory reaction oftissue.

Use of positivereinforcement train-ing before perform-ance of samplingprocedures.

Good practice, equip-ment/techniques.

Appropriate samplingvolumes not nor-mally limiting in thisspecies/design (fre-quent small volumesunlikely to cause anyphysiological dis-turbance).11 Use ofindwelling cathetersmay be preferred forperiod of frequentsampling to improvevenous access andprevent localreactions.

NA

Individual housing inmetabolism cagebecause of non-invasiveurine sampling by col-lection of spontaneousurine in one or two 24 hperiods)

Social isolation. Metabolismcage may prevent tactilecontact in a limited timeperiod but olfactory, visualand auditory contact is stillpresent. Below threshold ifshort period (metabolismcage� 24 h).

If longer than 24 h, it may beMild.

Promote visual, olfac-tory, auditory or lim-ited (e.g. nose-)tactile contact withconspecifics.

Frequent humansocialisation (notonly observations/procedures butinteraction/playingtime) or observation/treatment (if basedon veterinary rea-sons).

Non-interfering enrich-ment material.

NA

54 Laboratory Animals 52(1S)

Initial prospective analysis of severity. Animal is onlyexpected to experience MILD discomfort based on lim-ited food deprivation/water restriction, limited individ-ual housing, single dosing, and repeated blood sampling.Test substance effects are expected to be absent or verylimited due to levels considered pharmacological.

A prospective severity classification of MILD is there-

fore appropriate

Could the severity classification be MODERATE?

Single dosing of test substance and frequent bloodsampling in the dog is considered to be MILD based onspecies character/behaviour (social interaction withhumans), restraining method, and best practice dosing& sampling technique/volumes used. MODERATEseverity seems unlikely prospectively as not toxic, butpharmacologic, doses are to be used, but may be causedby unexpected test substance (or vehicle) effects or pos-sible complications during dosing and sampling.

Clinical observations. Veterinary examinations areperformed before start of study (at arrival from thesupplier or at periodic intervals in the animal stock)to ensure the good state of health and fitness for (re-)use of the animal for the procedure. Body weights aremeasured before each dosing period. When considerednecessary, veterinary examinations may be performedduring the study. During the study period, animals aremonitored on mortality/viability at least twice daily

and for clinical signs during dosing and the follow-upperiod at least once daily.12

An example of clinical score sheet based on an indi-vidual observation (applicable score grade rangesbetween brackets):

Severity assessment is performed by a combinationof general clinical observations (body weight, appear-ance, behaviour, cage environment) based on a clinicalscoring system used in toxicology. Time of onset, gradeand duration of observed signs are recorded. Signs aregraded for severity and the maximum grade is prede-fined at 3 or 4. Grades are coded as slight (grade 1),moderate (grade 2), severe (grade 3) and very severe(grade 4). For certain signs, only its presence (grade1) or absence (grade 0) are scored.

Results and assessment of actual severity. In thefirst period, no mortality occurred. In the first periodof intravenous dosing, one animal (no. 3 in the exam-ple) showed clinical signs like slight/moderate tremor,slight lethargy, hunched posture, vomiting of food andmucus, faeces containing mucus, and/or faeces with redparticles for a period of 2 h, followed by a quick andcomplete recovery. Assessment: MILD.

The mild but nevertheless clear clinical signs of rela-tively short duration after IV dosing could indicatesimilar signs for a longer duration after oral dosing.

Period: 1 2 etc.

Date: D1 D2 D3 D4 D1 D2 D3 D4

Body weight (in kg or g)

Piloerection (0–1)

Lethargy (0–3)

Restless (0–3)

Aggressive (0–3)

Food intake (in g)

Salivation (0–3)

Vomiting (of food) (0–1)

Vomiting of mucus (0–1)

Tachypnoea (0–1)

Tremor (0–3)

Hunched posture (0–1)

Abnormal posture (0–1)

Ventrolateral recumbency (0–1)

Abnormal gait (0–3)

Diarrhoea (0–1)

Faeces containing mucus (0–1)

Faeces with red particles (0–1)

Hyperthermia

Smith et al. 55

Therefore, the prospective assessment for periods oforal dosing was upgraded to MODERATE. Beforecontinuation of the study, the CA was notified on theupgrade of the prospective assessment.

In the second (oral dosing) period, the animalsshowed no clinical signs. Assessment: MILD. In thethird and fourth period (oral dosing with different cap-sules), one animal showed hyperthermia, vomiting offood, hunched/abnormal postures and gait, ventrolat-eral recumbency muscle switching, tremor and lethargy,

salivation, tachypnoea followed by recovery at the endof the day. Assessment: MODERATE.

Examples of clinical score sheets

The clinical scores per animal over the dosing periodsare a selection of signs observed in this study exampleand just a part of a complete list of clinical signs withtheir scores available.

Example of clinical scores in animal 1:

Period: 1 2 3 4

Date: D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4

Body weight (kg) 9.9 – – – 9.9 – – – 9.9 – – – 10 – – –

Vomiting (of food) (0–1) 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Tremor (0–3) 2 0 0 0 0 0 0 0 3 0 0 0 3 0 0 –

Faeces containing mucus (0–1) 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 –

Example of clinical scores in animal 3:

Period: 1 2 3 4

Date: D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4

Body weight (kg) 10.7 – – – 10.5 – – – 10.6 – – – 10.6 – – –

In heat (0–1) 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Example of clinical scores in animal 3:

Period: 1 2 3 4

Date: D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4 D1 D2 D3 D4

Body weight (kg) 10.3 – – – 10.0 – – – 10.0 – – – 9.9 – – –

Lethargy (0–3) 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Salivation (0–3) 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 –

Vomiting (of food) (0–1) 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Vomiting of mucus (0–1) 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 –

Tachypnoea (0–1) 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Tremor (0–3) 2 0 0 0 0 0 0 0 3 0 0 0 3 0 0 –

Muscle twitching (0–1) 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Hunched posture (0–1) 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Abnormal posture (0–1) 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 –

Ventrolateral recumbency (0–1) 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

Abnormal gait (0–3) 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 –

Faeces containing mucus (0–1) 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 –

Faeces with red particles (0–1) 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 –

Hyperthermia (0–1) 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 –

56 Laboratory Animals 52(1S)

Assessment of actual severity in the threeexamples

Cumulatively over the four dosing periods, duringactual severity assessment, two animals were consideredas MILD; one animal was considered as MODERATE

over all four dosing periods.

Actions

As clinical signs are usually noted just after (IV) and/orduring the first day after dosing (PO), closer monitoringmay be essential in case of unexpected moderate tosevere adverse effects, supported if necessary by veter-inary consultation, notification to CA before continu-ation and any intervention like humane killing in caseof humane end-points.13 If moderate to severe adverseeffects are noted in the first animal, a staggered start(some time interval between first and second or eachanimal, with consideration of expected duration of pos-sible clinical signs) and reconsideration of the dosage isconsidered good practice. Also any possible differencesbetween sexes need to be considered before the studyor, if unknown, in the study design (e.g. starting withone animal per sex, then following animals of each sex).

After the last dosing and observation period, in caseof mild up to moderate effects, the dogs are releasedfrom the study and monitored in stock until no furtherabnormalities are observed. In case of any planned re-use, it should be demonstrated that the general healthand well-being of the animal has been fully restoreduntil use for any next study will be considered. Thisassessment should be in accordance with veterinaryadvice and preferably supported by periodic clinicaland laboratory examinations. If necessary, based onthese findings and bioanalysis data (elimination half-time determined) in the study, an appropriate wash-out period between studies should be considered.

References

1. European Medicines Agency. Guidelines for the conduct of

pharmacokinetic studies in target animal species. Report

no. EMEA/CVMP/133/99, September 2000. London:

European Medicines Agency.2. European Community (EC). Annex of the EEC Directive

75/318/EEC, Part 3, section II.G, as amended with a view

to the granting of a marketing authorisation for a medicinal

product, 3BS11a: ‘‘pharmacokinetics and metabolic studies

in the safety evaluation of new medicinal products inanimals’’.

3. Environmental Protection Agency (EPA), Office of

Prevention, Pesticides and Toxic Substances (OPPTS).Health effects test guidelines: metabolism and pharmaco-kinetics. Paragraph 870.7485, August 1998. Washington,DC: EPA.

4. Environmental Protection Agency (EPA), Office ofPrevention, Pesticides and Toxic Substances (OPPTS).Health effects test guidelines: dermal penetration.

Paragraph 870.7600, August 1998. Washington, DC:EPA.

5. Environmental Protection Agency (EPA), Office of

Prevention, Pesticides and Toxic Substances (OPPTS).Residue chemistry test guidelines: nature of the residue –plants, livestock. Paragraph 860.1300, August 1996.

Washington, DC: EPA.6. ICH Secretariat. Note for guidance on toxicokinetics: the

assessment of systemic exposure in toxicity studies. ICHS3A, October 1994. Geneva: ICH Secretariat.

7. ICH Secretariat. Pharmacokinetics: guidance for repeateddose tissue distribution studies. ICH S3B, October 1994.Geneva: ICH Secretariat.

8. Organisation for Economic Co-operation andDevelopment (OECD). Test no. 417: toxicokinetics. In:OECD guidelines for the testing of chemicals, section 4:

health effects. Paris: OECD Publishing, 2010.9. Organisation for Economic Co-operation and

Development (OECD). Test no. 427: percutaneousabsorption: in vivo method. In: OECD guidelines for the

testing of chemicals, section 4: health effects. Paris: OECDPublishing, 2004.

10. Organisation for Economic Co-operation and

Development (OECD). Test no. 503: metabolism in live-stock. In: OECD guidelines for the testing of chemicals,section 5: other test guidelines. Paris: OECD Publishing,

2007.11. Diehl HK, Hull R, Morton D, et al. A good practice

guide to the administration of substances and removal

of blood, including routes and volumes. J Appl Toxicol2001; 21: 15–23.

12. Fentener van Vlissingen JM, Borrens M, Girod A, et al.The reporting of clinical signs in laboratory animals:

FELASA Working Group Report. Lab Anim 2015; 49:267–283.

13. Organisation for Economic Co-operation and

Development (OECD). Guidance document on the recog-nition, assessment and use of clinical signs as humane end-points for experimental animals used in safety evaluation.

Environmental Health and Safety Monograph Series onTesting and Assessment No. 19. Paris: OECD Publishing,2000.

Smith et al. 57