DR PENNY HAWKINS, RSPCA RESEARCH ANIMALS DEPARTMENT Severity assessment before, during and after the procedure

Requirements of the Directive ARTICLE 15: CLASSIFICATION OF SEVERITY OF PROCEDURES 1: Member States shall ensure that all procedures are classified as ‘non-recovery’, ‘mild’, ‘moderate’ or ‘severe’ on a case-by-case basis using the assignment criteria set out in Annex VIII 2: Subject to the use of a safeguard clause in Article 55(3), Member States shall ensure that a procedure is not performed if it involves severe pain, suffering or distress that is likely to be long-lasting and cannot be ameliorated

Requirements of the Directive ARTICLE 38: PROJECT EVALUATION 38(2): The project evaluation shall consist of the following: b)an assessment of the compliance of the project with the requirement of replacement, reduction and refinement; c)an assessment and assignment of the classification of the severity of procedures

Requirements of the Directive ARTICLE 39: RETROSPECTIVE ASSESSMENT 39(1): Member States shall ensure that when determined in accordance with Article 38(2)(f), the retrospective assessment shall be carried out by the competent authority which shall, on the basis of the necessary documentation submitted by the user, evaluate the following: b)The harm inflicted on the animals, including the numbers and species of animals used, and the severity of the procedures

Requirements of the Directive ARTICLE 54: REPORTING 2: Member States shall collect and make publicly available, on an annual basis, statistical information on the use of animals in procedures, including information on the actual severity of procedures and on the origin and species of non- human primates used in procedures

General considerations for a severity assessment The consideration of severity within a procedure should be a continuous process –beginning with initial study design, –through the study-specific day-to-day monitoring of animals during the project, –to the “actual” severity assessment upon completion of the study, which provides opportunities to identify further refinements for future studies By approaching in this manner, there is a greater opportunity to ensure that the Three Rs are considered and implemented throughout, that communication among all involved will be improved and that consistency will be enhanced

All aspects of the study that may cause pain, suffering, distress or lasting harm should be identified, and consideration given as to how their effects can be minimised, for example by consulting the literature, colleagues, animal technologists, the veterinarian and the Animal Welfare Body if appropriate The recommended prospective severity classification assigned to procedures should be based on the highest severity anticipated for any animal on the study 1. Severity assessment before the procedure AT THE PROJECT PLANNING STAGE

The factors related to the procedure shall include: Type of manipulation, handling Nature of pain, suffering, distress or lasting harm caused by (all elements of) the procedure, and its intensity, the duration, frequency and multiplicity of techniques employed Cumulative suffering within a procedure Prevention from expressing natural behaviour including restrictions on housing, husbandry and care standards

The following additional factors shall also be taken into account: Type of species and genotype Maturity, age and gender of the animal Training experience of the animal with respect to the procedure If the animal is to be reused, the actual severity of the previous procedures Humane end-points

Examples in Annex VIII

EAE example (EC, 2013) APPROACH FOR INITIAL PROSPECTIVE ASSESSMENT Prospective severity classification = severe

Severe, prolonged suffering and the ‘safeguard clause’ ARTICLE 55: SAFEGUARD CLAUSES 55(3): Where, for exceptional and scientifically justifiable reasons, a Member State deems it necessary to allow the use of a procedure involving severe pain, suffering or distress that is likely to be long- lasting and cannot be ameliorated... It may adopt a provisional measure to allow such procedure. Member States may decide not to allow the use of non-human primates in such procedures. 55(4): The Member State must immediately inform the Commission and other Member States, with reasons and evidence. The matter will then go before a Commission Committee within 30 days, after which the provisional measure may be authorised for a set time period or revoked.

When the EC Expert Working Group was discussing this, nobody could think of an example of ‘a procedure involving severe pain, suffering or distress that is likely to be long-lasting and cannot be ameliorated’ There was support for the wording because it meant that people had to strive to ameliorate suffering in severe procedures Practical implications

A plan for observing the animals that is suitable for and tailored to the study should be developed –Standardised terminology –Sufficient trained and competent staff –Take account of species, strain, individuals and procedure Protocol should indentify any adverse effects that may occur throughout the animal’s lifetime, including housing, husbandry and handling 2. Severity assessment during the procedure AT THE PROJECT PLANNING STAGE

A hierarchical approach HIGH LEVEL CATEGORIES AS A STARTING POINT Appearance Body functions Environment Behaviours Procedure-specific indicators Free observations

Photo: The Pennsylvania State University

Nest building behaviour Arras M et al. 2007; OR SIMPLY ‘TIME TO INCORPORATE NESTING MATERIAL’ (TIN) – OVER 10 MINUTES IF POST-OPERATIVE PAIN IS PRESENT

Orbital tightening Nose bulge Cheek bulge Ear position Whisker change Normal Increasing evidence of pain ‘Pain faces’: five facial ‘action units’ have been defined that can be used to assess levels of pain in mice Langford DJ et al. Nature Methods 2010, doi: /Nmeth.1455

‘Score sheets’ should be considered –Tailored to the species and type of study –Standardised language and terminology is recommended –May be numerical or ‘binary’ ‘Team approach’ to defining Effective training in using the system is essential for all relevant staff Monitoring tools FOR USE IN DAY-TO-DAY ASSESSMENTS

EAE Example - clinical score sheet Date AppearanceBody weight Coat condition Body functionBladder control Tail tone Respiration EnvironmentNest condition BehaviourSocial behaviour Gait Procedure-specific indicatorsSide resting position Righting time Paresis Other observations – free text

Filling in the table Score:1 = mild2 = moderate3 = severe Weight lossUp to 10 %10 to 20 %20 to 35 % Tail toneDiminished lifting or curling of tail Loss of tone in distal half of tail Loss of tone in entire tail Nest conditionSlightly disorganised Some attempt at nest but disorganised No nest

There should be a communication plan to ensure that all relevant staff know about the monitoring system and how to use it, including a mechanism to rapidly communicate unexpected outcomes to all appropriate individuals (and the Competent Authority if necessary) Procedures that may cause severe suffering need more frequent and detailed monitoring There should be clear criteria for intervention with respect to humane endpoints – all should know what these are, what to do and whom to contact Remember the primary purpose of day-to-day monitoring TO ENSURE THAT SUFFERING IS DETECTED AND RELIEVED

Required for statistical reporting Part of the retrospective assessment that is mandatory for all severe procedures and all procedures involving primates But it is good practice for all procedures to carry out a detailed review of the nature and level of suffering, what caused it, and how it could be better prevented or relieved in future 3. Severity assessment after the procedure ACTUAL SEVERITY ASSESSMENT

Conduct an individual assessment for each animal Use the observations taken from the animal during day-to-day monitoring –The actual severity may be different from the predicted severity classification Report an actual severity that is the highest level experienced during the course of the procedure Assessing actual severity MAIN PRINCIPLES

EAE example - actual severity USING THE FILLED-OUT CLINICAL SCORE SHEETS Two mice lost 8 % of their body weight following induction of EAE, had slightly unkempt fur and slow forelimb abduction, but score ‘2’ for all other indicators for the first 5 days. Their scores were then ‘1’ or ‘0’ for each indicator throughout the study –Severity = MODERATE Three mice lost between 22 and 32 % of their body weight and scored a combination of ‘3’ ‘2’ and ‘1’ throughout –Severity = SEVERE One mouse lost 37 % of his body weight post induction of EAE and was humanely killed –Severity = SEVERE

EAE - review of the protocol FOLLOWING ACTUAL SEVERITY ASSESSMENT AND CONSULTATION WITH COLLEAGUES AND THE LITERATURE Animals were pre-fed with high-energy supplement foods, such as jelly and condensed milk, before administering the adjuvant The dose of adjuvant was reduced The study protocol was altered so that the duration of the project could be reduced

Effective severity assessment is essential for better animal welfare and science It should be viewed as an opportunity to make continuous improvements to science and welfare, and to address public concerns about animal use A team approach, with adequate training and communication for all, will improve objectivity and consistency Keep up with the literature – this is constantly growing and there are many new approaches to try Concluding comments

Thank you