Humane endpoints and use of score sheets

1. Humane endpoints and use of score sheets Timo Nevalainen University of Eastern Finland

2. Humane endpoint? • The earliest indicator in an animal experiment of severe pain, severe distress, suffering, or impending death. • The ultimate purpose of the application of humane endpoints is to be able to accurately predict severe pain, severe distress, suffering, or impending death, before the animal experiences these effects. • These adverse conditions, once identified should be minimised or eliminated, either by humanely killing the animal or, in long-term studies by (temporary) termination of exposure, or by reduction of the test substance dose.

3. Death • Predictable Death • presence of clinical signs indicative of death before the planned end of the experiment; for example: inability to reach water or food (CD 22) • Impending Death • when moribund state or death is expected prior to the next planned time of observation. Signs indicative of this state in rodents could include convulsions (CD27), recumbency, and tremor (CD 26) • Moribund • being in state of dying or inability to survive, even if treated

7. Pain • Acute nociceptive pain: pain response evoked by a brief noxious stimulus which produces no tissue damage. This form of pain is not regarded as severe. • Example: pedal reflex • Persistent (chronic) inflammatory pain: the pain resulting from tissue damage lasting for the duration of the damage or the ensuing inflammatory process, and may persist after the local tissue damage has healed. This type of pain may be severe or distressing, particularly if long lasting or permanent. • Example: self mutilation, localised infection • Neuropathic pain: pain as a result of compromised function or abnormal activation of the peripheral or central nervous system. Neuropathic pain is always considered as severe and distressing pain. • Example: the presence of a large internal tumour that compresses nerves.

9. Distress • An aversive state resulting from maladaption or inability to adapt to stressors • Physical or behavioural alterations may be signs of stress. Acute stress is not regarded as a cause of distress; it may be necessary to optimise vigilance and to reduce the risk of boredom. Distress is usually associated with a change in motility or locomotion, and can result in stereotype behaviour (DC23). • The major stressors associated with distress are situations that may give rise to marked pain, fear, or anxiety • Retreat to the corner of the cage or excessive struggling or vocalising on dosing are examples of distress in anticipation of an experimental procedure.

11. Suffering • A negative emotional state that in human beings is produced by persistent pain and /or distress • It should be assumed that persistent pain or distress in animals leads to suffering of animals • If something is known to cause suffering in humans, it should be assumed to cause suffering in animals • ‘Absence of evidence is not evidence of absence’

12. Principles • Severe pain, suffering, or death are to be avoided as endpoints • Studies must be designed to minimise any pain, distress or suffering experienced by the animals, consistent with the scientific objective of the study • The earliest possible endpoints that are indicators of distress, severe pain, or impending death that should be used as indications for humanely killing the animals should be determined prior to the animals’ reaching a moribund state • Studies should be terminated prior to their anticipated termination time if the objectives of the study have been satisfied, or if it is obvious that they will not be achieved

13. Humane endpoints for toxicity studies • Deviations from an animal’s “normal state” -> correlation of these changes with the possibility and severity of pain and distress • observations to detect abnormal signs and conditions (behaviour, physiology, etc.), and other indicators of welfare problems • whether such observations are indicators of pain and distress, and whether pain and distress are severe • whether abnormal conditions not necessarily considered to be indicative of severe pain and/or distress, are indicative of an irreversible condition likely to lead to further deterioration (e.g., moribund condition; impending death; inability to eat or drink)

14. prolonged, impaired ambulation preventing the animal from reaching food or water, or prolonged anorexia excessive weight loss and/or extreme emaciation and/or severe dehydration significant blood loss evidence to suggest irreversible organ failure prolonged absence of voluntary responses to external stimuli persistent, difficult laboured breathing (CD13) prolonged inability to remain upright persistent convulsions (CD28) self-mutilation prolonged diarrhoea significant and sustained decrease in body temperature substantial tumours other treatment-related effects judged to be indicative of impending death Signs for impending death

16. Signs for pain and distress • Look for changes in • physical appearance (e.g., coat texture; hair soiled with urine or faeces, CD 11) • clinical signs (e.g., respiration rate; posture) • unprovoked behaviour (e.g., self mutilation; compulsive behaviour) • response to external stimuli (e.g., excitability; righting reflex, CD24) • body weight, and related changes in food and water consumption • clinical parameters (e.g., body temperature; heart and respiration rate; clinical chemistry and haematology)

19. abnormal vocalisation abnormal aggressiveness abnormal posture abnormal reaction to handling abnormal movements self-induced trauma open wounds or skin ulceration difficulties in respiration corneal ulceration bone fractures reluctance to move abnormal external appearance rapid weight loss or emaciation or severe dehydration significant bleeding or any other factor that suggests that the animal may be in pain or distress Signs for severe pain and distress

20. Scoring systems • Impossible to have general scoring sheet • Predicable outcomes are different • Sheets must be • Procedure specific • Species/strain specific • Pilot studies needed to • Identify cardinal signs • measurable (analogue) • qualitative (binary)

23. CD14 Jones et al. 1999

24. Jones et al. 1999

25. Example: Rabies vaccine trial Cussler et al. 1999

26. Cussler et al. 1999

27. Cussler et al. 1999

28. Rabies trial conclusions • Animals scoring 2 never recovered • Body weight • showed an early decrease before clinical signs • up to 30-40% by time of death • Establishment of humane endpoint • A decrease of body weight > 15% with • Clinical signs of neurological disorder • -> shortened the experiment by 3 days

29. More on the topic

30. Reference material • Morton, D.B. (1998) The importance of non-statistical design in refining animal experimentation.  ANZCCART Facts Sheet.  ANZCCART News 11, No. 2 June 1998 Insert.  pp12.  Publrs. ANZCCART PO Box 19 Glen Osmond, SA5064, Australia. • Hendriksen, CFM & D. B. Morton. (1999). Eds Humane Endpoints in Animal Experiments for Biomedical Research.  Proceedings of the Intnl Conference, 22-25 Nov 1998 Zeist, The Netherlands. pp 150.  ISBN 1-85315-429-6  Publrs Royal Soc Med. London WIM 8AE (http://www.lal.org.uk/endpoints1.html ) • ILAR (2000) Humane Endpoints for Animals used in Biomedical Research and testing. ILAR Journal 41 No 2. • UK Co-ordinating Committee on Cancer Research UKCCCR Guidelines http://www.ncrn.org.uk/csg/animal_guides_text.pdf