Washington State University INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE Policy #8: Policy for Humane Endpoints A. Purpose: This policy outlines the requirements for developing and defining humane endpoints for all animals used in research, teaching or testing at WSU. A humane endpoint is defined in the 8th edition of The Guide for the Care and Use of Laboratory Animals as the point at which pain or distress in an experimental animal is prevented, terminated, or relieved. These are different from experimental endpoints, which occur when scientific objectives have been met, but the two can be closely linked. The use of humane endpoints contributes to refinement by providing an alternative to experimental endpoints that result in unrelieved or severe animal pain and distress, including death, and should be relevant and reliable. The Principal Investigator (PI), who has precise knowledge of both the objectives of the study and the proposed model, should identify, explain, and include in the animal subject approval form (ASAF) a study endpoint that is both humane and scientifically sound. The determination of humane endpoints should involve the PI, veterinarian, and the IACUC, and must be defined prior to the start of the study. Unanticipated adverse events may require modification of the endpoints. B. Special Considerations: The 8th edition of the Guide (page 27) indicates that while all studies should employ endpoints that are humane, studies that commonly require special consideration include those that involve: tumor models (For examples see Appendix D: Tumor Burden Guidelines) infectious diseases vaccine challenge pain modeling trauma production of monoclonal antibodies (addressed in WSU IACUC policy #23) assessment of toxicological effects organ or system failure models of cardio-vascular shock
These models often cannot utilize common default humane endpoint guidelines and therefore should be developed on a case-by- case basis in conjunction with the IACUC and veterinarians. C. Protocol Components: The following items must be included when outlining humane endpoints in the IACUC protocol: 1. Definition of endpoints: Endpoints are anticipated adverse effects the research animal may experience. These include the likely time course, and progression of the adverse effects, the justification for the endpoint to meet scientific requirements of the study, and the response when that point is reached. This response could entail removal from the study until the condition has adequately improved, clinical treatment sufficient to allow experimentation to continue, or more commonly euthanasia. 2. Assessment criteria: Describe the parameters that will be measured during a study that will be indicative of an animal s general health and well-being or clinical condition. Examples include, but are not limited to, measuring body weight, food and water consumption, behavior, tumor size, imaging findings, blood chemistry, or activity level. Research staff must be adequately trained in recognition of species-specific behavior and, in particular, species-specific signs of pain, distress, and morbidity (for sample see Appendix A). Scoring systems are one way in which humane endpoints can be defined and implemented. The attached example of a scoring system (for sample see Appendix C) is based upon routine observations. Scoring systems may be developed or modified to fit individual protocols or animal models. 3. Frequency of monitoring: State how regular (e.g., number of times per day and per week), responsible personnel will observe the animal or measure the parameters identified as assessment criteria. Monitoring requirements may change through the course of a study as a condition worsens over time or experimental manipulations change. 4. Required response: Describe the intervention that must occur when the defined endpoint(s) has been reached. The intervention will typically be medical treatment or euthanasia and may be performed by research or veterinary staff in compliance with pre-set arrangements in the IACUCapproved protocol
5. Default Endpoints: The following criteria may be considered standard, default endpoints for low-risk animal models which would not generally be felt to have pain and distress under normal conditions, but may experience untoward, potentially severe, effects unpredictably. For models in which animals are expected to have pain and distress, these default endpoints may not generally be considered sufficient and additional refinement of the endpoints on a case-by-case basis may be required. The OCV veterinary staff are always available for consultation regarding an individual animal or proposed study.. a) Loss of >20% of body weight from baseline weight when assigned to the protocol. NOTE: A growth nomogram must be used to adjust the basal weight for young growing animals. b) Major organ failure or medical conditions unresponsive to treatment c) Surgical complications unresponsive to intervention d) Failure to eat or drink or severe vomiting and/or diarrhea for 24 48 hours (species dependent), resulting in significant dehydration or rapid weight loss/emaciation. e) Tumors arising from other than experimental means that grow in excess of 15% body weight or for rodents (mice >20mm and rats >40mm), impair movement, or ulcerate. f) Clinical or behavioral signs of significant stress that are unresponsive to intervention. g) Severely impaired ambulation h) Difficult labored breathing i) Persistent & severe abnormal posture or lameness j) Prolonged inability to remain upright k) Unresponsive to stimuli or is moribund l) Body condition score less than 2 (for sample see Appendix B) 6. Death as an endpoint: The use of death as an endpoint is generally discouraged. However, it is understood that in some special circumstances it is necessary or unavoidable and thus will be considered on a case-by-case basis. Approval requires adequate scientific justification for death as an
endpoint including why alternative endpoints or stress or pain-relieving drugs cannot be used. Approval also requires detailed plans for monitoring and supportive care including frequency of monitoring and record-keeping practices. 7. Veterinary Oversight: The Attending Veterinarian (AV) has the responsibility for oversight of the health and welfare of animals used for research, teaching and testing. The AV and designated veterinary staff, have the authority to euthanize an animal in the interest of animal welfare. All attempts will be made to reach an agreement with the PI and research staff when these decisions must be made. D. Required Documentation: Studies with defined monitoring parameters and humane endpoints are expected to maintain the following records and to be able to provide those to the IACUC or OCV on request. Sample assessment/animal scoring documents are provided (for sample see Appendix C, D & E) and may be modified for individual studies. Assessment of the animal for the parameters and at the frequency defined in the approved protocol should include: 1. Animal identification a. parameters assessed b. date of assessments c. initials of the personnel assessing the animal 2. Intervention including the type (euthanasia, treatment, etc.), date and personnel identification Approved 12.13.17
Appendix A: Indicators of Pain in Several Common Laboratory Animals (NRC 2003). Species General Behavior Appearance Other Rodents Decreased activity; excessive licking and scratching; self- mutilation; may be unusually aggressive; abnormal locomotion (stumbling, falling); writhing; does not make nest; hiding, social isolation Piloerection; rough/stained hair coat; abnormal stance or arched back; porphyrin staining (rats) Rapid, shallow respiration; decreased food/water consumption; tremors Rabbit Head pressing; teeth grinding; may become more aggressive; increased vocalizations; excessive licking and scratching; reluctant to locomote Excessive salivation; hunched posture Rapid, shallow respiration; decreased food/water consumption, decreased fecal production Dog Excessive licking; increased aggression; increased vocalizations, inclusive of whimpering, howling, and growling; excessive licking and scratching; self-mutilation, social isolation Stiff body movements; reluctant to move; trembling; guarding Decreased food/water consumption; increased respiration rate/panting Cat Hiding; increased vocalizations, inclusive of growling and hissing; excessive licking; increased aggression Stiff body movements; reluctant to move; hair coat appear rough, ungroomed; hunched posture; irritable tail twitching; flattened ears Decreased food/water consumption
Appendix B: Representative Body Condition Scoring (BCS) Charts for Rodents MICE BC 1 Mouse is emaciated Skeletal structure extremely prominent, little or no flesh cover Vertebrae distinctly segmented BC 2 Mouse is under-conditioned Segmentation of vertebral column evident Dorsal pelvic bones are readily palpable BC 3 Mouse is well-conditioned Vertebrae and dorsal pelvis not prominent, palpable with slight pressure BC 4 Mouse is over-conditioned Spine is a continuous column Vertebrae palpable only with firm pressure BC 5 Mouse is obese Mouse is smooth and bulky Bone structure disappears under flesh and subcutaneous fat RATS BC 1 Rat is emaciated Segmentation of vertebral column prominent if not visible Little or no flesh cover over dorsal pelvis, pins prominent if not visible Segmentation of caudal vertebrae prominent BC 2 Rat is under-conditioned Segmentation of vertebral column prominent Thin flesh cover over dorsal pelvis, little subcutaneous fat, pins easily palpable Thin flesh cover over caudal vertebrae, segmentation palpable with slight pressure BC 3 Rat is well-conditioned Segmentation of vertebral column easily palpable Moderate subcutaneous fat store over pelvis, pins easily palpable with slight pressure Moderate fat store around tail base, caudal vertebrae may be palpable but not segmented BC 4 Rat is over-conditioned Segmentation of vertebral column palpable with slight pressure Thick subcutaneous fat store over dorsal pelvis, pins of pelvis palpable with firm pressure Thick fat store over tail base, caudal vertebrae not palpable BC 5 Rat is obese Segmentation of vertebral column palpable with firm pressure, may be a continuous column Thick subcutaneous fat store over dorsal pelvis, pins of pelvis not palpable with firm pressure Thick fat store over tail base, caudal vertebrae not palpable
Appendix C: Representative Scoring System for Determining Humane Endpoints Variable Score Body Weight Changes 0 Normal 1 < 10 percent weight loss 2 10-15 percent weight loss 3 > 20 percent weight loss Body Condition Score (see diagram for details) 0 Body condition score >3 1 BCS >2 and < 3 2 BCS >1 and <2 3 BCS of 1 or less Physical Appearance 0 Normal 1 Lack of grooming 2 Rough coat, nasal/ocular discharge 3 Very rough coat, abnormal posture, enlarged pupils Measurable Clinical Signs 0 Normal 1 Small changes of potential significance 2 Body temp change of 1-2 C, cardiac and respiratory rates up to 30% 3 Body temp change of 1-2 C, cardiac and respiratory rates up to 50%, or markedly reduced Unprovoked Behavior 0 Normal 1 Minor changes 2 Abnormal, reduced mobility, decreased alertness, inactive 3 Unsolicited vocalizations, self-mutilation, either very restless or immobile Behavioral Responses to External Stimuli 0 Normal 1 Minor depression/exaggeration of response 2 Moderately abnormal responses 3 Violent reactions, or comatose TOTAL: Note: This scoring template should be modified for specific species and designed to fit each protocol and animal model. In this example, a score is assigned to each variable, 0 (normal or mild) to 3 (severe). The cumulative score gives an indication of the likelihood that the animal is experiencing pain or distress. Humane endpoints can be established based on these criteria. A total score of >5 or a score of 3 in any one variable, regardless of the total score should warrant mandatory evaluation/decision by a veterinarian or humane euthanasia.
Appendix D: Tumor Burden Guidelines A. Purpose: To establish guidelines that will allow investigators and OCV veterinary staff to objectively evaluate the health and welfare of animals carrying spontaneous or experimentally-induced tumors and determine when euthanasia might be warranted to alleviate pain and distress associated with these tumors. The recommendations presented in this document are intended to augment, not supersede, guidelines presented in the WSU IACUC Policy for Humane Endpoints B. General: In using animal models for the study of cancer, it is typically necessary that the animals utilized experience tumor growth and possibly metastatic disease. The determination of humane endpoints should involve the PI and veterinarian and must be approved by the IACUC prior to the start of the study. To maximize data acquisition and minimize pain and distress, the animal s health and welfare needs to be continually and comprehensively assessed and documented as tumors and associated disease progress. The PI and\or research staff are responsible for monitoring the animals for endpoint criteria. OCV veterinary personnel can assist in the development and assessment of appropriate endpoints. Unanticipated adverse events require report to the IACUC and may require modification of the endpoints. When assessing the general health of animals utilized in studies of cancer, the following clinical presentations warrant development of a management plan as they indicate diminished health status that may be associated with increasing tumor burden and metastasis: 1. Weight loss and decreasing body condition. 2. Hunched posture, Mobility or lethargy issues, lack of responsiveness, rough hair coat 3. Respiratory-associated symptoms such as increased respiratory rate, coughing, and nasal discharge 4. Icterus/Jaundice 5. Neurological signs such as circling or ataxia 6. Self-trauma 7. Difficulty with ambulation that might interfere with food and water acquisition 8. Ulceration and necrosis of visible tumors
Immediate action would be warranted for these clinical signs: 1. Distention of the abdomen with fluid or palpable mass resembling the size of a pregnant animal 2. Abnormal vocalization when touched or handled indicating severe distress 3. Labored breathing 4. Severe anemia (pale feet or ears or decreased packed cell volume) 5. Unresponsive to stimuli or moribund Criteria for Endpoints of Solid tumors (each listed as a standalone, not in addition): 1. Tumor burden (combined burden if more than one mass present) is greater than 15% body weight (Figure 1& 2) a. The weight reference is the weight of the animal on the day of tumor implantation b. The mass of the tumor is calculated from the following formulai. Mass (mg) = Tumor volume (mm3) = d 2 x D/2 where d and D are the shortest and longest diameter in mm, respectively. ii. Mass of tumor (g)/ Weight of the animal (g) x 100= % of body weight (see chart below) 2. Mean tumor diameter 20mm in adult mice (~25g) or 40mm in adult rats (~300g) were Mean = (d +D)/2. 3. Ulceration, infection or necrosis of tumor.
Examples of Solid Tumors in Rodents (how to calculate % body weight, mean tumor size, tumor burden) Figure 1: Mouse Tumors (25g animal) Tumor examples d=20mm D=20mm D d Mass of Tumor (mg) Tumor volume= d 2 x D/2 20 2 x 20/2 =4000mm 3 or 4g Mean tumor size=20mm Tumor burden=4g/25g=16% Meets Criteria for end point? Yes-exceeds 15% body weight and mean tumor size 20mm d=15mm D=25 mm D d 15 2 x 25/2 =2812mm 3 or 2.8g Mean tumor size=20mm Tumor burden=2.8g/25g=11% Yes-mean tumor size 20mm Figure 2: Rat Tumors (300g animal) Tumor examples d=40mm D=40mm D d Mass of Tumor (mg) Tumor volume= d 2 x D/2 40 2 x 40/2 =32,000mm 3 or 32g Mean tumor size=40mm Tumor burden=4g/300g=11% Meets Criteria for end point? Yes-exceeds mean tumor size 40mm d=35mm D=50 mm D d 30 2 x 50/2 =22,500mm 3 or 22.5g Mean tumor size=40mm Tumor burden=22.5g/300g=7.5% Yes-mean tumor size 40mm
Appendix E: Rodent health monitoring sheet {Fill In Study} Model Document all observations on the experimental health monitoring sheet. Alert the Attending Veterinarian of any ill or injured animals. PI: IACUC protocol: ANIMAL ID Experimental Procedure: Date of Experimental Procedure: Date Weight (g) Physical Appearance (0-3) Body Condition Score (0-3) Lethargy? Y/N Response to Stimuli (0-3) Mobility (0-3) Comments** Initials of observer *Weights on all animals will be x weekly unless an animals is exhibiting clinical signs (lethargy, Inappetance, diarrhea, jaundice) then the animal will be weighed immediately. ** If an animal is lethargic, weight loss is greater than 15% of starting weigh, or body condition less than 2 immediate evaluation/decision by veterinarian or the PI is warranted.