Humane endpoints

A broad definition of Humane endpoint is a concept for continuous refinement of animal studies (Hendriksen et al., 2010).

This broad definition is aligned with the 3R principles (Balls et al., 1995, Russell and Burch, 1959).

Continuous improvements or refinement is embedded in the directive 2010/63 (European Commission, 2010)

• Article 4.3 Member States shall ensure refinement of breeding, accommodation and care, and of methods used in procedures, eliminating or reducing to the minimum any possible pain, suffering, distress or lasting harm to the animals.

Refinement of study specific endpoints and alternatives to death/moribund as endpoint

	Focus of the Study	Proposed Endpoint	Reference
INFECTIONS, IMMUNE SYSTEM
	Infectious diseases	Acute phase responses	Olfert, E.D. and D.L. Godson, Humane endpoints for infectious disease animal models. ILAR J, 2000. 41(2): p. 99-104.
		Body Temperature	Olfert, E.D. and D.L. Godson, Humane endpoints for infectious disease animal models. ILAR J, 2000. 41(2): p. 99-104.
		Weight loss	Olfert, E.D. and D.L. Godson, Humane endpoints for infectious disease animal models. ILAR J, 2000. 41(2): p. 99-104.
	Shock by cecal ligations and puncture (CLP)	Observation of Loss of ability to ambulate. Body weight and temperature	Nemzek, J. A., Xiao, H. Y., Minard, A. E., BOLGOS, G. L. & REMICK, D. G. 2004. Humane endpoints in shock research. Shock, 21, 17-25.
	Rheumatoid arthritis in rodents	Swollen digits, knuckle, midfoot and ancle/wrist scoring, Ulceration, gait and posture analysis	Hawkins, P., et al., Applying refinement to the use of mice and rats in rheumatoid arthritis research. Inflammopharmacology, 2015. 23(4): p. 131-50.
	Tuberculosis vaccine studies	Bodyweight AND Breathing pattern AND activity during handling combined predict need for euthanasia 1 week ahead	Collymore, C., Kent, et al. 2018. Humane Endpoints for Guinea Pigs Used for Mycobacterium tuberculosis Vaccine Research. Comparative Medicine, 68, 41-47
	Inoculation with infectious organisms	Temperature AND Body weight	Trammell, R. A. & Toth, L. A. 2011. Markers for predicting death as an outcome for mice used in infectious disease research. Comp Med, 61, 492-8.
	Vaccine potency testing	Reduced temperature AND bodyweight	Hendriksen. (2011). Humane endpoints in vaccine potency testing. Procedia in Vaccinology, 5, 221–226. https://doi.org/10.1016/j.provac.2011.10.022
CANCER
	Early Cancer detection	Ultrasound	Wang, L. V. (2003). Ultrasound-mediated biophotonic imaging: a review of acousto-optical tomography and photo-acoustic tomography. Disease Markers, 19(2-3), 123–138. https://doi.org/10.1155/2004/478079
	Intracranial Glioma in rat	Body weight algorithm	Helgers, S.O.A., et al., Body weight algorithm predicts humane endpoint in an intracranial rat glioma model. Sci Rep, 2020. 10(1): p. 9020.
	Severe irradiations studies in NHP	Rapid decrease of body temperature for 3 consecutive days combined with general behaviour score	Bertho, J.M., et al., Humane endpoints in severe irradiation experiments using non-human primates: a retrospective analysis. Scandinavian Journal of Laboratory Animal Science, 2020. 46(1): p. 1-15
	Total-Body Irradiation in Mouse	Body weight, temperature by telemetry	Koch, A., et al., Establishment of Early Endpoints in Mouse Total-Body Irradiation Model. PLoS One, 2016. 11(8): p. e0161079.
	Colon cancer in rats	black feces or presence of fresh blood	Kobaek-Larsen, Morten, Fenger, Claus, & Ritskes-Hoitinga, Jelmera. (2004). Secondary effects induced by the colon carcinogen azoxymethane in BDIX rats. APMIS : Acta Pathologica, Microbiologica et Immunologica Scandinavica, 112(6), 319–329. https://doi.org/10.1111/j.1600-0463.2004.apm1120601.x
	Experimental liver metastases	Laparoscopy	Kobaek-Larsen, M., et al., Laparoscopy of rats with experimental liver metastases: a method to assess new humane endpoints. Lab Anim, 2004. 38(2): p. 162-8.
	Lung Cancer	Iincreases in respiratory rate	Winn, Hwang, S.-K., Morin, J., Bluette, C. T., Manickam, B., Jiang, Z. K., Giddabasappa, A., Liu, C.-N., & Matthews, K. (2021). Automated monitoring of respiratory rate as a novel humane endpoint: A refinement in mouse metastatic lung cancer models. PloS One, 16(9), e0257694. https://doi.org/10.1371/journal.pone.0257694
	Urinary Bladder Carcinogenesis	Hematuria, Reduced hematocrit, lower final body weight, increase mean drink intake, increased urinary output	Oliveira, Nascimento-Gonçalves, E., Silva, J., Oliveira, P. A., Ferreira, R., Antunes, L., Arantes-Rodrigues, R., & Faustino-Rocha, A. I. (2017). Implementation of Humane Endpoints in a Urinary Bladder Carcinogenesis Study in Rats. In Vivo (Athens), 31(6), 1073–1080.
CNS
	Amyotrophic Lateral Sclerosis (ALS)	Decline in motoric Function	Mead, R.J., et al., Optimised and rapid pre-clinical screening in the SOD1(G93A) transgenic mouse model of amyotrophic lateral sclerosis (ALS). PLoS One, 2011. 6(8): p. e23244.
		Home cage running wheel	Bennett, E.J., et al., Early detection of motor dysfunction in the SOD1G93A mouse model of Amyotrophic Lateral Sclerosis (ALS) using home cage running wheels. PLoS One, 2014. 9(9): p. e107918.
	Stroke	Weight change on the 1st and 3rd day after treatment. Core temperature change on the 3rd day after treatment (male, stroke model), or with Neuroscore, weight change, and core temperature change on the 2nd day after treatment (female, stroke model) by machine learning.	Mei, J., Banneke, S., et al. Refining humane endpoints in mouse models of disease by systematic review and machine learning-based endpoint definition. ALTEX, 36, 555-571.
TOX testing
	Alternatives to LD 50	Hypotermiadose50 (HD50) replaces LD50	Soothill, J. S., Morton, D. B. & Ahmad, A. 1992. The HID50 (hypothermia-inducing dose 50): an alternative to the LD50 for measurement of bacterial virulence. Int J Exp Pathol, 73, 95-8.
SURGERY
	Post laparotomy	Telemetric recording of heart rate	Arras, M., et al., Assessment of post-laparotomy pain in laboratory mice by telemetric recording of heart rate and heart rate variability. BMC Vet Res, 2007. 3: p. 16.
OTHER ORGAN SYSTEMS
	Murine model for cholestasis	Burrowing activity	Zhang, X., et al., A rational approach of early humane endpoint determination in a murine model for cholestasis. ALTEX, 2020. 37(2): p. 197-207.
	Anti-urolithiasis activity of test compounds by zinc disc implantation	Imaging (X-ray radiographs) of the bladder deposits	Singh, P.K., et al., Zinc disc implantation model of urinary bladder calculi and humane endpoints. Lab Anim, 2010. 44(3): p. 226-30.
	Ocular Herpes virus Infection	Combined Body weight reduction (>0.05 g/day) AND Body Temperature less than 34,5°C measured by s.c implanted temperature probe. - Predicted death in 98% of mice.	Hankenson, F. C., Ruskoski et al , 2013. Weight Loss and Reduced Body Temperature Determine Humane Endpoints in a Mouse Model of Ocular Herpesvirus Infection. Journal of the American Association for Laboratory Animal Science, 52, 277-285.
AGING STUDIES
	Identify objective criteria that predict death in aged mice	Combined Body weight reduction (>0.05 g/day) AND Body Temperature	Trammell, Cox, L., & Toth, L. A. (2012). Markers for Heightened Monitoring, Imminent Death, and Euthanasia in Aged Inbred Mice. Comparative Medicine, 62(3), 172–178.
	Improve care, husbandry and management of aging mice	Enhanced clinical monitoring of age related parameters	Wilkinson, Selman, C., McLaughlin, L., Horan, L., Hamilton, L., Gilbert, C., Chadwick, C., & Flynn, J. N. (2019). Progressing the care, husbandry and management of ageing mice used in scientific studies. Laboratory Animals (London), 54(3), 23677219865291–238. https://doi.org/10.1177/0023677219865291
Literature surveys
	Lung injury	Temperature AND Body weight	McGinn, Fergusson, D. A., Stewart, D. J., Kristof, A. S., Barron, C. C., Thebaud, B., McIntyre, L., Stacey, D., Liepmann, M., Dodelet-Devillers, A., Zhang, H., Renlund, R., Lilley, E., Downey, G. P., Brown, E. G., Côté, L., Dos Santos, C. C., Fox-Robichaud, A. E., Hussain, S. N. A., … Lalu, M. M. (2021). Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians. Critical Care Medicine, 49(2), 311–323. https://doi.org/10.1097/CCM.0000000000004734
	Acute lung injury	Temperature AND Body weight	Zingarelli. (2021). First Do No Harm: A Proposal of an Expert-Guided Framework of Surrogate Humane Endpoints in Preclinical Models of Acute Lung Injury. Critical Care Medicine, 49(2), 373–375. https://doi.org/10.1097/CCM.0000000000004758

OTHER OBSERVATION STRATEGIES WITH POTENTIAL TO BE USED FOR DEFINING ENDPOINTS

In-Tank Underwater Cameras can Refine Monitoring of Laboratory Fish	Ellis, T; Rimmer, GSE; Parker, S-J; Joiner, Claire; Sebire, Marion, Verner-Jeffreys, David, Lines, Jeffrey 2019/05/01, 191, 203, In-Tank Underwater Cameras can Refine Monitoring of Laboratory Fish DO  - 10.7120/09627286.28.2.191, Animal Welfare

Many or few endpoint criteria

Some authors stress that endpoints can rarely be generalized. Using too many and too general endpoints for scoring is labor intensive. Researchers might be required to evaluate variables that might appear arbitrary or unrelated to the animal’s condition. Compliance may be increased if the measurement of the variables is related to research objectives. Other authors underline that humane endpoint cannot rely on a single variable, suggesting to use a combination of variables and recommend a combination of assessment parameters for a robust welfare assessment in laboratory animals   and early predictor of death. For example, the combined reduction of body weight and body temperature, has shown to be a reliable predictor of death in studies including:

• Nemzek JA, Xiao HY, Minard AE, Bolgos GL, Remick DG. Humane endpoints in shock research. Shock. 2004;21(1):17-25.
• McGinn R, Fergusson DA, Stewart DJ, Kristof AS, Barron CC, Thebaud B, et al. Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians. Crit Care Med. 2021;49(2):311-23.
• Zingarelli B. First Do No Harm: A Proposal of an Expert-Guided Framework of Surrogate Humane Endpoints in Preclinical Models of Acute Lung Injury. Crit Care Med. 2021;49(2):373-5.
• Trammell RA, Toth LA. Markers for predicting death as an outcome for mice used in infectious disease research. Comp Med. 2011;61(6):492-8.
• Hendriksen C. Humane endpoints in vaccine potency testing. Procedia in Vaccinology 5 (2011) 2011;5:221 – 6.
• Hankenson FC, Ruskoski N, van Saun M, Ying GS, Oh J, Fraser NW. Weight Loss and Reduced Body Temperature Determine Humane Endpoints in a Mouse Model of Ocular Herpesvirus Infection. J Am Assoc Lab Anim. 2013;52(3):277-85.
• Trammell, Cox, L., & Toth, L. A. (2012). Markers for Heightened Monitoring, Imminent Death, and Euthanasia in Aged Inbred Mice. Comparative Medicine, 62(3), 172–178.