Unjustifiability of moribund animal testing

The use of animals for scientific knowledge is still essential in many areas. The European Directive 2010/63/EU, which was implemented in Austria by the Animal Experiments Act 2012 (TVG 2012), creates a legal framework whose primary goal is to ensure the "welfare of animals" as a "value of the Union". Accordingly, a project will only be approved which is "justified from a scientific or educational point of view or required by law". At the same time, the law prescribes that "possible pain, suffering, distress or permanent damage must be eliminated or reduced to a minimum." Furthermore, it applies that "death as an endpoint of a procedure is to be avoided if possible" as well as "to be replaced by early and, if possible, painless endpoints. "

Within biomedical research, there are some areas in which experiments are likely to involve a higher degree of stress on the laboratory animals used. These include, for example, cancer and infection studies, as well as models to test the efficacy and toxicity of a drug. Human endpoints represent termination criteria that must lead to the immediate release of an affected animal in an experiment. These must be precisely defined in the project application prior to the start of the trial. Apart from a legal and additionally moral obligation to prevent any suffering in animal experiments, it is also in the interest of science to ensure timely termination criteria in order to obtain the best possible results. After all, if sufficiently meaningful data have been obtained and the intended experimental goal has been achieved, there is no need to continue the experiment and animals can be released or relieved from pain in a timely manner. The moribund outcome of an experiment is not only cruel and against any animal welfare, but additionally to be considered as a loss of knowledge. If an animal is euthanized at a certain and fixed time, a lot of pathological data can still be collected. An animal that perishes unnoticed at some point during the experiment is useless for many investigations due to autolytic processes. Furthermore, the physical and psychological health status of the test animal can influence the results, which could even lead to missing the test objective.

Score sheets are a suitable tool for assessing the health status of an animal. In principle, there are a wide variety of factors that serve to classify the condition and thus to establish human endpoints. Orientation is provided by clinical symptoms, all pathophysiological changes as well as behavioral changes, but also biochemical and hormonal changes. In addition, a distinction can be made between preclinical and non-clinical endpoints. Preclinical means that an assessment can be made before clinical symptoms appear. Data can be collected using non-invasive measurement methods already in the preclinical stage. An example of this would be the use of molecular biological methods, such as bioluminescence labeling of tumor cells, whereby the metastasis of a tumor in the body can be observed. Thus, termination of the trial prior to the appearance of clinical symptoms is made possible. Non-clinical endpoints include the use of methods that lead to the replacement of methods that cause clinical symptoms. A typical example is vaccine production. In order to test a new vaccine, animals were infected with the virulent pathogen or its toxin after vaccination to verify the efficacy of the immunization. Thanks to new methodologies, it is now possible to obtain the desired information in the production of many vaccine types without infecting the test animals. Thus, data on vaccination protection can be determined by means of antibody titer determination from blood. All of the above measures for early detection of pain and other stresses in the animals provide the opportunity to act in the spirit of refinement, one of Russell and Burch's 3Rs.

The following articles address methods for establishing a human endpoint in animal studies:

• Gavin HE, Satchell KJF. Surface hypothermia predicts murine mortality in the intragastric Vibrio vulnificus infection model. BMC Microbiol. 2017 Jun 19;17(1):136. doi: 10.1186/s12866-017-1045-z. PMID: 28629317; PMCID: PMC5477130.
• Koch A, Gulani J, King G, Hieber K, Chappell M, Ossetrova N. Establishment of Early Endpoints in Mouse Total-Body Irradiation Model. PLoS One. 2016 Aug 31;11(8):e0161079. doi: 10.1371/journal.pone.0161079. PMID: 27579862; PMCID: PMC5007026.
• Ernest D. Olfert, Dale L. Godson, Humane Endpoints for Infectious Disease Animal Models, ILAR Journal, Volume 41, Issue 2, 2000, Pages 99–104, https://doi.org/10.1093/ilar.41.2.99 
• Coenraad F. M. Hendriksen, Björn Steen, Refinement of Vaccine Potency Testing with the Use of Humane Endpoints, ILAR Journal, Volume 41, Issue 2, 2000, Pages 105–113, https://doi.org/10.1093/ilar.41.2.105
• Kort WJ, Hekking-Weijma JM, Tenkate MT, Sorm V, VanStrik R. A microchip implant system as a method to determine body temperature of terminally ill rats and mice. Laboratory Animals. 1998;32(3):260-269. doi:10.1258/002367798780559329 
• Kobaek-Larsen M, Rud L, Oestergaard Soerensen F, Ritskes-Hoitinga J. Laparoscopy of rats with experimental liver metastases: a method to assess new humane endpoints. Laboratory Animals. 2004;38(2):162-168. doi:10.1258/002367704322968849
• Hickman DL, Swan M. Use of a body condition score technique to assess health status in a rat model of polycystic kidney disease. J Am Assoc Lab Anim Sci. 2010 Mar;49(2):155-9. PMID: 20353688; PMCID: PMC2846001.

The publications listed here show that many research groups have already implemented various practices to prevent the moribund outcome of animal experiments. Nevertheless, there is still a need to optimize current means and to develop further techniques to replace moribundity in experiments. It is the responsibility of future scientists to contribute to the achievement of this goal and thus to ensure the welfare of the animals used.

Sources:
RL 2010/63/EU
https://www.humane-endpoints.info/de/zu-dieser-website/zweck-der-website, accesed on 16.03.2023
Morton, David. (1998). Humane endpoints in animal experimentation for biomedical research: ethical, legal and practical aspects. Humane Endpoints in Animal Experiments for Biomedical Research.