The study of snake venom toxicity and evaluation of the neutralisation ability of antivenoms and novel therapeutic agents for envenoming has largely relied on animal tests, especially the analysis of the lethal activity of venoms and its neutralisation. In addition, other animal-based assays are used to assess pathology-specific effects of venoms. Despite their demonstrated value, these assays have several limitations. They involve acute stress and pain in animals, and their validity, vis-à-vis the characteristics of human snakebite envenoming, is limited owing to the nature of the assays. Therefore, urgent innovations are required in this field. Ensuring the implementation of the 3Rs (Replacement, Reduction, and Refinement) is essential when animal studies are required. These include: (a) refinement of the tests (e.g., use of analgesia, reduction of assay duration, and refining of humane endpoints); (b) reduction in the number of animals used by more thorough in vitro assessment of antivenoms prior to in vivo assays, and improved statistical analyses and group sizing; and, most importantly, and (c) replacement of in vivo rodent tests with validated in vitro approaches, such as immunoassays, enzymatic and cell-based assays, the use of invertebrate models, and the implementation of New Approach Methodologies (NAMs), i.e., novel in vitro or in silico techniques that model complex pathophysiological processes of envenoming. A roadmap involving researchers, manufacturers, quality control groups, regulators, and funding agencies is proposed, with the aim of driving significant changes in this field.
Ainsworth S, Casewell NR, Sartim MA, Marriott AE, Gutiérrez JM (2026) Animal models in venom and antivenom research: The need to align academic discovery with manufacturing and regulatory expectations. PLoS Negl Trop Dis 20(7): e0014537. https://doi.org/10.1371/journal.pntd.0014537
