Reducing time in lethality assay (LD50) for Bothrops jararaca and Crotalus durissus terrificus venoms and lethality neutralizing assay (ED50) for their respective antivenoms: A 3Rs-based retrospective data validation

Abstract

Mouse lethality assays (MLA) remain the gold standard for evaluating antivenom potency despite ethical concerns regarding animal welfare. This study assessed whether reducing observation periods from 48 to 24 h in MLA for determining median lethal dose (LD₅₀) and median effective dose (ED₅₀) of Bothrops jararaca and Crotalus durissus terrificus venoms and antivenoms maintains scientific validity while improving animal welfare. Through retrospective analysis of 518 quality control assays conducted between 2009-2023 at INCQS, including 334 potency assays and 27 median lethal dose assays for B. jararaca, and 134 ED₅₀ assays and 23 LD₅₀ assays for C. d. terrificus, we found that over 98% of deaths occurred within the first 24 h post-injection for all LD₅₀ determinations on venoms and all ED₅₀ determinations on antivenoms for both species. Statistical analyses demonstrated exceptional agreement between 24-hour and 48-hour endpoints, with concordance correlation coefficients exceeding 0.96 for all assays. Bland-Altman analysis revealed narrow limits of agreement with minimal systematic bias, particularly for antivenom potency measurements. Classification performance metrics showed excellent accuracy (98.5%-100%) in identifying satisfactory antivenoms at 24 h compared to the standard 48-hour protocol, with weighted kappa coefficients exceeding 0.98, indicating near-perfect agreement. For C. d. terrificus antivenom, the 24-hour assay demonstrated 100% concordance with the 48-hour classification, while B. jararaca antivenom showed 98.5% accuracy with only 6 discordant results among 334 assays. Kaplan-Meier survival analysis confirmed statistically equivalent survival probabilities between both time points. These findings provide robust evidence supporting the adoption of a 24-hour observation period as an ethically superior refinement that maintains scientific integrity while significantly reducing animal distress. This refinement aligns with the 3Rs principles and could enhance testing efficiency in antivenom quality control, establishing a precedent for similar refinements in other biological assays and supporting the revision of regulatory guidelines to incorporate more humane testing protocols.

Keywords: 3Rs principles; Antivenom neutralizing potency; Bothrops jararaca; Crotalus durissus terrificus; Mouse lethality assay; Quality control of antivenoms.