Analytical equivalence between standard and refined lethality neutralization protocols for snake antivenoms

Improving animal welfare in murine lethality neutralization assays is desirable in snake antivenom quality control, but any refinement must preserve analytical reliability. We evaluated the impact of a refined protocol incorporating analgesia (tramadol, 50 mg/kg) and a shortened 6-h endpoint on the performance of median lethal dose (LD₅₀) and median effective dose (ED₅₀) determinations, compared with the Standard Protocol (without analgesia and with a 48-h endpoint). LD₅₀ values obtained at 6 h were slightly higher than those at 48 h, but only one venom showed a statistically significant difference. When ED₅₀ assays were conducted using challenge doses derived from the LD₅₀ corresponding to each endpoint, no significant differences were observed between ED₅₀ values determined at 6 h and 48 h across 22 antivenom batches representing three products, multiple venoms, two administration routes, and two different mouse weight ranges. ED₅₀ values obtained under the Standard and Refined Protocols showed a strong correlation. The Refined Protocol also met analytical validation criteria, demonstrating adequate linearity (R² = 0.986), accuracy (84-119% recovery), and intermediate precision (maximum RSD 11.7%). These findings show that the Refined Protocol provides potency estimates equivalent to the Standard Protocol while reducing animal pain and distress, supporting its adoption in routine antivenom potency testing.