Assessing a 6-h endpoint observation time in the lethality neutralization assay used to evaluate the preclinical efficacy of snake antivenoms. (November 2021)

• Record Type: Journal Article
• Authors: Durán, Gina; Solano, Gabriela; Gómez, Aarón; Cordero, Daniel; Sánchez, Adriana; Villalta, Mauren; Sánchez, Melvin; Díaz, Cecilia; Gutiérrez, José María; León, Guillermo
• Is Part Of: Toxicon. Volume 12(2021)
• Publication Date: 2021-11
• Subjects: Antivenom potency -- Lethality neutralization assay -- Median effective dose -- Median lethal dose -- Replacement -- Reduction -- And refinement principle -- Snake antivenom
• DOI: 10.1016/j.toxcx.2021.100087
• Languages: English
• ISSNs: 2590-1710

Abstract

Abstract: The lethality neutralization assay in mice is the gold standard for the evaluation of the preclinical efficacy and specification fulfillment of snake antivenoms. However, owing to the animal suffering involved, this assay is a candidate to be replaced by in vitro alternatives or, at least, improved by the reduction of the number of animals used per experiment, the introduction of analgesia, and the refinement of the test. Since these tests are usually run for 24 or 48 h, one possibility to refine it is to shorten the endpoint observation time of the assay and so limiting the duration of suffering. To assess the effect of this modification of the standard procedure on the analytical properties of the assay, we compared the median lethal dose (LD50 ) and median effective dose (ED50 ) values, estimated through observation times of 6, 24 and 48 h. We used African and Latin American snake venoms and several batches of two polyspecific antivenoms. A significant correlation was found between LD50 and ED50 values estimated at the three observation times. Although some LD50 and ED50 values were significantly different at these time points, results of 6 h were robust enough to be used in the characterization of new antivenoms, the verification of specification compliance, and the parallel comparison of formulations. Our observations support the modification of the standard procedures used for assessing neutralizing ability of antivenoms by carrying out the observations at 6 h instead of 24 or 48 h, with the consequent reduction in the suffering inflicted upon mice during these assays. However, the shortening of the observation time in the lethality tests must be validated for each venom and antivenom before its introduction in the routine procedures. Graphical abstract: Image 1 Highlights: The lethality neutralization assay in mice is the gold standard for the preclinical evaluation of snake antivenoms. This assay is a candidate to be improved by the 3Rs philosophy (i.e., replacement, reduction and refinement). The effect of the reduction of the endpoint observation time on the analytical properties of the test was assessed. A significant correlation was found between LD50 and ED50 values estimated at 6 h and 24 or 48 h. A 6-h observation time limits the duration of the suffering without reducing the analytical quality of the test.