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

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 (LD 50 ) and median effective dose (ED 50 ) 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 LD 50 and ED 50 values estimated at the three observation times. Although some LD 50 and ED 50 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.

Two snakebite antivenoms have potential to reduce Eswatini’s dependency upon a single, increasingly unavailable product: Results of preclinical efficacy testing

“…Antivenoms were compared for venom neutralising efficacy using ED 50 and eMND assays. A refined version of the WHO recommended antivenom ED 50 was used [ 3 , 17 – 20 ], in which 3 x or 5 x the LD 50 dose was pre-incubated at 37°C for 30 minutes with varying amounts of each antivenom, prepared in a total volume of 200 μL with PBS and the venom/antivenom mixtures injected intravenously. In line with WHO guidelines, our target venom challenge dose was 5 x LD 50 s but when this proved 100% lethal when mixed with the highest possible volume of antivenom, we reduced the venom challenge dose to 3 x LD 50 s. Due to an error, 5.3 x LD 50 dose was used for D .…”

Intrageneric cross-reactivity of monospecific rabbit antisera against venoms of mamba (Elapidae: Dendroaspis spp.) snakes

“…Fifteen minutes afterward, mice received an intraperitoneal (IP) route of 0.5 mL of 0.12 M NaCl, 0.04 M phosphate, pH 7.2, solution (PBS) containing different amounts of venom. The number of deaths occurring during the following 6 h was recorded ( Durán et al, 2021 ) and used to estimate the median lethal dose (LD 50 , i.e., the amount of venom that results in the death of 50% of the injected mice) by Probits ( Finney, 1971 ). The observation time of 6 h, instead of 48 h, is justified for ethical considerations, and on the basis of previous observations ( Durán et al, 2021 ).…”

African polyvalent antivenom can maintain pharmacological stability and ability to neutralise murine venom lethality for decades post-expiry: evidence for increasing antivenom shelf life to aid in alleviating chronic shortages

“…The rationale for using 3× LD 50 challenge doses for N. nigricollis and D. polylepis , rather than the conventional 5× LD 50 , was a refinement due to experience with these particular venoms demonstrating the use of 5× LD 50 resulted in poor resolution of dose groups outcomes. The number of resulting deaths was recorded at 6 hours 38. The ED 50 and the corresponding 95% CIs were calculated by Probit analysis 32.…”