Comparison of Zebrafish Embryo Toxicity and L929 Cytotoxicity Assays for Preliminary Quality-Control Assessment of Veterinary Autogenous Vaccine Matrices

Background: Veterinary autogenous vaccines (AVs) are emergency tools produced in a short timeframe from bacterial/viral/fungal isolates when commercial vaccines are not available and undergo strict quality assurance and safety tests before release. In accordance with the European Directive 2010/63/EU and the 3R principles, we evaluated for the first time the possible substitution of the final toxicity in vivo test (abnormal toxicity test) with an alternative cell-based and embryo-based assay. Methods: The five most requested different autogenous matrices (adopted to produce a customizable number of lots requested from customers) were tested in vitro in parallel for residual toxicity by in vitro fish embryo acute toxicity (FET) test and cytotoxicity assay (MTS) using serial dilutions, together with the authorized in vivo test. Results: Statistical analysis revealed different outcomes between the two in vitro methods, highlighting the greater discriminatory power of the MTS assay compared to the other, with results more like the observed in vivo ATT outcome (pass/negative). Conclusions: In this preliminary first dataset, FET appeared poorly compatible with several complex autogenous vaccine matrices, while the L929 MTS assay yielded interpretable viability thresholds for all tested matrices. These findings support further qualification of MTS as part of an integrated non-animal batch characterization strategy, but they do not yet establish replacement of the in vivo test for veterinary autogenous vaccine quality control.