Cytotoxicity assay as a potential alternative method for animal testing for batch release of Italian fish autogeneous vaccines

BACKGROUND: Vaccination is widely used in fish aquaculture for three primary reasons: to prevent bacterial disease spreading, minimize antibiotic use and fight antibiotic resistance. Vaccine production is an expensive and consuming process, mainly in terms of money, resources, and animals for quality control. The replace, reduce, and refine (3Rs) philosophy suggests developing and validates alternative methods to animal testing for scientific purposes, even for biologicals and vaccines. AIM: The current study explored the potential use of mouse and fish cells in the in vitro toxicity grade assessment through different methods, as an alternative assay to in vivo residual toxicity tests for autogenous fish vaccine control. METHODS: BF2 and L929 cell lines were exposed to vaccine dilutions in two different ways of administration and toxicity grade was recorded by MTS assay, compared to the in vivo gold standard test. RESULTS: Autogenous vaccines (AVs) caused no reactions in the in vivo test. In the in vitro assay, the different toxicity grade recorded was statistically significant between the cell lines adopted and the AVs way of administration. CONCLUSIONS: Data obtained represent the first application of 3Rs method to fish AVs produced in Italy, more investigations are needed to collect solid results and standardize new in vitro methods for vaccine quality control.