Botulinum neurotoxin: Tracking the transition from lethal dose to in vitro models

Botulinum neurotoxin (BoNT), commonly referred to as 'Botox', is widely used in medical and aesthetic applications across the globe. As a biological product, BoNT requires rigorous batch potency testing to ensure safety, and is currently reliant on the ethically and scientifically contentious Mouse Lethality Bioassay (MLB). Despite severe suffering of the mice, the reproducibility crisis, and translatability issues of this Lethal Dose (LD50) test, its use persists due to regulatory inertia and other systemic barriers. Recent advancements in new approach methodologies (NAMs), particularly cell-based assays (CBAs), offer promising non-animal alternatives for regulatory potency testing. This paper seeks to provide an update on the transition to non-animal NAMs with a primary focus on Europe as a key player in the production of BoNT products. Namely, it describes the (accepted) alternatives to the MLB, the distribution of BoNT products and use of mice in the European Union and United Kingdom, and attempts to understand the number of mice used per year for BoNT alone, by analyzing Non-Technical Summaries (NTSs) from known testing facilities across the UK, Germany, and Ireland. This paper synthesizes findings from publicly available resources to provide actionable insights into overcoming barriers to implementing ethical and scientifically robust alternatives for regulatory testing of BoNT products. Based on NTS data, the MLB has likely ceased in Germany but persists in the UK and Ireland. Our findings show that even when companies have developed CBAs, MLB use may continue for various factors such as testing for reference standards, comparability testing/validation of CBAs, a lack of universal alternatives, and compliance with regulations for market authorizations of new BoNT products. Finally, predicting the number of mice used for BoNT potency testing was strikingly difficult, especially given the severity of the LD50 test, demonstrating the need for greater transparency in actual animal use for (severe) procedures and the limited applicability of public data to predict actual animal use for more specific research questions.