Saxitoxin-Equivalent Toxicity of Gymnodinium catenatum and Toxicity Equivalence Factors of GC Toxins Determined by the Neuro-2a Assay.

Gymnodinium catenatum is a paralytic shellfish toxin (PST)-producing dinoflagellate that produces classical PSTs and hydroxybenzoate analogues known as GC toxins. Toxicological evaluation of GC toxins remains limited because certified reference materials (CRMs) and toxicity data are unavailable. This study applied the Neuro-2a (N2a) cell-based assay to evaluate PST functional toxicity in a Korean G. catenatum strain. The algal extract was analyzed by dose-response curve fitting to estimate total saxitoxin (STX)-equivalent toxicity, while GC1 & 2, GC3, and GC4 & 5, isolated from large-scale culture and structurally confirmed by NMR and LC-MRM-MS/MS, were evaluated to determine toxicity equivalence factors (TEFs). A clear sigmoidal curve yielded a functional toxicity of 3.7275 pg STX eq cell-1, and TEFs followed the order GC3 > GC4 & 5 > GC1 & 2, with potency gaps considerably larger than previously reported receptor-binding differences. These findings support the N2a assay as a complementary approach for PST analogues lacking CRMs and suggest that receptor-binding affinity alone does not fully predict cellular functional potency. Although the proposed TEFs, obtained from single assays per preparation, are preliminary and assay-specific, this study provides the first functional toxicity estimates for GC toxins, contributing to the toxicological characterization of novel PST analogues.