Tetrodotoxin (TTX) is starting to appear in molluscs from the European waters and is a hazard to seafood consumers. This toxin blocks sodium channels resulting in neuromuscular paralysis and even death. As a part of the risk assessment process leading to a safe seafood level for TTX, oral toxicity data are required. In this study, a 4-level Up and Down Procedure was designed in order to determine for the first time the oral lethal dose 50 (LD50) and the No Observed Adverse Effect Level (NOAEL) in mice by using an accurate well-characterized TTX standard.