Necessity of redefinition and revision of mouse unit for tetrodotoxin: Relationship between lethal time and mouse unit of tetrodotoxin in mouse bioassay.

Background: An assay that utilizes the relationship between the amount of toxin administered and the lethal time of mice is called a mouse bioassay (MBA) and has been used as an assay for marine neurotoxins such as paralytic shellfish poison toxins and pufferfish toxins. The MBA for tetrodotoxin (TTX), which is possessed by pufferfish, has been used for many years in Japan. In the Japanese reference method, equivalent to the official method, the toxicity of TTX is expressed in mouse units (M.U.), and 1 M.U. is defined as the amount of the toxins required to kill a 20 g mouse in 30 min and is stated as 0.22 μg of TTX. The regulation limit in food is also expressed in M.U. in Japan.

Objectives: In the previous studies, we have questioned the definition of 1 M.U. of TTX in the currently used Japanese reference method. In this study, we investigated the dose-lethal time relationship of TTX in mice.

Methods: Followed by the Japanese reference method, various doses of TTX were inoculated intraperitoneally into mice (n = 3/each dose) and the time from the end of the injection to the last gasp of breath was measured.

Results: The dose-lethal time relationship curve obtained in this study showed quite similar curves described in the reference method, but lethal time was always shorter than the time in the reference method. Also, the amount of 1 M.U. of TTX could not be determined based on the definition because most of the mice died earlier than 30 min after injection or survived.

Discussion: Based on the dose-lethal time relationship curve obtained in this study, 1 M.U. might be 0.1727 μg of TTX, which was about 22 % smaller than 0.22 μg. Furthermore, based on the definition, it was impossible to determine the amount of TTX for 1 M.U. The author asserts that the definition of M.U. and the amount of TTX for 1 M.U. should be reviewed.