The lethality after OA inoculation was 70%–100% at 24 hours after injection in both experiments (Table 1 and Table 2). Fisher’s exact test showed no significant differences (p < 0.05) in the lethality among the groups in both experiments.

The survival curves of the mice after OA inoculation are shown in Figure 1 and Figure 2. For survival analysis, both log-rank test and Gehan–Wilcoxon test were used. It is generally considered that the log-rank test is more standard, and the Gehan–Wilcoxon test gives more weight to deaths at earlier time points. In the first experiment, no statistically significant differences were found among any groups of mice (Table 3), although the median survival time was slightly longer in ICR male and female mice compared with ddY male and female mice (Table 1). On the other hand, in the second experiment, statistically significant difference was found between ICR female mice and ddY female mice (Table 4), but not among other groups. The median survival time of ICR female mice was twice as long as that of ddY female mice (Table 4).

The results of each sex and strain of mice were statistically compared between the first and second experiments. Significant differences were found only between the first and second experiments of ICR female mice (Table 5).

Specific-pathogen-free, 4 weeks old, male and female mice of strains ICR and ddY were purchased from Japan SLC Inc. (Shizuoka, Japan). These 2 strains are designated to be used in the Japanese official MBA method. The mice were adapted to our animal facility for 1 day and used at 18–20 g body weight. The room lighting was 12 hours light (09:00~21:00)–12 hours dark (21:00~09:00) cycle. The mice were housed in plastic cages with wood chip bedding and commercial pellets (CRF-1; Charles River Japan Inc., Kanagawa, Japan) and tap water were provided ad libitum. All animal experiments were conducted with the approval of the Animal Care and Use Committee of the National Institute of Health Sciences, Japan.

The ddY strain is a closed-colony outbred mouse strain and widely used in Japan, but not in other countries of the world. This strain was first established as the dd strain, probably German Swiss albino mice origin, at the Institute of Infectious Diseases of Tokyo University (Denken) in 1943. Then, dd mice were provided to, maintained and finally subdivided as ddY mice at the National Institute of Health (Yoken) in 1953. The strain name, ddY, stands for the capital letters of Deutschland (Germany), Denken, and Yoken [8].

OA was purchased from LC Laboratories (Woburn, MA, U.S.A.). OA inocula were prepared as described previously [9]. Briefly, OA was first dissolved in acetone and then mixed with soybean oil. Soybean oil was used as a non-toxic vehicle for the lipophilic toxin. Acetone was removed by evaporation and the residue was suspended in 1% Tween 60 saline and sonicated. The final inocula contained 10% soybean oil. An inoculum prepared in one tube and mixed well was used in each experiment.

A lethal dose of OA (4 μg/mL/mouse) [6,10] was injected i.p. into each mouse. Each group consisted of 10 mice. The mice were observed every 10 minutes until 12 hours after injection, and then every 30 minutes until 24 hours after injection. During the dark period, red lights were used for observation. No negative (vehicle) control (injected only 1% Tween 60 saline contained 10% soybean oil) was examined in this experiment, but no deaths of the negative control group were observed in our other experiments. The experiments were repeated twice. The experiment design basically followed the official MBA method for DSP toxins of Japan (and the EU). Humane endpoints were not applied in these experiments, because humane endpoints are not provided in the official methods.

The lethality was statistically compared among the groups using Fisher’s exact test. Survival analysis was conducted using log-rank test and Wilcoxon–Gehan test. All statistical analyses were performed using the statistical package R version 2.13.0 [11].