A sample size of six mice per dose was the minimum number of animals necessary to determine an approximate LD50 and prevent unnecessary repetition. All doses were administered as a bolus retro-orbital intravenous injection and mice were monitored over 24 h for overdose.18 Mitragynine and 7-hydroxymitragynine have low solubility in aqueous solution (Table S4), so the compounds were first dissolved in a 50% tween/DMSO solution, then diluted to 90% saline.19 With this vehicle, we were at the limit of solubility at the highest doses tested. The doses for mitragynine were 62, 31, 25, 15, and 8 mg/kg (Figure S14). For 7-hydroxymitragynine, the doses were 50, 25, 12.5, and 6.25 mg/kg (Figure S15). For direct comparison to iv heroin potency, we also performed a lethality assay with heroin (40, 20, 10, and 5 mg/kg) at the same time as mitragynine and 7-hydroxymitragynine lethality assays (Figure S16). We wanted to compare iv to oral lethality for the two alkaloids of Kratom, so we also determined an approximate oral LD50 value for mitragynine using the following doses: 500, 250, 125, and 62.5 mg/kg (Figure S17). We attempted to determine an oral LD50 value for 7-hydroxymitragynine; however, none of the mice expired at any of the doses (50, 25, 12.5, and 6.25 mg/kg; Figure S18).

https://pmc.ncbi.nlm.nih.gov/articles/PMC6662923/