Generally, the determination of maximum tolerated dose (MTD) is performed in a way that conserves compound and minimizes the number of animals sacrificed. Thus, a single mouse is given a single injection (IP, IV, SC, IM or PO) of 400 mg/kg (or lower if the compound is anticipated to be extremely potent, e.g. natural products); a second mouse receives a dose of 200 mg/kg and a third mouse receives a single dose of 100 mg/kg. The mice are observed for a period of 2 weeks. They are sacrificed if they lose more than 20% of their body weight or if there are other signs of significant toxicity. If all 3 mice must be sacrificed, the next 3 dose levels (50, 35 and 12.5 mg/kg) are tested in a similar manner. This process is repeated until a tolerated dose is found. This dose is then designated the MTD and is used to calculate the amount of material administered to mice during anti-tumor testing. The mice are allowed ad libitum feed and water. Injections are most commonly administered IP, but SC, PO and IV dosing may be required on occasion. Dose volumes are generally 0.1 mL/10 grams body weight but may be up to 0.2 mL/10 grams of body weight for IP, IV, SC and PO routes.
For the standard hollow fiber assay (HFA), the high and low dose levels are determined using the MTD as determined above using the formula below.
High dose = [MTD x 1.5]/4
Low dose = 0.67 x high dose
The standard vehicle used for both acute toxicity testing and HFA is 10% DMSO in saline/0.05% Tween 80.