Determination of maximum tolerated dose (MTD) of NSC23925b and evaluation of acute safety profiles in rodents

The MTD determination assay was composed of sighting study and main study^26,35,36,37. For the sighting study of the determination maximum tolerable dose (MTD) of NSC23925b, one of each animal (one female rat, one male rat, one female mouse, and one male mouse) was used at each I.V. and I.P. injection dose level. The starting dose was selected from the fixed dose levels (5, 50, 500, 2000 mg/kg) as a dose expected to produce evident toxicity. The first animal was dosed and observed. Time of appearance, degree, and lasting/recovery time of signs were recorded in details. Afterwards, animals were observed twice on the dosing day and then changed to daily observation. The observation lasted at least 14 days. If the first animal died or appeared impaired, the second animal received a middle dose of the two doses and vice versa. The sighting study was completed when a decision on the starting dose for the main study was made.

During the main study, a total of five animals of one sex were used for each dose level investigated. The five animals were made up of one animal from the sighting study dosed at the selected dose level together with an additional four animals. Each group received one of the fixed doses by I.V. and I.P. based on the sighting study. If the first animal died or appeared impaired, the second animal received a middle dose of the two doses and vice versa.

Animals were fasted overnight prior to dosing. Following the period of fasting, the animals were weighed and the dose was calculated according to the body weight. After the animals were administrated a dose volume of 10 mL/kg, food was withheld for another 3–4 hours. Individual weights of animals were determined shortly before the test substance was administered and twice weekly thereafter. Weight changes were calculated and recorded. Animals injected with saline were the control group for the weight alteration.