**4.1 Mass balance study**

Due to the high expense, complete ADME profiling (non-radiolabeled) does not typically occur until a strong candidate for IND submission has been identified. The *in vitro* metabolism, tissue distribution analyses, and *in vivo* PK studies performed in the prior development stages are used to inform the investigators regarding the potential route of clearance, the tissues to be collected and quantitated, and if a major metabolite should be investigated as part of the study. The goals of these studies are to definitively identify the route of elimination (including biliary excretion), assess tissue distribution, and if applicable, characterize metabolite exposure relative to parent exposures. The animals in these studies are typically split into two groups where one group has their bile duct cannulated in order to assess biliary excretion from the liver. The animals are individually housed in metabolism cages that collect the urine and feces excreted by each animal. Depending on the study design, each animal may be sacrificed at various time points in order to assess critical tissues for exposure to the drug entity.
