Comparative analysis of Rapid Equilibrium Dialysis (RED) and solid phase micro-extraction (SPME) methods for In Vitro-In Vivo extrapolation of environmental chemicals.

In vitro to in vivo extrapolation (IVIVE) is a critical component of the efforts to prioritize and assess environmental chemicals using high-throughput in vitro assays. The plasma unbound fraction (F(ub)) is a key toxicokinetic parameter in IVIVE, and is usually measured via the Rapid Equilibrium Dialysis (RED) assay widely used for pharmaceuticals. However, pharmaceuticals have a narrower range of physicochemical properties than environmental chemicals. Motivated by the observation that high LogK(OW) compounds appeared to have disproportionately low F(ub) measurements using RED, we added a protein-free control in order to verify equilibration to 100% unbound in the absence of proteins. We found that many high LogK(OW) non-pharmaceuticals fail to equilibrate in RED in protein-free controls, and thus had apparent values of F(ub) = 0 in plasma. In these cases, Solid Phase Microextraction (SPME) as an alternative method provided an accurate, though more time-consuming, alternative to accurately determine F(ub). We propose an updated IVIVE workflow that adds a protein-free control to the RED protocol, with the use of alternative approaches, such as SPME, in cases where compounds fail to adequately equilibrate. These refinements will provide additional confidence in the use of IVIVE as part of high-throughput screening programs of chemicals.