Characterization of Complex Substances Used in Biological Profiling Through Determination of the Free Concentration Within In Vitro Assays
Abstract
Application of traditional high through-put (HTP) in vitro screening, coupled with advanced mass spectrometric and separation techniques, to substances of unknown or variable composition, complex reaction products, and biological materials (UVCBs) has made monumental progress toward biological and analytical profiling, facilitating regulatory read across and decision making for these challenging substances. However, limited understanding of protein and non-specific binding effects to the chemical composition of screening extracts may cause misinterpretations of potential in vivo effects. Conventional in vitro to in vivo extrapolation (IVIVE) approaches could alleviate these concerns, but current techniques only evaluate single-constituents. Furthermore, applied in vitro pharmacokinetic assays measuring protein binding, specifically equilibrium dialysis, are limited to hydrophilic chemicals.
This study clearly defines chemical in vitro equilibrium dialysis assay limitations through application of environmental chemicals with a range of octanol/water partition coefficients (logKOW) and identifies solid phase micro-extraction (SPME) as a suitable alternative for environmental chemicals not suitable for equilibrium dialysis. This alternative technique is used to characterize the chemical composition of complex substances, used in biological profiling, by evaluating chemical protein binding of these substances within cell medium.
Lastly, this study discusses the utility of traditional chemical composition analysis, via GC-MS, to effectively group or “fingerprint” complex substances, specifically petroleum products, for regulatory read across application. This is accomplished by evaluating its ability to effectively group similar petroleum refinement classes compared to more advanced analytical techniques (GCxGC-FID and IM-MS).
Subject
complex substancesequilibrium dialysis
solid phase micro-extraction
protein binding
in vitro to in vivo extrapolation
regulatory read across
Citation
Ferguson, Kyle Charles (2018). Characterization of Complex Substances Used in Biological Profiling Through Determination of the Free Concentration Within In Vitro Assays. Master's thesis, Texas A & M University. Available electronically from https : / /hdl .handle .net /1969 .1 /173389.