The CITEPro module #5 Exposure assessment in bioassays aims at the direct combination of the bioassay testing in modules #1-4 with instrumental analysis. The effects measured in in vitro bioassays are typically reported as nominal effect concentrations. However, various processes, e.g. sorption to plastic well plates, binding to medium components, metabolism, and volatilization can influence the bioavailability of the test chemicals in in vitro bioassays.
The free concentration in the assay medium is regarded a better dose metric than the nominal concentration. In serum-free media the free concentration can be determined directly, while a solid phase microextraction (SPME) method is used for the cell-based bioassays of module #1 to remove the sample matrix. All samples are analyzed using an LC-MS/MS system from Agilent.
The experimental exposure assessment is accompanied by physicochemical characterization of the test chemicals. A Sirius T3 from Pion Inc. is used for the determination of acidity constants, pH dependent solubility and octanol-water distribution ratios of ionizable test chemicals.
Free concentration measurement in cell-based bioassays for quantitative in vitro to in vivo extrapolation and determination of partition constants.
Literature referencesHenneberger, L.; Mühlenbrink, M.; Fischer, F. C.; Escher, B. I., C18-coated solid phase microextraction fibers for the quantification of partitioning of organic acids to biological materials. Chemical Research in Toxicology, in press.
Henneberger, L.; Mühlenbrink, M.; Fischer, F. C.; Escher, B. I., Using a combination of serum-mediated passive dosing and SPME measurements for controlling the dose of chemicals in in vitro bioassays. Manuscript in preparation.
Fischer, F. C.; Henneberger, L.; König, M.; Bittermann, K.; Linden, L.; Goss, K. U.; Escher, B. I., Modeling Exposure in the Tox21 in Vitro Bioassays. Chem Res Toxicol 2017, 30, (5), 1197-1208.