Measuring, modeling, and increasing the free Concentration of test Chemicals in cell assays

Difficulties arise when extrapolating in vitro derived toxicity data to in vivo acute toxicity data because in vitro results are highly variable and occasionally less sensitive. Differences in the free concentration of a test chemical between in vitro and in vivo systems and within in vitro systems may in part explain this variability and sensitivity difference. Generally, only the free concentration of a chemical is considered available to tissues. However, in vitro effect concentrations are normally expressed as ‘nominal’ or total concentrations. The measurement or estimation of the free concentration in these systems has thus far been very limited. The aim of this thesis was to determine what assay components and physicochemical properties influence the free concentration of organic chemicals in basal cytotoxicity assays and how differences in the free concentration affect the in vitro-in vivo acute toxicity extrapolation. This thesis found a good correlation between basal cytotoxicity and acute toxicity data from fathead minnow of 82 industrial organic chemicals. However, significant improvements to the correlation could be made if a) multiple cell types and endpoints are included in an in vitro test battery and b) the free concentration is determined for hydrophobic and volatile chemicals. Indeed, the free concentration of volatile and hydrophobic polycyclic aromatic hydrocarbons (PAHs) was found to be significantly lower than the nominal concentration because these chemicals strongly bind to serum protein, sorb to microtiter plate plastic, partition into cells, and evaporate. By measuring the sorption coefficients of PAHs to the various assay components and by correlating these coefficients to the volatility and hydrophobicity of the PAH, the free concentration of PAHs in a basal cytotoxicity assay could be successfully modelled. Moreover, the loss of free concentrations in vitro could be adequately reduced by using solid phase dosing instead of conventional dosing