The aims of this research are to measure and model the freely dissolved concentration of organic test compounds in cytotoxicity assays and to determine the importance of this free concentration in in vitro-in vivo acute toxicity extrapolations.

To determine what factors influence the correlation between in vitro and in vivo toxicity data, basal cytotoxicity and acute toxicity data from fathead minnow of more than eighty industrial organic chemicals, spanning a wide range of physicochemical properties and toxicities are collected from the Halle Registry of Cytotoxicity and the U.S. EPA Fathead Minnow Database and statistically analysed.

To measure and model what assay components and physicochemical properties influence the free concentration of test chemicalsin vitro, polycyclic aromatic hydrocarbons (PAHs) are tested for cytotoxicity on mouse fibroblasts and fish gill cells. Solid phase microextraction (SPME) is used to measure the free concentration directly in the assay and to measure the partition coefficients of the compounds to serum, well plate plastic, cells and headspace. These partition coefficients may then be correlated with physicochemical properties. These relationships can then be used to model the free concentration of the compound in vitro.

To improve the predictability of free concentrations of volatile and/or hydrophobic compounds in cell assays, a passive dosing is being developed in which cell assays can be dosed with the chemical via a solvent-free partition controlled delivery system, which maintain a constant medium concentration.

The toxicological risk assessment paradigm has traditionally relied on a black-box animal based approach. However, the surge in demands for the regulatory toxicity testing of chemicals by new EU legislation and the public's unfavorable attitude towards animal testing provides a major impetus for the incorporation of novel methodologies like in vitro assays into risk assessment. The potential of refining, reducing and ultimately replacing animal testing with such methodologies, however, depends on our understanding of the factors that influence the power of in vitro derived toxicity data to predict the in vivo situation.

Indeed, although good correlations exist between in vitro and in vivo derived toxicity data, data obtained from cell tests have shown to be variable and less sensitive in a number of cases. Differences in the free concentration of the test compound between in vitro and in vivo systems and within in vitro systems may help explain this variability and sensitivity difference. The free, unbound concentration is considered to determine toxicity in cells and organisms. Processes like serum protein binding, well plate plastic binding and evaporation may reduce the free concentration in a cell assay and thus reduce the observed cytotoxicity. This aspect, however, has been generally overlooked in in vitro toxicology.

CEllSens, Predict-IV, Doerenkamp-Zbinden