Biokinetics and repeated exposure in in vitro assays : A detailed study into the behaviour of chlorpromazine and diazepam in different cell systems

The potential risk of compounds is commonly assessed with animal experimentation and extrapolation of these data to assess human health effects. The use of Integrated Testing o Strategies combines different methods, including in vitro tests and in silico methods, to perform risk assessment with less costs and less animal experiments. Although alternatives to animal tests have been developed and validated, research into alternatives for certain toxicological endpoints is yet limited. One of these endpoints is systemic toxicology after repeated dosing. In addition, difficulties arise when extrapolating in vitro data to the in vivo situation. In vitro data can be highly variable and it is difficult to compare nominal concentrations in vitro with doses in vivo. One aspect that is often missing in in vitro assays is information on exposure concentrations. These exposure concentrations are highly influenced by the in vitro biokinetics of the test compound. Therefore, the aim of the work presented in this thesis was to study in vitro biokinetics in different cell systems. This was done for both single as well as repeated exposure studies in vitro. The biokinetic behaviour of chlorpromazine was studied in an intestinal permeability assay, cytotoxicity assays with different cell lines and after repeated exposure in different liver cell systems and a primary neuronal cell system. The biokinetic behaviour of diazepam was studied in a repeated exposure experiment with primary neuronal cells. In all these assays, samples were taken from the medium, cells and well plastic to determine the amount of compound in each of these compartments. In addition, the free concentration of chlorpromazine in the medium was measured by a negligible depletion-solid phase microextraction method which was optimised for this compound. The data presented in this thesis show that by expressing in vitro data based on a nominal concentration one ignores the different processes that can take place in vitro and that could influence the actual exposure concentration. By studying the in vitro biokinetics of CPZ in different cell systems, we found that the compound distributes over the different compartments present in an in vitro system: medium (freely dissolved and protein bound), cells and plastic. Although in some cases reasonably good correlations have already been found between in vitro and in vivo dat