Defining and Controlling Exposure During In Vitro Toxicity Testing and the Potential of Passive Dosing

Abstract

Toxicity testing using in vitro bioassays is assuming an increasingly important role. Nevertheless, several issues remain with regard to their proper application, which mainly relate to the proper definition and control of the test chemical(s) concentrations to which the cells or tissues are exposed. This has fundamental implications for understanding the underlying relationship between the in vitro exposure regime and response, and leads to uncertainty in the resulting bioassay data. This chapter covers the definition and control of exposure of hydrophobic organic chemicals (HOCs) in in vitro bioassays aimed at measuring their toxicity. A review of the fate of HOCs in typical in vitro set-ups is followed by a discussion of how to define the test exposure. Currently applied approaches for introducing HOCs into in vitro bioassays are then related to these different definitions of test exposure. Finally, passive dosing as one possible approach for giving defined and constant dissolved concentrations of HOCs in in vitro toxicity tests is introduced, using examples taken from the literature, and how this might be better integrated into high throughput in vitro toxicity testing is discussed.

Bioanalytical Tools in Water Quality Assessment

“…Passive dosing techniques have been advocated in the past to overcome the issue of loss processes (Smith and Schäfer, 2016). While passive dosing might be very useful for testing of very hydrophobic chemicals to overcome solubility issues and to obtain effect concentrations expressed as freely dissolved or cellular concentrations, we do not recommend passive dosing for testing of water extracts that also contain many hydrophilic and charged chemicals.…”

“…In passive dosing, the chemical or sample is partitioned into a polymer. The loaded polymer, once inserted in the bioassay well, slowly releases the chemical to the medium from which it can be taken up by the cells (Smith et al, 2015;Smith and Schäfer, 2016). In case of adherent cells, the set-up might be modified, that the cells do not touch the passive dosing device, for example, by growing the cells on inserts hung into the dosing plate (Kramer et al, 2010).…”

Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays—Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction

“…In high-throughput toxicity screening tests, the optimal scenario is to design in vitro test systems where the exposure is constant and controlled during the test . Passive dosing is a promising approach for this .…”

“…Passive dosing is a promising approach for this . It has mainly been used in larger volumes such as microplates with 24 wells and vials. ,,− However, designing passive dosing systems for in vitro tests is not straightforward because the tests require specific conditions relating to growth surface, growth medium, and CO 2 level and because contact between the cells and the passive dosing phase should be avoided (direct contact will make it hard to determine the exposure level). No such design has yet been demonstrated for well plates with 96, 384, or 1536 wells.…”

“…Solid phase microextraction (SPME) is currently the most promising method for determining C free of hydrophobic chemicals. 16 It has successfully been used in microplates with 24 wells to determine C free of phenanthrene using the negligible depletion SPME regime. 1 However, in the small volumes of wells in microplates with 96 or more wells, it is generally not possible to fulfil the negligible depletion requirement, and the C free can then not be measured directly in the wells.…”