The Prediction of Human Acute Systemic Toxicity by the EDIT/MEIC In Vitro Test Battery: The Importance of Protein Binding and of Partitioning into Lipids

Abstract

The aim of the two studies presented in this paper was to further improve the predictability of the original Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) in vitro test battery for acute systemic toxicity. In the first study, whether a protein-free cytotoxicity assay could improve the prediction of human acute systemic toxicity was investigated. The cytotoxicity of 39 MEIC reference chemicals was measured by the neutral red uptake inhibition test after 30 minutes in phosphate-buffered saline (PBS), with hepatoma-derived Fa32 cells. The results were compared with the corresponding values obtained in complete culture medium, including 10% fetal calf serum. Mercuric chloride and hexachlorophene were much more cytotoxic in PBS, as was the case, to a lesser extent, for seven other chemicals. Potassium cyanide and eight other chemicals were less cytotoxic in PBS than in complete culture medium, probably because of poor physiological conditions. The correlation between the cytotoxicity measured in PBS and human acute toxicity was rather low, but became of the same order as for other assays, when mercuric chloride and hexachlorophene were withdrawn from the comparison. In the second study, modelling of human lethal blood concentrations by using the results of the three cell line tests of the original MEIC test battery were complemented by logP (octanol–water partition coefficient) values. The introduction of logP into the modelling did not improve the correlations, but some improvement of both R2 and Q2 was obtained by expanding the logP values with logP2 values. The highest R2 (0.84) and Q2 (0.80) values were obtained for a model in which both experimental and calculated (ambiguous) logP values were used. When only experimental logP values were used, the corresponding values were 0.80 and 0.78. These two studies showed that including protein binding and the partition of chemicals in the MEIC in vitro test battery is important, in order to improve the predictability of the results obtained.

Toward More Useful In Vitro Toxicity Data with Measured Free Concentrations

“…Apparatus and Reagents. [2,4,6, Cell Culture. Human embryonal kidney 293 (HEK293) cells were obtained from the American Type Culture Col-lection (ATCC; Rockville, MD).…”

“…cell number), the serum (protein) content in the test medium, and the volatility and hydrophobicity of the compound. Serum content of the cell culture medium is known to vary between laboratories, and this can lead to a different outcome of the same test in different laboratories (2). Such a variety in outcome complicates a comparison of the potencies of biologically active chemicals.…”

Influence of Co-Dosed Lipids from Biota Extracts on the Availability of Chemicals in In Vitro Cell-Based Bioassays

“…Bioanalytical tools are widely used to assess the burden of chemical mixtures, among others in biota extracts, including mussel, , fish, , and marine mammals. , In vitro cell-based reporter gene bioassays are highly sensitive, target specific modes of actions, allow high-throughput applications, and can be used for the effect characterization of the chemical mixture present in an environmental sample . However, the bioavailability of chemicals in the bioassay system can be influenced by various processes, such as binding to medium constituents (lipids and proteins), , sorption to the well plate, , and, in the case of biota samples, binding to co-extracted lipids , that form droplets on the medium surface. Co-dosed lipids add an additional partitioning phase to the bioassay system and can thus reduce the bioavailability of chemicals.…”

The Use of the Integrated Discrete Multiple Organ Co-culture (IdMOC®) System for the Evaluation of Multiple Organ Toxicity

“…Experimental approaches for the evaluation of in vitro toxicity include the use of tissue slices (1, 2), primary cells (3)(4)(5)(6)(7)(8)(9) and cell lines (10)(11)(12). They are generally viewed as useful for initial toxicity screening and for the mechanistic evaluation of toxic effects observed in vivo.…”