Early consideration of the multiplicity of factors that govern the biological fate of foreign compounds in living systems is a necessary prerequisite for the quantitative in vitro-in vivo extrapolation (QIVIVE) of toxicity data. Substantial technological advances in in vitro methodologies have facilitated the study of in vitro metabolism and the further use of such data for in vivo prediction. However, extrapolation to in vivo with a comfortable degree of confidence, requires continuous progress in the field to address challenges such as e.g., in vitro evaluation of chemical-chemical interactions, accounting for individual variability but also analytical challenges for ensuring sensitive measurement technologies. This paper discusses the current status of in vitro metabolism studies for QIVIVE extrapolation, serving today's hazard and risk assessment needs. A short overview of the methodologies for in vitro metabolism studies is given. Furthermore, recommendations for priority research and other activities are provided to ensure further widespread uptake of in vitro metabolism methods in 21st century toxicology. The need for more streamlined and explicitly described integrated approaches to reflect the physiology and the related dynamic and kinetic processes of the human body is highlighted i.e., using in vitro data in combination with in silico approaches.
CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of
in vitro
test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two
in vitro
methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells.
The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals.
The two methods were found to be reliable and relevant
in vitro
tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed.
Embryonic midbrain micromass cultures were exposed for five days to ochratoxin A (OTA) at seven concentrations (ranging from 0.16 to 10 µg/mL). Cell viability was assessed in neutral red uptake test (NRU), and differentiation -by immunoenzymatic determination of structural proteins (β III -tubulin, MAP2, GFAP) expression level as well as by computer image analysis. Dose dependent decrease in cell number and differentiation was observed. Concentration-response curves were analysed and the mean inhibition concentrations (µg/mL) for cytotoxicity (IC 50 ) and differentiation (ID 50 ) were calculated. There were no significant differences in the sensitivity of neurons in early and late stage of differentiation and astrocytes to the toxic activity of this compound. For all endpoints ID 50 value was very low (< 10 µg/mL) so OTA was classified as a strong teratogen. IC 50 / ID 50 ratios <2 pointed out that with harmful action of OTA the basic cytotoxicity should be connected.
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