Test No. 445A: Determination of Cytochrome P450 (CYP) Induction using Differentiated Human Hepatic Cells

This Test Guideline describes the use of human-derived metabolic competent hepatic test systems (e.g. cryopreserved differentiated HepaRGTM cells) to assess the potential of test chemicals to induce (i.e. increase the synthesis and activity) three Phase I biotransformation enzymes: the cytochrome P450 (CYP)3A4, CYP1A2 and with less certainty CYP2B6 subfamily which are susceptible to induction and are highly expressed in human liver. Besides detoxifying chemicals or increasing their toxicity due to formation of toxic metabolites, CYP enzymes play a key role in the biosynthesis of endogenous substrates (e.g. steroid hormones). Chemical CYP enzyme activity induction may therefore cause dysregulation of normal metabolism and homeostasis, with potential toxicological effects. In one plate, cells are exposed to (I) test chemical(s) at least at six concentrations (II) reference chemicals at set concentrations (providing experimental positive controls) and (III) solvent-containing medium (e.g. 0.1% DMSO if nécessary) serving as the negative control. After the required incubation period, CYP enzyme activity is determined by applying fresh medium containing a combination (“cocktail”) of the CYP-selective probe substrates: phenacetin (CYP1A2), bupropion (CYP2B6) and midazolam (CYP3A subfamily). CYP1A2, CYP2B6 and CYP3A subfamily enzymes present in the test system will metabolise these probe substrates into the known metabolites acetaminophen, hydroxy bupropion and 1-hydroxy midazolam, which can be quantified with an appropriate analytical technique, such as liquid chromatography coupled to mass spectrometry (LC/MS). The amount of metabolites formed per unit of time and normalised to the protein content is a direct measurement of CYP enzyme activity.