Table 3:

Part of the published EURL ECVAM interactive questionnaire for detecting standards that users of in vitro human hepatic metabolic clearance method might apply within their laboratories. In the second column examples of received replies are presented

Question	Example of replies
For which of the following purpose(s) you apply the submitted method in your test facility?	Screening, development of a new compound, regulatory use, incorporation into a PBPK model, ranking chemicals from low to high clearance, species selection for safety studies etc.
Which of the following test systems is employed in the in vitro method/ SOP?	Human fresh hepatocytes (single donor or pooled), cryopreserved human hepatocytes (single donor or pooled), S9, microsomes, liver slices, cytosol etc.
Which is the test system configuration?	Suspension, 2D cultured, 3D cultured, sandwich cultured etc
Which parameter is measured?	Parent compound depletion rate, metabolite formation rate, both parent compound depletion and metabolite formation rate
Are there any internal standards or reference compounds, for which human in vivo behaviour is known, included in the in vitro method/SOP?	27% replied No, 73% replied Yes (e.g. Midazolam, Verapamil, Naloxone, Piroxicam)
Are acceptance criteria for the biological system (e.g. cell line, tissue model etc) being used and are they specified in the in vitro method/ SOP? An example of acceptance criterion could be a sufficient metabolic activity of CYP3A4, or the transporter competence of the biological system	55% replied No, 45% replied Yes (e.g. metabolic activities, cell viabilities at the conclusion of the assays intrinsic clearance data relative to historical data, historical data batch wise corrected, the hepatocyte pools were characterized for metabolism (CYP1A2, CYP2C9, CYP2D6, CYP3A4/5, CYP2C19, 7-ethoxycoumarin glucuronidation and 7-ethoxycoumarin sulfation) and viability (Trypan Blue exclusion). The pools were used if they fell within acceptable ranges compared with historical quality control limits, as determined by cell provider, basal activity of CYP 1A2, 3A4, should be greater than the median given by historical data of the provider and viability greater than 75%.)
Do you correct results for unbound concentrations in the incubation, either by experimental measurement (e.g. Equilibrium dialysis) or by use of a log D prediction? If yes, please describe the approach you follow.	46% replied No, 27% replied that it is case dependent (e.g. correction is performed sometimes using ultrafiltration, depending on the purpose of the analysis correction might be performed, depending on serum concentration etc) 27% relied YES (e.g. using equilibrium dialysis)