The Innovation and Quality Induction Working Group presents an assessment of best practice for data interpretation of in vitro induction, specifically, response thresholds, variability, application of controls, and translation to clinical risk assessment with focus on CYP3A4 mRNA. Single concentration control data and Emax/EC data for prototypical CYP3A4 inducers were compiled from many human hepatocyte donors in different laboratories. Clinical CYP3A induction and in vitro data were gathered for 51 compounds, 16 of which were proprietary. A large degree of variability was observed in both the clinical and in vitro induction responses; however, analysis confirmed in vitro data are able to predict clinical induction risk. Following extensive examination of this large data set, the following recommendations are proposed. a) Cytochrome P450 induction should continue to be evaluated in three separate human donors in vitro. b) In light of empirically divergent responses in rifampicin control and most test inducers, normalization of data to percent positive control appears to be of limited benefit. c) With concentration dependence, 2-fold induction is an acceptable threshold for positive identification of in vitro CYP3A4 mRNA induction. d) To reduce the risk of false positives, in the absence of a concentration-dependent response, induction ≥ 2-fold should be observed in more than one donor to classify a compound as an in vitro inducer. e) If qualifying a compound as negative for CYP3A4 mRNA induction, the magnitude of maximal rifampicin response in that donor should be ≥ 10-fold. f) Inclusion of a negative control adds no value beyond that of the vehicle control.

The human constitutive androstane receptor (CAR, CAR1) regulates the expression of genes involved in xenobiotic metabolism in the liver. The CAR gene uses multiple alternative splicing events during pre-mRNA processing, thereby enhancing the CAR transcriptome. Previous reports have identified two prominent human CAR variants, CAR2 and CAR3, that possess fourand five-amino acid insertions in their ligand binding domains, respectively. Unlike the constitutively active reference form of the receptor, we now demonstrate that CAR2 is a ligandactivated receptor and comprises approximately 30% of the reference transcript level in human liver tissues in human hepatocytes. Furthermore, we identify the common plasticizer, di(2-ethylhexyl) phthalate (DEHP), as a highly potent and uniquely selective agonist of CAR2. Results from reporter transactivation and mammalian two-hybrid assays reveal that DEHP activates CAR2 at low nanomolar concentrations, results further supported by analysis of CAR target gene expression in primary human hepatocytes. In addition, comparative genomic analyses show that the typical mouse, rat, and marmoset models of DEHP toxicity cannot accurately profile potential human toxicity because of these species' inability to generate a CAR2-like transcript. The discovery that CAR2 is an ultimate human DEHP receptor identifies a novel pathway modulating human DEHP toxicity with potential clinical implications for a subset of patients undergoing critical care medical interventions.