Comparative multispecies CAR1 and CAR3 reporter assays conducted with prototypical and experimental ligands. The CAR3 variants created in each of the respective species exhibit negligible constitutive activity allowing for detection of chemical dose-response relationships that are not apparent in the CAR1 assays. Species selective CAR ligands were used as controls (human, 5μM 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO); mouse, 0.5μM 1,4-bis-[2-(3,5,-dichloropyridyloxy)] benzene (TCPOBOP); rat, 10μM clotrimazole (CLOT); and dog, 10μM CLOT). The indirect CAR activator phenobarbital (PB) was tested at a single concentration (500μM) and was predicted not to be functional in this direct CAR activation assay. The remaining test chemicals, meclizine (MECL), etaconazole (ETA), itraconazole (ITRA), artemisinin (ART) and cervistatin (CERV), were each evaluated at three doses: 1, 10, and 30μM. Transactivation assays were performed in COS-1 cells using CAR expression plasmids and a CYP2B6-derived PBREM/XREM luciferase reporter as indicated in the figure and as described under “Materials and Methods”. Data are presented as fold change, setting the DMSO vehicle response as equal to 1. Each data point represents the mean ± SD of four separate transfections. Panels A, C, E, and G: transactivation of the reference form of CAR for each species. Panels B, D, F, and H: transactivation of the CAR3 variant form for each species. *Denotes data points that were significantly different from their respective DMSO control as determined by ANOVA in combination with a Dunnett’s multiple comparison posttest (p < 0.01).