Structure Activity Relationship Read Across and Transcriptomics for Branched Carboxylic Acids. Structure Activity Relationship Read Across and Transcriptomics for Branched Carboxylic Acids

The purpose of this study was to use chemical similarity evaluations, transcriptional profiling, in vitro toxicokinetic data and physiologically based pharmacokinetic (PBPK) models to support read across for a series of branched carboxylic acids using valproic acid (VPA), a known developmental toxicant, as a comparator. The chemicals included 2-propylpentanoic acid (VPA), 2-ethylbutanoic acid (EBA), 2-ethylhexanoic acid (EHA), 2-methylnonanoic acid (MNA), 2-hexyldecanoic acid (HDA), 2-propylnonanoic acid (PNA), dipentyl acetic acid (DPA) or 2-pentylheptanoic acid (PHA), octanoic acid (OA, a straight chain alkyl acid) and 2-ethylhexanol. Transcriptomics was evaluated in four cell types (A549, HepG2, MCF7 and iCell cardiomyocytes) 6 hours after exposure to 3 concentrations of the compounds, using the L1000 platform. The transcriptional profiling data indicate that two- or three-carbon alkyl substituents at the alpha position of the carboxylic acid (EHA and PNA) elicit a transcriptional profile similar to the one elicited by VPA. The transcriptional profile is different for the other chemicals tested, which provides support for limiting read across from VPA to much shorter and longer acids. Molecular docking models for histone deacetylases, the putative target of VPA, provides a possible mechanistic explanation for the activity cliff elucidated by transcriptomics. In vitro toxicokinetic data was utilized in a PBPK model to estimate internal dosimetry. The PBPK modeling data show that as the branched chain increases, predicted plasma Cmax decreases. This work demonstrates how transcriptomics and other mode of action-based methods can improve read across. Overall design: Four cell types were used for the transcriptomic experiments: MCF-7 (breast epithelial adenocarcinoma), A549 (lung epithelial carcinoma), HepG2 (hepatocellular carcinoma) and iCell cardiomyocytes (derived from induced pluripotent stem cells, FujiFilm Cellular Dynamics, Madison, WI). MCF-7, A549 and HepG2 cells were purchased from American Type Culture Collection (Manassas, VA) and grown in phenol red-free DMEM media containing 10% serum (Invitrogen, Carlsbad, CA). The iCell cardiomyocytes were grown in a proprietary maintenance medium (FujiFilm Cellular Dynamics, Inc.) for 168 h before chemical treatment. All cell cultures were performed in 96-well plates with 12 DMSO controls on each plate. Cells were seeded on 96 well plates and treated with 3 concentrations of each chemical DMSO for 6 h. Chemical samples were randomized across the plate and DMSO were placed on fixed well locations for each plate. Following 6 h treatment, Cells were lysed with 50 μl of Genometry Lysis Buffer to each well, sealed and stored at -80⁰C. Cell lysate plates were shipped frozen to Genometry for L1000 assasy.