Background and Purpose: The etiology of orofacial clefts is complex with most cases being of unknown origin. To facilitate the study of the Sonic Hedgehog (SHH) signaling to improve understanding of normal and abnormal orofacial development, a 3D microphysiological model was engineered [1]. This model is designed as a throughput compatible model of the SHH gradient between the epithelial and mesenchyme tissues of the developing facial processes and is well suited for drug and chemical screening. SHH is sensitive to chemical disruption at multiple locations along the signaling cascade and has critical windows of exposure during development that can lead to OFCs. To organize the state of evidence linking SHH disruption to OFCs in an opensource and regulatory relevant manner an Adverse Outcome Pathway (AOP) network is being developed. The creation of AOP 460: antagonism of Smoothened receptor leading to OFC has identified data gaps in dose response for cellular proliferation and tissue outgrowth. This work focuses on improving the human relevance of the MPM and applying it to generate additional data to fill the gaps identified in the AOP. Human relevance will be increased by the incorporation of migratory cranial neural crest cells (CNCCs) and the transition to a human derived mesenchyme. The generation of additional data to fill these gaps using the MPM can increase the confidence in the AOP and adds to the usefulness of the AOP to be applied to risk assessment of drugs and chemicals as well as improves the fields understanding of the etiology of OFC. Methods: This AOP Network is being developed through the OECD’s AOP development program (EAGMST workplan project 1.101.) in accordance with OECD AOP development guidelines. To identify sources and data for each key event relationship (KER), Pubmed and MeSH terms were used. Initially results were screened for relevance off title/abstract and any of suspected relevance were reviewed in full to determine their applicability for the KER. Each KER includes a table of relevant search information (date, search terms, citations, etc) and weight of evidence (WoE) assessments were performed. MPMs are constructed via CNC micromachining of standard 96 well ANSI microplates as previously described [2]. Devices are designed to incorporate 3 wells to create a microtissue well flanked with a side channel separated by a phase guide. Devices are loaded with a dense mesenchymal (3T3 SHH lightII or HEPM LUC) embedded in a hyaluronic acid hydrogel (Hystem-C). Once polymerized, the tissue is overlaid with an oral epithelial cell line modified to over secrete SHH (GMSM-K RFP SHH) by flowing a cell suspension down the flanking side channels. CNCCs are derived from RFP tagged S1 hIPSCs [3]. These CNCCs are incorporated into the MPM by placing 500 cells on top of the center of the microtissue after polymerization has occurred. Results: To date, three of the six AOPs are actively underdevelopment or developed in the AOP wiki (AOPs 460, 491, 502). AOP 460 is complete on the wiki and is currently under scientific review. AOP 460 has identified multiple data gaps for dose response for cellular proliferation, tissue outgrowth, and apoptosis. The key event relationships for proliferation, tissue outgrowth, and apoptosis were all determined to have both a low level of evidence and low quantitative understanding. MPMs have been successfully cultured and response to epithelial secreted SHH ligand has been quantified via GLI1 luminescence (foldchange of 22.6±0.27). The model has been validated to respond to both SHH agonists and antagonists (e.g. SAG and Vismodegib respectively). Humanization is improved by transitioning the mesenchymal cells to the human derived HEPM (human embryonic palatal mesenchyme) and adding a migratory cranial neural crest to the MPM. CNCCs have been successfully differentiated from hIPSCs and incorporated into the MPM. Differentiation is verified via qPCR for SOX10 and TFA2A. The migration of the RFP tagged CNCCs has been tracked via time lapse imaging using confocal microscopy. A new version of the MPM has been designed and successfully prototyped to increase throughput from 20 to 40 tissues while also enabling tissues to grow next to each other facilitating the study of outgrowth and fusion. Conclusions: Combining the findings of an AOP with an MPM to generate additional regulatory relevant data creates a broadly useful new approach method to generate and organize regulatory relevant data. The successful integration of CNCCs into the MPM enhances the human relevance of the model, providing a more accurate representation of human craniofacial development. The data generated from this study will fill critical gaps identified in AOP 460, increasing the confidence in the AOP and its applicability for risk assessment of drugs and chemicals. This approach not only advances the understanding of the etiology of OFCs but also provides a valuable tool for regulatory agencies to assess the safety of drug and/or chemical exposures during development. 1. Johnson, B.P., et al., A Microphysiological Approach to Evaluate Effectors of Intercellular Hedgehog Signaling in Development. Front Cell Dev Biol, 2021. 9: p. 621442. 2. Reynolds, J.I., et al., Engineering EpithelialMesenchymal Microtissues to Study Cell-Cell Interactions in Development, in Craniofacial Development: Methods and Protocols, S. Dworkin, Editor. 2022, Springer US: New York, NY. p. 201-213. 3. Hackland, J.O.S., et al., Top-Down Inhibition of BMP Signaling Enables Robust Induction of hPSCs Into Neural Crest in Fully Defined, Xenofree Conditions. Stem Cell Reports, 2017. 9(4): p. 1043-1052.