Sonic hedgehog (SHH) is a major intercellular signaling pathway involved in the orchestration of embryogenesis, including orofacial morphogenesis. The SHH pathway is sensitive to disruption, including both genetic predisposition and chemical-induced disruption at multiple molecular targets including antagonism of the SHH signal transducer Smoothened (SMO). Here we report the adverse outcome pathway (AOP) 460 describing the linkage between antagonism of the SMO receptor, a key intermediate in the hedgehog signaling, and orofacial clefts (OFCs). Multiple antagonists of SMO have been identified including natural compounds, synthetic pharmaceuticals, and a common pesticide synergist. Activation of the SHH pathway causes a signaling cascade that culminates with the transcription of genes driven by glioma-associated oncogene (GLI) transcription factors. When SMO is antagonized during normal development, the cascade is disrupted causing myriad phenotypes at different critical windows of exposure, ranging from major structural defects and spontaneous abortion early in gestation to reduced outgrowth of the facial prominences and the formation of an OFC later in development. There is high evidence that antagonism of SMO causes OFCs that include a dose response relationship with incidence of clefting. Several emerging new approach methodologies (NAMs) offer the ability to monitor intermediate key events and test for temporal and dose response relationships in vitro. While most data used to support this AOP were generated using mouse (Mus musculus) models during embryonic development, SHH and the development of the face are largely conserved between mouse and human, making this AOP able to be extrapolated to risk assessment for human exposures.