Abstract This Opinion assesses the biological relevance of the non-monotonic dose responses (NMDR) identified in a previous EFSA External Report (Beausoleil et al., 2016) produced under GP/EFSA/SCER/2014/01 and the follow-up probabilistic assessment (Chevillotte et al., 2017a,b), focusing on the in vivo data sets fulfilling most of the checkpoints of the visual/statistical-based analysis identified in Beausoleil et al. (2016). The evaluation was completed with cases discussed in EFSA assessments and the update of the scientific literature. Observations of NMDR were confirmed in certain studies and are particularly relevant for receptor-mediated effects. Based on the results of the evaluation, the Opinion proposes an approach to be applied during the risk assessment process when apparent non-monotonicity is observed, also providing advice on specific elements to be considered to facilitate the assessment of NMDR in EFSA risk assessments. The proposed approach was applied to two case studies, Bisphenol A and bis(2-ethylhexyl phthalate (DEHP) and these evaluations are reported in dedicated annexes. Considering the potential impact of NMDRs in regulatory risk assessment, the Scientific Committee recommends a concerted international effort on developing internationally agreed guidance and harmonised frameworks for identifying and addressing NMDRs in the risk assessment process.