Next Generation Risk Assessment (NGRA) can use the Dietary Comparator Ratio (DCR) to evaluate the safety of a defined exposure to a compound with an identified mode of action. The DCR compares the Exposure Activity Ratio (EAR) for the compound of interest, to the EAR of an established safe level of human exposure to a comparator compound with the same mode of action. A DCR ≤ 1 indicates the exposure evaluated is safe. We have aimed to define adequate and safe comparator compound exposures for evaluation of endocrine effects. 3,3-Diindolylmethane (DIM), from cruciferous vegetables, and the anti-androgenic drug bicalutamide (BIC) were used to evaluate anti-androgenic effects and the EAR values for these comparator compounds were defined using an in vitro androgenic reporter gene assay. The isoflavone genistein (GEN) was used to evaluate estrogenic effects and its EAR value was set using in vitro estrogenic proliferation and reporter gene assays. The EAR values of DIM and BIC were evaluated using physiologically based kinetic (PBK) modelling and comparison to in vivo reported safe and therapeutic-active levels, respectively. The EAR values of GEN were evaluated by comparison with in vivo reported safe internal levels of GEN following different diets. The adequacy of the comparator EAR values was further evaluated by comparing the generated DCRs of a series of test compound exposures to actual knowledge of their safety regarding in vivo anti-androgenicity or estrogenicity. Exposure scenarios of nine anti-androgenic pesticides, phenols, and drugs and of 12 estrogenic hormones, phytoestrogens, pesticides, phenols, parabens, phthalates, and drugs were included. Results obtained supported the use of the in vitro bioactivity assay-derived comparator EARs for DCR-based NGRA for putative anti-androgenic and estrogenic compounds. This further validates the DCR approach as an important animal free tool in NGRA and will further support the safety assessment of endocrine acting substances.