<p>The Fish Short Term Reproduction Assay (TG229) evaluates endocrine disruption inducing reproductive dysfunction in fish (medaka, fathead minnow and zebrafish). TG229 evaluates effects on spawning, vitellogenin (vtg), gonadal histopathology and secondary sex characteristics (SSC). Four actions are detectable based on their effects: estrogenic, anti-estrogenic, androgenic, and steroidogenesis inhibition, but TG229 is not supposed to reveal specific actions. In the four actions, Adverse Outcome Pathways (AOP) has already been established, with reduced spawning and changes in vtg being key events. Therefore, the four actions can be discriminated to some extent due to the results of TG229. Estrogenic action can be identified by increased vtg in males. Anti-estrogenic action and steroidogenesis inhibition can be inferred from decreased vtg and spawning, but these are indistinguishable. Androgenic action can be identified by the appearance of male-like SSC in females. SSC are gross morphological changes that are indicator of masculinization: the formation of papillary processes on the anal fin in medaka and nuptial tubercles on head in fathead minnow. The appearance of male-like SSC is regulated by androgens and has been reported to be a more sensitive indicator of androgen substance than changes in vtg and spawning. On the other hands, zebrafish does not possess quantifiable SSC. This presentation will describe the characteristics of the indicators that should be evaluated for TG229 in terms of AOP and sensitivity.</p>