Summary of reference chemicals evaluated by the fish short‐term reproduction assay, OECD TG229, using Japanese Medaka,Oryzias latipes

Abstract

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARβ of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

Summary of 17 chemicals evaluated by OECD TG229 using Japanese Medaka,Oryzias latipesin EXTEND 2016

“…Also, the applicability of OECD TG229 (Fish Short-Term Reproduction Assay, FSTRA) using Japanese medaka (OECD, 2012) in Tier 1 in vivo screening was demonstrated for identifying the effects of chemicals with Er agonistic and antagonistic activity, and Ar agonistic activity, potency on the reproduction of fish, as well as steroidogenesis inhibitory activity. In addition, the reference chemical studies demonstrated that the effects of the progestin that masculinized and reduced reproductive activity in females by activating Ar (Onishi et al, 2021) were similar to the reports on other small test fish (Ellestad et al, 2014;Frankel et al, 2016;Svensson et al, 2013). Regarding the endpoints adopted for the in vivo fish assay, vitellogenin (VTG) and secondary sex characteristics (SSC) in Japanese medaka were sensitive to Esr agonistic and antagonistic, and Ar agonistic, activities of chemicals (Onishi et al, 2021).…”

“…were closer to the LOECs on VTG induction in males than to the LOECs on reproduction in the FSTRA (TG229 study), for instance the ER agonists (E2 and EE2) (Flynn et al, 2017;MOE, 2006;Onishi et al, 2021). In the 17 chemicals of the present study, as shown in Table 9, the LOEC mVTG /MEC ratios of 4NP, E1, and 4tOP were relatively smaller than other chemicals, indicating that there is a concern of adverse effects on reproduction in long-term exposure through the full life cycle history of fish.…”

“…1NT 90-15-3 WAKO, 100% (A)(D) ; TCI, 99.9% (B)(C) WAKO, 100% 4NP 84852-15-3 SIGMA, 97.8% (A)(B)(D) ; SIGMA, 92.9% (C) KNTC, 99.7% 4tOP 140-66-9 TCI, 97.2% (A)(B)(C)(D) TCI, 97.2% PDM 40487-42-1 KNTC, 98.8% (A)(B)(C)(D) FUJI, 99.3% 4tPP 80-46-6 WAKO, >97.0% (A) ; TCI, 99.9% (B)(C)(D) TCI, 99.9% PHT 57-41-0 TCI, 100% (A)(D) ; WAKO, 99.3% (B)(C) TCI, >98% TCS 3380-34-5 TCI, 99.8% (A)(B)(C)(D) WAKO, 98.0% TPP 115-86-6 TCI, 99.9% (A)(B)(D) ; TCI, 100% agonist and the antagonist assay, respectively. The details of methods of both mEsr1 and mArβ RGAs can be found in Onishi et al (2021).…”

“…In addition, the reference chemical studies demonstrated that the effects of the progestin that masculinized and reduced reproductive activity in females by activating Ar (Onishi et al, 2021) were similar to the reports on other small test fish (Ellestad et al, 2014;Frankel et al, 2016;Svensson et al, 2013). Regarding the endpoints adopted for the in vivo fish assay, vitellogenin (VTG) and secondary sex characteristics (SSC) in Japanese medaka were sensitive to Esr agonistic and antagonistic, and Ar agonistic, activities of chemicals (Onishi et al, 2021). On the other hand, the TG229 assay using Japanese medaka was less sensitive in the detection of antiandrogenic (Ar antagonistic) activity of chemicals (OECD, 2012;Onishi et al, 2021).…”

“…And the TG229 assay using Japanese medaka was conducted for twenty chemicals which had estrogenic, anti-estrogenic, androgenic, and/or anti-androgenic activities as suggested by RGAs using medaka Esr1 (mEsr1, known as ERα in mammals) or mArβ (known as ARβ in mammals, Ogino et al, 2016). This manuscript describes the results of the Tier 1 screening assays for 17 chemicals, except three chemicals previously reported (Onishi et al, 2021). Chemicals of Law of PRTR system were included.…”