Approach to Endocrine Disruptor Screening Program of Environmental Protection Agency Sumitomo Chemical Co., Ltd. Environmental Health Science Laboratory Kenta MINAMI Keiko OSE Takafumi YAMAGUCHI The existence of endocrine disrupting chemicals (EDCs) was suggested in the late 1990s, and concern about their effects on humans and wild animals has gathered a lot of public attention. This resulted in a worldwide reinforcement of regulations for EDCs, and the U. S. Environmental Protection Agency published their Endocrine Disruptor Screening Program (EDSP) and started an evaluation of EDCs. In this article, we introduce our approach to and achievements in the evaluation of EDCs. In addition, we provide an overview of EDSP and the result of an EDSP assessment of Sumitomo Chemical s compound,. Endocrine disrupting chemicals ED WHO2002 IPCSED 1) ED ED 1996 Our Stolen Future 2) ED ED ED ED ED 1996 Environmental Protection Agency EPA1998 Endocrine Disruptor Screening Program EDSP 3) 200911 EDSP EPA EDSP EDSP 2015EPA WHO ED 2016
ED ED ED Gonads Thyroids HypothalamusPituitary HPG HPT Fig. 1 Fig. 1 Hypothalamus Gonadotropin-releasing hormone (GnRH) Thyrotropin-releasing hormone (TRH) Pituitary Gonadotropin Thyroid-stimulating hormone (TSH) Thyroids Thyroid hormone Gonads (e.g. Ovary, Testis ) Estrogen Androgen Representative organs on endocrine system and typical hormones OECD 1998 ED HPG HPT ED EDSP ED ED 1990 ED ED OECD1998 OECD TG 440Hershberger OECD TG 44128 OECD TG 4073 in vitro OECD 4) 6) ER OECD TG 455 herα-hela-9903 2016
in vitro in vivo 7) 8) EPA 1998EDSP EDSP EPA 1996 EPA EDSTAC EDSTAC 9) Tier 1 Tier 2 2 Tier 1ED Tier 2 Fig. 2 OECD EDSP EDSP 200967 List 1 ED 10) Tier 1EPA Test order Test order Tier 1 Tier 1 Test order2list 1 Tier 1EPA 2015 6 11) List 2 EDSP No more analysis required Tier 1 EPA HPGHPT 2009 Tier 1 11Fig. 3 Tier 1 in vitro in vivo (1) in vitro No No Chemicals Tier 1 Test Does it have the potential to ED? Yes Tier 2 Test Does it have any adverse ED related effects? Yes Risk Assessment Fig. 2 Flow chart for EDSP evaluation (Tier 1 and Tier 2) ER OPPTS TG 890.1250 AROPPTS TG 890.1150 2016
HPG axis HPT axis Estrogen related Androgen related Thyroid hormone related ER binding (890.1250*) AR binding (890.1150*) in vitro assay ER transcriptional activation (890.1300*) Steroidogenesis (890.1550*) Steroidogenesis (890.1550*) Aromatase (890.1200*) Uterotrophic (890.1600*) Hershberger (890.1400*) in vivo assay Fish short-term reproduction (890.1350*) Pubertal male and female (890.1500*, 890.1450*) Amphibian metamorphosis (890.1100*) HPG: Hypothalamus-Pituitary-Gonad HPT: Hypothalamus-Pituitary-Thyroid *: Guideline No. Fig. 3 Overview of the EDSP Tier 1 Tests EROPPTS TG 890.1300 ER ER HeLa herα-hela-9903 OPPTS TG 890.1550 H295R OPPTS TG 890.1200 CYP19 (2) in vivo OPPTS TG 890.1600 HPG 3 Hershberger OPPTS TG 890.1400 HPG 10 OPPTS TG 890.1500 31 2016
OPPTS TG 890.1450 21 OPPTS TG 890.1350 21 OPPTS TG 890.1100 HPT 12) Fig. 4 HPT Tier 1 in vitro in vivo in vitro Tier 1 Tier 1 Tier 1 Tier 1 Tier 2Tier 2 2 OECD TG 416 1 OECD TG 443 OCSPP TG 890.2300 1 OCSPP TG 890.2200 2 OCSPP TG 890.21002 List 1 2009EDSP Tier 1 Test order Control Anti-thyroid agent (PTU) Thyroid hormone Anti-thyroid agent induced thyroid grand hypertrophy, follicular cell hypertrophy and hyperplasia (middle), Thyroid hormone induced thyroid grand atrophy and decreased follicular lumen area (right). Fig. 4 Thyroid histopathology of African clawed frogs (tadpoles) 2016
2010 Other Scientific Relevant Information OSRI Tier 1 ER ER AR Hershberger ED EPA EPA Tier 1 (1) in vitro ER AR ER AR ER 13), 14) in vivo H295R in vivo (2) in vivo 1000 mg/kg Hershberger 1000 mg/kg 23500 1000 mg/kg 31 Fig. 5 15) Testosterone levels (ng/ml) 4 3 2 1 0 Fig. 5 Control * : p<0.01 Serum testosterone levels of the pyriproxyfen administrated male rats in the pubertal assay UDP UGT TSH 16) * 500 mg/kg * 1000 mg/kg 2016
Liver weight per body weight (g%) 10 8 6 4 2 0 Fig. 6 pmol/min/mg S9 protein pmol/min/mg S9 protein 140 120 100 80 60 40 20 0 6000 5000 4000 3000 2000 1000 0 Fig. 7 Control * 500 mg/kg 1000 mg/kg * : p<0.01 Relative liver weight of the pyriproxyfen administrated male rats in the additional study Control Control CYP2B activity ** 500 mg/kg CYP3A activity * 500 mg/kg * ** 1000 mg/kg ** 1000 mg/kg CYP2B CYP3A 17) 19) Fig. 6 Fig. 7 17β-HSD Fig. 8Tier 1 Fig. 9 * : p<0.05 **: p<0.01 Fig. 8 Testicular enzyme activity of the Hepatic enzyme activities of the pyriproxyfen administrated male rats in pyriproxyfen administrated male rats in the additional study the additional study pmol/min/mg testis homogenate 140 120 100 80 60 40 20 0 Control 17β-HSD activity 500 mg/kg 1000 mg/kg AR binding Steroidogenesis Hershberger Liver Secondary effect Increased activity of metabolism enzyme HPG axis Induction of testosterone metabolism Anti-androgenic effect Inhibit testosterone synthesis etc. Direct effect Decreased serum testosterone levels Fig. 9 Decreased serum testosterone levels caused via liver effect 2016
22500 1000 mg/kg 21 2 300 µg/l 60 110 60 8.6 µg/l in vitro in vitro Tier 2 ED Tier 2 2015 EPA Tier 2 EDSP List 167 Tier 152 14 17 18 1 4 13 5 Tier 2 List 1 Tier 1EDSP HP 11) 300 µg/l HPT EDSP Tier 1 EDSP 2011 10000 20) List 1 67 EPA Test order 2009Tier 12015 6 EPA EDSP EDSP21 20) 2016
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