Approach to Endocrine Disruptor Screening Program of Environmental Protection Agency

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1 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) EDSP EPA EDSP EDSP 2015EPA WHO ED 2016

2 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 OECD TG 4073 in vitro OECD 4) 6) ER OECD TG 455 herα-hela

3 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 List 1 ED 10) Tier 1EPA Test order Test order Tier 1 Tier 1 Test order2list 1 Tier 1EPA ) 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 AROPPTS TG

4 HPG axis HPT axis Estrogen related Androgen related Thyroid hormone related ER binding ( *) AR binding ( *) in vitro assay ER transcriptional activation ( *) Steroidogenesis ( *) Steroidogenesis ( *) Aromatase ( *) Uterotrophic ( *) Hershberger ( *) in vivo assay Fish short-term reproduction ( *) Pubertal male and female ( *, *) Amphibian metamorphosis ( *) HPG: Hypothalamus-Pituitary-Gonad HPT: Hypothalamus-Pituitary-Thyroid *: Guideline No. Fig. 3 Overview of the EDSP Tier 1 Tests EROPPTS TG ER ER HeLa herα-hela-9903 OPPTS TG H295R OPPTS TG CYP19 (2) in vivo OPPTS TG HPG 3 Hershberger OPPTS TG HPG 10 OPPTS TG

5 OPPTS TG OPPTS TG OPPTS TG 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 OECD TG 443 OCSPP TG OCSPP TG OCSPP TG List EDSP 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

6 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 mg/kg 31 Fig. 5 15) Testosterone levels (ng/ml) 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

7 Liver weight per body weight (g%) Fig. 6 pmol/min/mg S9 protein pmol/min/mg S9 protein 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 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

8 mg/kg µg/l µg/l in vitro in vitro Tier 2 ED Tier EPA Tier 2 EDSP List 167 Tier Tier 2 List 1 Tier 1EDSP HP 11) 300 µg/l HPT EDSP Tier 1 EDSP ) List 1 67 EPA Test order 2009Tier EPA EDSP EDSP21 20) 2016

9 in vitro HTP Tier 1 HTP Fig. 10 Integrated Bioactivity Exposure Ranking IBER IBER ED HTP EDSP Tier 1 21) HTPEDSP Tier 1 HTPin vitro Fig. 10 Chemicals Tier 1 Test Tier 2 Test Prediction of ED potential using HTP assay Prioritization (short-term objective) Alternatives to Tier 1 Test (long-term objective) Future scheme of the EDSP proposed in the EDSP21 EPA EDSP ED EDSP21 EUED 22) 23) ED HPG HPT ED HPG HPT OMICS 1) WHO, International Programme on Chemical Safety, Global Assessment of the State-of-the-Science of Endocrine Disruptors (2002). 2) T. Colborn, D. Dumanoski and J. P. Myers, Our Stolen Future, Dutton/Penguin Books (1996). 3) U. S. Environmental Protection Agency, Federal Register, Vol. 63, No. 248, Endocrine Disruptor Screening Program; Proposed Statement of Policy (1998). 4) T. Yamada, O. Sunami, T. Kunimatsu, Y. Kamita, Y. Okuno, T. Seki, I. Nakatsuka and M. Matsuo, Toxicology, 162, 103 (2001). 5) T. Yamada, T. Kunimatsu, K. Miyata, S. Yabushita, T. Sukata, S. Kawamura, T. Seki, Y. Okuno and N. Mikami, Toxicol. Sci., 79, 64 (2004). 6) T. Kunimatsu, T. Yamada, K. Miyata, S. Yabushita, T. Seki, Y. Okuno and M. Matsuo, Toxicology, 200, 77 (2004). 7),,, 2012, 51 (2012). 8),,,, 2011-I, 49 (2011). 9) U. S. Environmental Protection Agency, Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) Final Report (1998). 10) U. S. Environmental Protection Agency, Federal Register, Vol. 74, No. 71, Final List of Initial Pesticide Active Ingredients and Pesticide Inert Ingredients to be Screened Under the Federal Food, Drug, and Cosmetic Act (2009). 11) U. S. Environmental Protection Agency, Endocrine Disruptor Screening Program Tier 1 Screening Determinations and Associated Data Evaluation 2016

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