The Endocrine Disruptor Screening Program What Can Screening

The Endocrine Disruptor Screening Program: What Can Screening Results Tell Us About Potential Adverse Endocrine Effects? Assessing Cumulative Effects of Endocrine Active Substances Robert Golden, Ph. D Tox. Logic LLC Potomac, MD September 10, 2009

Topics in Presentation • An approach for assessing the cumulative effects of endocrine active substances – Emphasis on weakly estrogenic endocrine disruptors (WEEDs) – Would likely apply to other receptor-mediated responses • Use of human DES data to calibrate risk assessment for WEEDs • Other endocrine-related issues Tox. Logic 2

Endocrine Disruptor Screening Program (EDSP) • ED a new health endpoint of potential concern – Contrast with cancer & non cancer effects with established regulatory paradigm for each • How will EPA assess & communicate results to the public? • Are current Rf. Ds & Rf. Cs “protective” for ED effects? • A few publications broadly discuss some approaches • No cogent strategy for risk assessment and/or assessing relevance to humans from rodent data • Need for consideration of biological plausibility Tox. Logic 3

Cumulative Effects of Endocrine Active Substances • Focus on estrogen-mediated effects – Concepts similar for other receptor-mediated effects • Model on TEF/TEQ approach for Ah. R agonists, e. g. , TCDD, PCB 126 and less potent congeners – Exposure to most potent member of class, i. e. , TCDD; basis for clear health concern – For weakly estrogenic endocrine disruptors (WEEDs) no exogenous exposure to most potent members of class, e. g. , estrogen; concern? Tox. Logic 4

Cumulative Effects of Endocrine Active Substances • Regulatory framework established to assess cumulative exposure/risk to Ah. R agonists of vastly differing potency (i. e. , TEFs) – 1. 0, 0. 5, 0. 1, 0. 003, 0. 001, 0. 0003, 0. 0001 • Considerations of receptor biology assumed similar for Ah. R- and ER-mediated effects – Demise of “holy grail” of synergism? – Additivity at receptor based on potency x exposure (dose) – TEQ value of concern = ? ? (i. e. , 2, 5, 10, 15) Tox. Logic 5

Proposed Regulatory Scheme for WEEDs Follow same guidelines/rules as for TEF/TEQ Ligands for ER (ERα & ERβ)? Base on in vitro or in vivo data? Full D-R curves for each WEED and reference compound (e. g. , E 2 and/or DES)? • Order of magnitude values vs. actual potency? • • Tox. Logic 6

Proposed Regulatory Scheme for WEEDs • Relevant end point affected by both studied and reference compound • TEQ = ΣTEFs (i. e. , potency) of individual WEEDs X concentration (or dose) in mixture(s) • Consider non-additivity, i. e. , < additivity based on antagonism (anti-estrogenicity of TCDD)? • Application to all assayed WEEDs? – Collectively (i. e. , running total), body burdens? chemical structure/class? Other approaches? Tox. Logic 7

Comparative Potency of Xenoestrogens in Rodent Uterotropic Assay Substance Effective dose (mg/kg/day) Relative potency EE=1, 000 DES 0. 0001 3, 000 Ethinylestradiol (EE) 0. 0003 1, 000 Estrone 0. 0012 250, 000 Coumestrol 0. 24 1250 Genistein 8 37 Daidzein 11 27 Isobutylparaben 73 4 Butylparaben 600 0. 5 Benzylparaben 2500 0. 12 Tox. Logic 8

Hypothetical Illustration of TEF/TEQ Approach for WEEDs in Drinking Water Chemical MCL (mg/l) Hypothetical Potency (TEF) Intake TEQ Dioxin 0. 00000003 0. 000004 0. 000012 Lindane 0. 002 0. 0038 0. 00000152 Methoxychlor 0. 04 0. 000016 0. 00000128 Atrazine 0. 003 0. 00005 0. 0000003 PCBs 0. 005 0. 0000018 0. 00000018 Toxaphene 0. 003 0. 008 0. 000048 Simazine 0. 1 0. 00002 Endrin 0. 002 0. 0009 0. 000036 Chlordane 0. 002 0. 00001 0. 000004 Heptaclor 0. 0004 0. 0003 0. 000024 B(a)P 0. 0002 0. 0006 0. 0000024 Total 0. 00014 Tox. Logic 9

Hypothetical Illustration of TEF/TEQ Approach for WEEDs in Drinking Water Chemical MCL (mg/l) Hypothetical Potency (TEF) Intake TEQ Dioxin 0. 00000003 0. 000004 0. 000012 Lindane 0. 002 0. 0038 0. 00000152 Methoxychlor 0. 04 0. 000016 0. 00000128 Atrazine 0. 003 0. 00005 0. 0000003 PCBs 0. 005 0. 0000018 0. 00000018 Toxaphene 0. 003 0. 008 0. 000048 Simazine 0. 1 0. 06 0. 012 Endrin 0. 002 0. 0009 0. 000036 Chlordane 0. 002 0. 00001 0. 000004 Heptaclor 0. 0004 0. 0003 0. 000024 B(a)P 0. 0002 0. 0006 0. 0000024 Total 0. 013 Tox. Logic 10

Implications of Dioxin TEF/TEQ Analogy • TEF/TEQ scheme an exposure/potency bookkeeping system • For dioxin, exposures to TCDD, the most potent member of class (= legitimate concern) • For WEEDs, exposure to least potent members of class (= ? concern) • Potency-wise, WEEDs exact opposite of dioxin • Need to consider potency differences with DES & E 2 Tox. Logic 11

“New” Concepts From NAS Which May Complicate Risk Assessment for WEEDs • Cumulative risk assessment of similarly acting compounds (ER, AR & TH? ) • Additivity to background exposures or disease processes (endogenous E 2, androgen or TH? ) • Lack of population thresholds for cancer & non-cancer effects • Overall effects on risk assessment unknown or uncertain Tox. Logic 12

Other Considerations • BPA a clarion call - Nightmarish disaster with irreproduceable results driving debate • Risk-no risk scenario - Baby bottle (BPA = bad) - Soy-based formula (isoflavones = good) • Critical need to consider potency • Dioxin TEF/TEQ scheme lacking one key element…. NO CALIBRATION WITH HUMAN DATA • Need to calibrate WEEDs risk assessment/communication with human data (i. e. , DES and/or estrogen) where possible Tox. Logic 13

EROD Humans Less Sensitive to TCDD and PCBs Concentration (Log M) Ah. R- mediated CYP 1 A enzyme induction assay: • If species equally sensitive, all lines should overlap • Human less sensitive to TCDD & even less sensitive to PCB • PCB 126 TEF = 0. 1; based on human data TEF = 0. 0005 Silkworth et al. , Tox. Sci. , 2005 14

Background • Arnold et al. (1996) Synergistic activation of ER with binary combinations of environmental chemicals. – endosulfan, dieldrin, toxaphene & chlordane with estrogenic potency 160 -1600 X > than additivity • Ramamoorthy et al. (1997 a, b) & Ashby et al. (1997) confirmed additivity only; no synergism observed • Mc. Lachlan (1997) Synergistic effect of environmental estrogen: Report withdrawn. Tox. Logic 15

Background • Fisch et al. (2009) study of hypospadias incidence in NY State; no increase from 1992 -2005 • Results suggest that testicular dysgenesis syndrome (i. e. , decreased sperm count, infertility, hypospadias, testicular cancer) may not be a problem in humans, contrary to earlier concerns. • Palmer et al. (2005); no increased risk of hypospadias in sons of women exposed to DES in utero Tox. Logic 16

Background • Endocrine Society Scientific Statement on endocrine disrupting chemicals (2009) • “Content is evidence-based to the extent possible” • “Key to understanding the mechanisms of action and consequences of exposure… include age at exposure, latency from exposure, the mixture of chemicals, dose-response dynamics, and long-term latent effects. ” • “Potency” only used once in 50 page document with 485 references! (is “potency “ evidence? ) Tox. Logic 17

“Estrogen Hypothesis” • Sharpe (1993) proposed the “estrogen hypothesis” for testicular dysgenesis syndrome • Male reproductive-tract disorders (e. g. , testicular cancer, cryptorchidism, hypospadias & ↓sperm counts) resulted from in utero or neonatal exposure to a variety of weakly estrogenic compounds from the environment • In 2003 Sharpe offered a candid reassessment of the hypothesis • Sharpe (2003) not cited in Endocrine Society scientific statement Tox. Logic 18

Reassessment of Estrogen Hypothesis • “…reasonably clear…that all of the identified “environmental estrogens” possess weak or very weak intrinsic estrogenic activity when measured by conventional in vitro and in vivo assays for estrogenicity. . . By comparison with the potency of DES, for which there [are] both human and rodent data on incidence of male reproductive developmental disorders following in utero exposure…unlikely that any of the identified environmental compounds could [cause any listed effects]…Based on estrogenic potency, human exposure to the most potent environmental estrogens would need to be at least 1000 -fold higher than this level for adverse effects relevant to the human male to be induced, and such levels of exposure are remote. ” [emphasis added] Tox. Logic 19

Implications of Estrogen Hypothesis • The identical logic would also pertain to other potential in utero/neonatal effects reported and/or alleged for weakly estrogenic compounds and for which there are human data on DES including: – – – Breast and testicular cancer Female reproductive tract abnormalities Effects on brain and behavior Effects on mammary and prostate gland Effects on puberty Effects on immune system Tox. Logic 20

Diethylstilbestrol (DES) General Information • DES ranges from slightly less to several times more potent than 17 -estradiol (E 2) depending on assay • 100 s– 1000 s X more potent than most weakly estrogenic naturally occurring and/or environmental chemicals • Effects in animals & humans following in utero exposure show dose-response & apparent no-effect maternal intake doses for some endpoints • DES a worst case scenario, e. g. , E 2 typically bound to sex hormone binding globulin (SHGB) which decreases free E 2 available to fetus Tox. Logic 21

Use of DES in Humans • Widely prescribed to 4 -5, 000 pregnant women until 1972 in mistaken belief that it would prevent miscarriage • Large numbers of males & females exposed in utero to widely differing dosing protocols • Use discontinued in 1972 with discovery that small number of women developed vaginal adenocarcinoma • 100 s of clinical studies on DES-exposed men and women • Ongoing investigations of DES-exposed cohorts; documentation/verification of exposure a critical element • Most exposures during 1 st trimester during critical window of sensitivity Tox. Logic 22

Human Dosing With DES • During peak years of use, in different clinical centers, dosing regimens varied widely • In utero exposure to wide range of DES doses Institution Estimated Mean Total Maternal Dose (g) Mayo Clinic Stanford Univ. Boston Univ. DES Efficacy trial Univ. Chicago British Medical Res. Council 1. 4 3. 5 6. 4 10+ 12 18 Tox. Logic 23

Endpoints Assessed in Follow-Up Studies of DES -Exposed Cohorts • • • Breast, testicular & prostate cancer Male & female reproductive tract abnormalities Male & female fertility Effects on brain & behavior (including sexual orientation) Age of puberty & menopause Immune system Thyroid effects (obesity? ) Endometriosis Epigenetic effects (i. e. , offspring of offspring) Tox. Logic 24

Effects of DES in Animals • DES extensively studied in animals – Numerous effects on offspring following in utero exposure – D-R relationships well characterized • Spectrum of effects; mainly on reproductive tract Effects in Mice Following in utero Exposure to DES Effects in males Sperm abnormalities Decrease sperm counts Epididymal cysts Cryptorchid testes Microphallus Hypospadias Testicular tumors Prostatic tumors Infertility Immune dysfunction Effects in females Ovarian cysts Ovarian tumors Structural abnor. uterus Malformations cervical canal Vaginal adenocarcinoma Vaginal adenosis Salpingitis of oviduct Uterine tumors Infertility Immune dysfunction 25

Extrapolation from Animals to Humans • Experimental mouse model qualitatively predicts/confirms adverse effects in humans from in utero exposure to DES • Quantitative aspects of extrapolating from rodents to humans less certain • Qualitative & quantitative species differences between rodents & humans for hormonally-mediated effects • Animals appear more sensitive to DES-induced effects – – Vaginal adenosis Cryptorchidism Epidymal cysts Impaired fertility Tox. Logic 26

Adverse Effects of DES on Male & Female Reproductive Tract • University of Chicago; total mean maternal DES dose ≈ 12, 000 mg • Males – Decreased sperm counts & penis size, cryptorchidism – No decrease in fertility • Females – Endometriosis, reproductive tract malformations, infertility, vaginal adenocarcinoma Tox. Logic 27

Use of DES Data for Hypothetical Potency Comparisons with Weakly Estrogenic Chemicals • Mayo Clinic: total mean maternal DES dose ≈ 1430 mg; median exposure = 101 days; no association with male reproductive tract effects ≈ 14 mg/day or 0. 3 mg/kg/day for 50 kg women • ≈ 0. 3 mg/kg/day may represent ≈ NOAEL for in utero DES effects on male reproductive tract • Need to account for NOAEL of test compound (e. g. , male repro. tract) & potency compared to reference compound, i. e. , estradiol and/or DES • Need to compare with actual intake to assess MOE Tox. Logic 28

Hypothetical Potency Comparison of a Weakly Estrogenic Chemical with DES • Butylparaben effect level for male repro tract = 600 mg/kg/d (600 times > DES repro tract effect level of 1. 0 mg/kg/day) • Paraben estrogenic potencies ≈ 6000 to 3, 300, 000 times less than estradiol • Assume estradiol & DES equipotent & butylparaben 6000 -fold below DES LOAEL for male repro tract effects (1 mg/kg/day) • Maxium butylparaben dermal dose = 0. 12 -0. 41 mg/kg/day • Daily intake by pregnant women ≈ 15, 000 to 50, 000 -fold less than equivalent DES LOAEL for repro tract effects from in utero exposure Tox. Logic 29

DES Data for Risk Assessment Calibration • Extensive 1998 review of DES database; coauthors • Maternal/in utero E-R data & some ≈NOAELs • Inform risk decisions based on rodent data • Mouse model provides basis for interspecies dose/potency comparisons • Human & animal DES data (plus substantial database on BC estrogen) permits dose/potency comparisons for WEEDs Tox. Logic 30

DES Data for Risk Assessment Calibration • Basing exposure/risk conclusions on comparative potency is biologically plausible • Only way to bring human data into risk assessment process. • If important for public health, how can potential risk decisions be made without “grounding” with human data? • If “good science” is a driver DES & other relevant human data must be considered Tox. Logic 31

Other Issues to Consider: Thyroid Effects • EDSP screening for potential effects on thyroid hormone system • Substantial species differences between rodents & humans – Thyroxine-binding globulin – T 4 Half-life – T 3 Half-life – T 4 Production rate – TSH Tox. Logic 32

Rodent-Human Differences in Thyroid Effects • Rodent thyroid effects of questionable relevance in humans • Many drugs with adverse effects on rodent thyroid function but none in humans – – – – – Phenobarbital Spironolactone Doxylamine succinate Oxazepam Clofibrate Griseofulvin Nicarpidine Sulfonamides Resorcinol Lithium salts Tox. Logic 33