Effects of mixtures of phthalates and other toxicants on sexual differentiation in rats: A risk assessment framework based upon disruption of common developing systems USEPAUSEPA scientistscientist grapplesgrapples withwith difficultdifficult environmentalenvironmental issuesissues L Earl Gray Jr. This presentation does not necessarily reflect USEPA policy, but rather represents the author’s current view on the state of the science Reproductive Toxicology Branch, NHEERL, ORD, USEPA EstrogensEstrogens Developmental MethoxychlorMethoxychlor Reproductive Toxicants EthinylEthinyl EstradiolEstradiol BisphenolBisphenol AA InhibitorsInhibitors ofof fetalfetal FetalFetal GermGerm CellCell ToxicantsToxicants ARAR AntagonistsAntagonists androgenandrogen synthesissynthesis BusulfanBusulfan DiazoDiazo dyesdyes CompeteCompete withwith naturalnatural PreventPrevent thethe synthesissynthesis ofof hormoneshormones TT andand DHTDHT forfor AR,AR, naturalnatural hormoneshormones TT andand SteroidogenesisSteroidogenesis inhibitorsinhibitors prevent AR-DNA binding in prevent AR-DNA binding in DHTDHT andand cancan induceinduce ProchlorazProchloraz vitro,vitro, inhibitinhibit AR-dependentAR-dependent malformations in male malformations in male Linuron genegene expressionexpression inin vivo,vivo, andand reproductivereproductive tracttract andand Linuron maymay induceinduce malformationsmalformations inin delaydelay pubertypuberty inin malemale ratrat KetoconazoleKetoconazole malemale reproductivereproductive tracttract andand FenarimolFenarimol delaydelay pubertypuberty inin malemale ratrat DEHPDEHP VinclozolinVinclozolin DPP,DPP, DCHPDCHP AndrogensAndrogens ProcymidoneProcymidone BBP,BBP, DBP,DBP, DiBPDiBP TestosteroneTestosterone LinuronLinuron DiHP,DiHP, DHP,DHePDHP,DHeP TrenboloneTrenbolone ProchlorazProchloraz DINPDINP p,p'p,p' DDEDDE andand otherother LinuronLinuron DioxinsDioxins andand PCBsPCBs o,p'-o,p'- andand p,p'p,p' DDTDDT ProchlorazProchloraz DioxinDioxin metabolitesmetabolites PCBPCB 169169 congenercongener •In Utero exposure and measure Hormone dependent endpoints male rat offspring as adults •Anogenital distance at birth •Nipple/ areolar numbers in infants •Reproductive Malformations •Undescended testes •Gubernacular abnormalities •Epididymal agenesis •Ventral prostate agenesis •Seminal vesicle agenesis •Vas deferens agenesis •Nipples •Hypospadias •Vaginal pouch •Reproductive Organ Weights •Glans penis •Ventral prostate •Seminal vesicle •Testes •Epididymides •Levator ani bulbocavernosus •Cowper’s glands •Testis and epididymal histopathology F1 male rat offspring: Reproductive endpoints sensitive to lower dosage levels of phthalates in utero. Critical “adverse effects”: % with Phthalate Syndrome and Fetal androgen production. DEHP DR Gray et al 2009 No Hypopadias 100 Testis weight Epididymal weight Seminal vesicle weight 80 Testis/EPI malfs NOT AGD 2 60 % no NIPS 13 Fetal t Production KLH % no PhSyn Percent of control of Percent 40 0 0 0 50 00 5 50 0 1 1 200 2 3 DEHP mg/kg/d PE Dose Response reproductive effects on F1 Male rat offspring indicate that DEHP, DBP, DiBP, BBP and Dihexyl phthalate are equipotent. DPP is more potent and DINP is less potent than this group of PEs TESTIS MALFS OR HISTOPATHOLOGY Testis weights are reduced by PE in utero treatments. Logistic regressin with shared slope 100 DEHP NTP 80 DBP WINE DEHP NTP BBP NAGAO 100 DBP WINE 60 BBP TYL BBP NAGAO BBP TYL DIBP SAILLENFAIT 80 40 DnHexyl P SAILLENFAIT DIBP SAILLENFAIT DnHexyl P SAILLENFAIT 20 BBP ASO DEHP GRAY 60 BBP ASO 0 DBP MYL 1999 DEHP GRAY Percent Affected 10 100 1000 control of Percent 40 DBP MYL 1999 mg/kg/d PE 10 100 1000 mg/kg/d PE Epididymal Weight Reduction Seminal Vesicle Weight Reduction 100 DEHP NTP 110 DEHP NTP BBP NAGAO DBP WINE BBP TYL 100 BBP NAGAO 80 DIBP SAILLENFAIT 90 BBP TYL DnHexyl P SAILLENFAIT DIBP SAILLENFAIT 80 60 BBP ASO DnHexyl P SAILLENFAIT DEHP GRAY 70 BBP ASO DBP MYL 1999 60 DEHP GRAY Percent of Control 40 Affected Percent DBP MYL 1999 10 100 1000 10 100 1000 mg/kg/d PE mg/kg/d PE Testis/EPI malfs data pooled studies Adult male F1 epididymal weight data pooled from several rat studies 100 80 LogEC50 2.617 100 HillSlope 2.572 60 EC50 414.0 80 40 Bottom = 0.0 Top = 100.0 20 LogEC50 2.984 60 HillSlope -1.805 Percent Affected Percent 0 EC50 964.7 0.01 0.1 1 10 100 100010000 Percent of control 40 mg/kg/d PE 10 100 1000 mg/kg/d PE Adult male F1 rat testis weight data Adult male F1 seminal weight data pooled from several rat studies pooled from several rat studies 100 100 80 80 Bottom = 0.0 Bottom = 0.0 Top = 100.0 Top = 100.0 60 LogEC50 2.971 60 LogEC50 3.068 HillSlope -2.438 HillSlope -1.740 EC50 935.1 EC50 1170 Percent control of 40 40 10 100 1000 10 100 1000 mg/kg/d PE mg/kg/d PE Fetal endocrine changes demonstrate the relative potency (RP) of individual PEs is consistent with their RP to induce reproductive tract alterations seen in postnatal life. Thus, changes in fetal androgen levels can be used to “screen” phthalates for their potential to induce malformations and possibly as a “critical effect” in individual and cumulative risk assessments. Fetal Phthalate screen (FPS) Dose dam with PE at a single high dosage group Gestational exposure during critical period of sexual differentiation On Gestational Day 18 necropsy dam Measure testis fetal testis Testosterone (T) production Testis gene expression STAR Insl3 Cyp11a For “Positives” Run dose response studies Use potency factors to execute mixture studies For “important” PEs with data gaps run a postnatal study FPSFPS ResultsResults toto datedate Positives Negatives DINP – both CAS #s * Diethyl phthalate Diheptyl phthalate Dioctyl terephthalate Diisoheptyl phthalate Hexamoll DINCH Dihexyl phthalate Di-2-ethylhexyl Dibutyl phthalate * tetrabromo phthalate Diisobutyl phthalate * Benzylbutyl phthalate Future studies Dicyclohexyl phthalate DMP, Diproply P, Dipentyl phthalate * DIDP, DPHP, others * FPS DOSE RESPONSE STUDIES ONGOING Fetal T production is more sensitive to Phthlate ester (PE) disruption than are testis genes of postnatal effects (epididymal malformations) and T prod is less variable resulting in lower NOELs and BMDs. Below is a generic example. PE IN UTERO EFFECTS ON FETAL AND POSTNATAL MALE RATS 120 100 Endpoint ED50 80 T Prod 231 T Production Extracted Testis T 275 60 Extracted Testis T STAR mRNA 280 40 STAR mRNA insl3 mRNA 372 Insl3 mRNA CYP 11a 431 20 CYP11a Epididymal agenesis 567 NORMAL EPI Percent of control of Percent 0 10 100 1000 In utero mixture studies Summary Similar cellular and molecular mechanisms of action Binary mixtures – pairs of chemicals Mixture of 5 phthalates Diverse cellular and molecular mechanisms of action Binary mixtures – pairs of chemicals Mixture of 7 chemicals including pesticides and phthalates Mixture of 10 chemicals Initial Step in a Cumulative Risk Assessment as outlined by the USEPA and other regulator agencies. Identification of a group of chemicals to be included in a Common Mechanism Group chemicals that induce a common toxic effect by a common mechanism of toxicity. Key Question: Should chemicals that disrupt differentiation of the same reproductive tissue but by different molecular mechanisms of action in different tissues be included in a Common Mechanism Group? The default answer has been: NO because such a mixture would not be expected to produce adverse effects if each chemical is administered below the NOAEL Response addition (RA) 0 + 0……+ 0 = No Adverse Effects However if this assumption is incorrect Dose addition (DA) 0 + 0 +…….+ 0 = 100% Malformations ObjectivesObjectives ofof ourour researchresearch Determine how chemicals with similar and dissimilar mechanisms of toxicity interact during sexual differentiation To provide a framework for deciding what chemicals to include in a cumulative risk assessment Working Hypothesis: Chemicals that disrupt the development of a common reproductive tissue/system during sexual differentiation will produce dose additive responses, regardless of the molecular mechanism or the signaling pathway that is disrupted Cumulative effects: Common mechanisms of toxicity AR antagonist mixture study 100 •Vinclozolin and Procymidone 95.8 •We90 nowHypospadias: know that – both 10+0=96% fungicides 80 Vaginal Pouch: 0+0=54% •Bind AR and antagonize androgen action in vitro 70 Hypospadias Vaginal Pouch 60•Inhibit androgen-stimulated tissue growth in vivo 54.2 50•Induce a common phenotype of male rat 40reproductive tract malformations 30 •Alter the same array of genes in the reproductive 20tract 10.3 10 0 0000 Percent of males aaffected 0 Control Vinclozolin Procymidone Combination Cumulative effects of Phthalates A Common Mechanism of Toxicity: Altered fetal Leydig cell differentiation and reduced hormone synthesis DBP plus BBP mixture study. PhthalatesHypospadias: with a0+0=50% common Epididymalmetabolite agenesis: 9+23=100% Hypospadias DBP plus DEHP mixture study 100 100 100 Epididymal Agenesis phthalates with no common Gubernacular agenesis 80 metabolite (Howdeshell et al 2007) 60 50 100 90 40 80 Hypospadias 75 23 Epididymal Agenesis 20 70 65 Gubernacular agenesis Percent of males aaffected 9 7.7 60 0 0000 00 50 Control BBP DBP Combination 40 30 26 27 25 20 Percent of males aaffected 10 3 3 4.7 Hypospadias: 3+0=25% 0 0 0 0 0 Epididymal agenesis: 26+5=65% Control DEHP DBP Combination Common mechanism of Toxicity Effects of a mixture of five phthalates on fetal testosterone production in the rat (Howdeshell et al, 2008) Individual studies DBP, DiBP,