Complex Mixtures of Anti-Androgens at Concentrations Below Individual Chemical Effect Levels Produces Reproductive Tract Malformations in the Male Rat

Home , Linuron

Complex mixtures of anti-androgens at concentrations below individual chemical effect levels produces reproductive tract malformations in the male rat

Justin Conley ORD/NHEERL/TAD/RTB

SOT RASS-MixSS Webinar September 13, 2017 Disclosure/Disclaimer

No conflicts of interest to disclose

This presentation does not necessarily reflect the views or policy of USEPA Declines in male reproductive health

Skakkebaeck et al. 2016 Exposures are mixtures

Examples of multiple residue detections in CDC-NHANES

17%

Pumarega et al. 2016 Qian et al. 2015 2003/2004 Cohort 2007/2008 Cohort n = 4,793 n = 2,604 OCs, PCBs, PBDEs, PCDDs/Fs, PFCs DEHP, DiBP, DBP, DINP, BBP, DIDP Chemical mixtures research and risk assessment in federal laws

Food Quality Protection Act Safe Drinking Water Act How do we group chemicals for cumulative risk assessment? Anti-androgen AOP

Reproductive tract malformations Androgen Reduced AR receptor dependent mRNA/protein antagonism synthesis Abnormal cell Reduced apoptosis/ androgen proliferation dependent tissue weights

Reduced sperm Infertility production

Suppressed development of Undescended Reproductive gubernacular testes tract cancers cords

Molecular Adverse Adverse Key Events Key Events Initiating Events Outcomes Outcomes “Anti-androgen” AOP network

5α-reductase Reduced DHT inhibition synthesis Reproductive tract malformations Androgen Reduced AR receptor dependent mRNA/protein antagonism synthesis Abnormal cell Reduced apoptosis/ androgen proliferation dependent Inhibition of tissue weights CYP450 Reduced steroidogenic testosterone synthesis enzymes Reduced sperm Infertility production

Inhibition of Reduced HMG-CoA cholesterol reductase synthesis

Suppressed Unknown MIE development of Undescended Reproductive gubernacular testes tract cancers cords

Molecular Adverse Adverse Key Events Key Events Initiating Events Outcomes Outcomes Hypothesis

Complex mixtures of many chemicals with multiple “anti- androgenic” molecular initiating events act additively at low doses to disrupt male reproductive tract development

“Low”: below individual chemical effect levels (LOAEL or NOAEL) Linuron Prochloraz Vinclozolin Procymidone Pyrifluquinazon p,p’-DDE 330-55-2 67747-09-5 50471-44-8 32809-16-8 337458-27-2 72-55-9

Ultramix

Simvastatin Flutamide Finasteride 79902-63-9 13311-84-7 98319-26-7

diethylhexyl phthalate dibutyl phthalate benzyl butyl phthalate diisobutyl phthalate 117-81-7 84-74-2 85-68-7 84-69-5

diisoheptyl phthalate 71888-89-6

dicyclohexyl phthalate dihexyl phthalate diheptyl phthalate dipentyl phthalate 84-61-7 84-75-3 3648-21-3 131-18-0 chemspider.com “Anti-androgen” AOP network

Finasteride 5α-reductase Reduced DHT inhibition synthesis Reproductive tract malformations p’p’-DDE Androgen Reduced AR Pyrifluquinazon receptor dependent mRNA/protein antagonism Procymidone synthesis Vinclozolin Abnormal cell Reduced Flutamide apoptosis/ androgen proliferation dependent Inhibition of tissue weights Prochloraz P450 Reduced Linuron steroidogenic testosterone synthesis enzymes Reduced sperm Infertility production

Inhibition of Reduced Simvastatin HMG-CoA cholesterol reductase synthesis

Suppressed Unknown MIE development of Undescended Reproductive Phthalates gubernacular testes tract cancers cords

Molecular Adverse Adverse Key Events Key Events Initiating Events Outcomes Outcomes Individual chemical effect levels LOAEL Chemical (mg/kg/d) Effect Reference Finasteride 0.03 AGD red., Nipple ret. Clark 1993 Flutamide 2.0 Hypospadias Welsh 2009 Vinclozolin 6.0 VP wt., AGD red., Nipple ret. Gray 1999; Hellwig 2000 Diethylhexyl phth. 11.0 Various malf. Gray 2009; Blystone 2010 Linuron 12.5 Testis & epididymal malf. McIntyre 2000 Procymidone 25.0 AGD red., Nipple ret., Various malf., Ostby 1999 Pyrifluquinazon 25.0 Various malf. Unpublished Prochloraz 31.3 Testis malf., T prod. Blystone 2007; Noriega 2005 Dipentyl phth. 33.0 T prod. Hannas 2011; Furr 2014 Simvastatin 62.5 Testis malf., T prod. Beverly 2014 Dibutyl phth. 100.0 Nipple ret. Mylchreest 2000 p,p’-DDE 100.0 AGD red., Nipple ret., PPS delay Kelce 1995 Diisoheptyl phth. 227.0 Red. sperm count McKee 2006 Dicyclohexyl phth. 250.0 AGD red. Saillenfait 2009 Benzylbutyl phth. 250.0 AGD red. Tyl 2004 Diisobutyl phth. 250.0 AGD red. Saillenfait 2008 Dihexyl phth. 250.0 AGD red.; Nipple ret. Saillenfait 2009 Diheptyl phth. 500.0 AGD red. Saillenfait 2011 1/5 1/10 1/20 1/40 1/80 LOAEL 100% 50% 25% 12.5% 6.3% Chemical (mg/kg/d) (mg/kg/d) (mg/kg/d) (mg/kg/d) (mg/kg/d) (mg/kg/d) Finasteride 0.03 0.006 0.003 0.001 0.0007 0.0003 Flutamide 2.0 0.5 0.2 0.1 0.06 0.03 Vinclozolin 6.0 1.2 0.6 0.3 0.1 0.07 Diethylhexyl phth. 11.0 2.2 1.1 0.5 0.2 0.1 Linuron 12.5 2.5 1.2 0.6 0.3 0.1 Procymidone 25.0 5.0 2.5 1.2 0.6 0.3 Pyrifluquinazon 25.0 5.0 2.5 1.2 0.6 0.3 Prochloraz 31.3 7.5 3.7 1.8 0.9 0.4 Dipentyl phth. 33.0 6.6 3.3 1.6 0.8 0.4 Simvastatin 62.5 12.5 6.2 3.1 1.5 0.7 Dibutyl phth. 100.0 20.0 10.0 5.0 2.5 1.2 p,p’-DDE 100.0 20.0 10.0 5.0 2.5 1.2 Diisoheptyl phth. 227.0 45.4 22.7 11.4 5.6 2.8 Dicyclohexyl phth. 250.0 50.0 25.0 12.5 6.2 3.1 Benzylbutyl phth. 250.0 50.0 25.0 12.5 6.2 3.1 Diisobutyl phth. 250.0 50.0 25.0 12.5 6.2 3.1 Dihexyl phth. 250.0 50.0 25.0 12.5 6.2 3.1 Diheptyl phth. 500.0 100.0 50.0 25.0 12.5 6.2 Postnatal reproductive development

GD0 GD2 GD14 GD18 GD22 PND23

F Necropsy F0 DOSING 0 Masculinizing window PND2 PND13 PND34 PND90

F1 Necropsy

F1 VO PND41 PND120

F1 Necropsy

AGD NR PPS

-Charles River Sprague-Dawley AGD = anogenital distance -Oral gavage administration NR = nipple retention -5 dams/litters per dose group VO = vaginal opening -Corn oil negative control PPS = preputial separation # nipples per m ale AGD (mm) 10 12 1 2 3 4 5 0 2 4 6 8

Control Control Earlyendpoints postnatal

LO AEL/80 LO AEL/80 * LO AEL/40 LO AEL/40 ** ** LO AEL/20 LO AEL/20 **

L O A E L /1 0 LO AEL/10 **** ****

L O A E L /5 L O A E L /5 Adult reproductive tissue weights

1000 160

** ** 5 ** 800 140

**** ** ** 600 120 4

400 100

200 80 P a ire d te s tis w t (g ) G lans penis wt (m wt g) penis G lans Ventral prostate w t (m w g) t prostate Ventral

p=0.063 p=0.055 p=0.061 0 60 2 Control Control Control L O A E L / 5 L O A E L / 5 L O A E L / 5 LO AEL/80 LO AEL/40 LO AEL/20 L O A E L / 1 0 LO AEL/80 LO AEL/40 LO AEL/20 L O A E L / 1 0 LO AEL/80 LO AEL/40 LO AEL/20 L O A E L / 1 0

1800 2 .8 * ** ***

2 .0 1600 ** 2 .4 *** ** *** **** ** * 1400

2 .0 **** 1 .5

1200

1 .6

1000 LABC wt (g) wt LABC 1 .0

1 .2 800 Paired epididym is w t w(m g) t is epididym Paired p=0.052 p=0.064 p=0.061 p=0.060 Paired sem inal vesicle (g) w t vesicle sem inal Paired 600 0 .8 0 .5 Control Control Control L O A E L / 5 L O A E L / 5 L O A E L / 5 LO AEL/80 LO AEL/40 LO AEL/20 L O A E L / 1 0 LO AEL/80 LO AEL/40 LO AEL/20 L O A E L / 1 0 LO AEL/80 LO AEL/40 LO AEL/20 L O A E L / 1 0 Ultramix Summary

• Cumulative effects of many “anti-androgens”

• Effects appeared to conform to dose addition

• Adverse reproductive tract effects occurred low doses (LOAEL/80)

• Concerns:

• No doses in ultra low dose range (non-monotonic effects)

• Very high potency of drugs – finasteride/flutamide

• Additional questions:

• Accuracy of mixture models (Dose Addition vs. Response Addition)

• Benchmark Dose Modeling Supermix

Linuron Prochloraz Vinclozolin Procymidone Pyrifluquinazon p,p’-DDE 330-55-2 67747-09-5 50471-44-8 32809-16-8 337458-27-2 72-55-9

diethylhexyl phthalate dibutyl phthalate benzyl butyl phthalate diisobutyl phthalate 117-81-7 84-74-2 85-68-7 84-69-5

diisoheptyl phthalate 71888-89-6

dicyclohexyl phthalate dihexyl phthalate diheptyl phthalate dipentyl phthalate 84-61-7 84-75-3 3648-21-3 131-18-0 chemspider.com Supermix dosing

Dose [Chemical] 100% Dose NOAELx2

50% Dose NOAEL

25% Dose NOAEL/2

12.5% Dose NOAEL/4

6.25% Dose NOAEL/8

3.3% Dose NOAEL/15

0.5% Dose NOAEL/100

0.05% Dose NOAEL/1000 AGD (mm) 1 2 3 4 5

C o n tro l

N O A E L/1000

NOAEL/100

NOAEL/15 Earlyendpoints postnatal

NOAEL/8

NOAEL/4 ****

NOAEL/2 ****

NOAEL ****

N O A E L x 2

# nipples per m ale 10 12 0 2 4 6 8

Control

N O A E L/1000

N O A E L /1 0 0

N O A E L /1 5

N O A E L /8 ****

NOAEL/4 ****

N O A E L / 2 ****

NOAEL ****

N O A E L x 2 Adult reproductive tissue weights

1200 200 5

1000 4 **** 150

800

600 100 ****

**** 2 400

P a ire d te s tis w1 t (g ) 200 (m wt g) penis G lans Ventral prostate w t (m w g) t prostate Ventral **** N /A N /A 0 0 0 NOAEL NOAEL NOAEL Control Control Control NOAEL/8 N O A E L / 4 N O A E L / 2 NOAEL/8 NOAEL/4 N O A E L / 2 NOAEL/8 NOAEL/4 N O A E L / 2 2 x N O A E L 2 x N O A E L 2 x N O A E L NOAEL/15 NOAEL/15 NOAEL/15 NOAEL/100 NOAEL/100 NOAEL/100 N O A EL/1000 N O A EL/1000 N O A EL/1000

2 .8 2 .0 1600

2 .4 1400 ****

1 .5 1200 **** 2 .0

1000 1 .6 **** **** 1 .0 800 1 .2

600

LABC wt (g) wt LABC **** 0 .8 400 0 .5

0 .4 200 ardeiiy us(m w g) epididym t Paired **** **** Paired sem inal vesicle (g) w t vesicle sem inal Paired 0 0 .0 0 .0 NOAEL NOAEL NOAEL Control Control Control NOAEL/8 NOAEL/4 N O A E L / 2 NOAEL/8 N O A E L / 4 N O A E L / 2 NOAEL/8 NOAEL/4 N O A E L / 2 2 x N O A E L 2 x N O A E L 2 x N O A E L NOAEL/15 NOAEL/15 NOAEL/15 NOAEL/100 NOAEL/100 NOAEL/100 N O A EL/1000 N O A EL/1000 N O A EL/1000 Adult external malformations

**** **** **** **** **** **** 100 100

80 80

60 60

40 40

20 20 %nipples m with ales H litter) (% ypospadias of

0 0 NOAEL NOAEL C o n tro l Control N O A E L /8 NOAEL/4 N O A E L / 2 N O A E L /8 NOAEL/4 N O A E L / 2 N O A E L x 2 N O A E L x 2 NOAEL/15 N O A E L /1 5 N O A E L /1 0 0 N O A E L /1 0 0 N O A E L/1000 N O A E L/1000 Adult internal malformations

Supermix 50% and 100% doses Control (Chemicals at NOAEL and 2xNOAEL)

Seminal vesicles

Vas deferens

Testes

*Male sex accessory tissues absent in: 95% of males at 100% dose Epididymis Ventral prostate 54% of males at 50% dose Mixture models of AGD

120

100

40 AG control) D (% of 20

0 0.01 0 .1 1 10 100

T re a tm e n t (% o f to p d o s e )

Superm ix male observed

ED50: 32.6-44.2% (95% C I)

Dose addition m odel

ED50: 41.3%

Response addition m odel

ED50: 52.4% Mixture models of Hypospadias

100 100

80 80

60 60

40 40

20 20 H litter) (% ypospadias of H litter) (% ypospadias of 0 0

0 20 40 60 80 100 0 0.01 0 .1 1 10 100

T re a tm e n t (% o f to p d o s e ) T re a tm e n t (% o f to p d o s e )

Superm ix male observed

ED50: 22.8-25.9% (95% C I)

Dose addition m odel

ED50: 38.4%

Response addition m odel

ED50: 624.8% Benchmark Dose estimation

120 BMDL BMD

100

40 AG control) D (% of 20

0 0.01 0 .1 1 10 100

T re a tm e n t (% o f to p d o s e )

BMD90 = 8.9 %top

BMDL90 = 6.2 %top ≈ NOAEL/8 Summary

• Chemicals with multiple “anti-androgenic” molecular initiating events act cumulatively to disrupt male reproductive tract development in the male rat

• Effects occur when component chemicals are at low individual doses • Significant reductions in tissue weights in Ultramix at LOAEL/80 • Significant reductions in fetal T production in Supermix at NOAEL/8

• Utility of AOPs for grouping chemicals that affect similar biological pathway • Grouping based on common MIE is too restrictive • Overlap of critical KEs can identify chemicals for cumulative risk assessment

• Dose addition model more accurate than Response addition model

• Some of the most complex developmental studies on reproductive toxicants to date • Very few researchers/laboratories are studying in utero exposure to mixtures

• Exposure to environmental chemicals that disrupt androgen signaling may be contributing to adverse male reproductive human health effects at doses lower than previous estimated Research Team

Earl Vickie Phill Gray Wilson Hartig

Mary Christy Nicki Elizabeth Cardon Lambright Evans Medlock-Kakaley

Hunter Johnathan Sampson* Furr* *no longer at USEPA Questions? Image References

Slide 1 & 14: Sprague Dawley rat image from http://www.criver.com/products-services/basic-research/find-a-model/cd-igs-rat?loc=US

Slide 3: Skaekkebaek NE, Rajpert-De Meyts E, Louis GMB, Toppari J, Andersson AM, Eisenberg ML, Jensen TK, Jorgensen N, Swann SH, Sapra KJ, Ziebe S., Priskorn L, Juul, A. (2016) Male reproductive disorders and fertility trends: Influences of environment and genetic susceptibility. Physiol Rev 96: 55-97

Slide 4: Pumarega J, Gasull M, Lee DH, Lopez T, Porta M. (2016) Number of persistent organic pollutants detected at high concentrations in blood samples of the United States population. PLoS One 11(8):e0160432

Slide 4: Qian H, Chen M, Kransler KM, Zaleski RT (2015) Assessment of chemical coexposure patterns based upon phthalate biomonitoring data within the 2007/2008 National Health and Nutrition Examination Survey. J Exposure Sci Environ Epidemiol 25: 249-255

Slide 22: Noriega NC, Ostby J, Lambright C, Wilson VS, Gray Jr LE. (2005) Late gestational exposure to the fungicide prochloraz delays the onset of parturition and causes reproductive tract malformations in male but not female rat offspring. Biol of Reproduction 72:1324-1335

Slide 29: Sprague Dawley rat image from http://www.criver.com/products-services/basic-research/find-a-model/sprague-dawley-rat?loc=US