RASS SOT Webinar

Are Nonmonotonic Dose Response Curves

(NMDRCs) Common after Estrogen or

Androgen Signaling Pathway Disruption:

Fact or Falderal?

L. Earl Gray Jr., PhD

This presentation does not necessarily reflect USEPA policy, but rather

represents the author’s current view on the state of the science

USEPA scientist USEPA scientist

grapples with difficult grapples with difficult

environmental issuesenvironmental issues

Estrogen and Androgen signaling

pathway toxicant literature review

Questions

Shape of the dose response curve

over a broad range of doses

Sensitivity of Endpoints to low

dose effects

Impact on EDC screening and

testing for E and A disruption

EDC Chemicals reviewed for the Shape of the Dose Response In

the “Low” Dose Range. Threshold, LNT, or NMDR?

Androgen signaling pathway

AR antagonists

Flutamide

Vinclozolin

Procymidone

Steroid hormone synthesis Inhibitors

Phthalates, DBP and DEHP

Finasteride – inhibition of DHT synthesis

Unknown EDC mechanism, if any

Semicarbazide – an EDC with a NMDRC?

Pesticides that disrupt the Androgen signaling pathway via multiple mechanisms of toxicity

Prochloraz

Linuron

Androgen agonists

Trenbolone

Testosterone

Selective Androgen Receptor Agonists

Estrogen signaling pathway Estrogens

Ethinyl estradiol, Estradiol,

Diethylstilbestrol

Genistein

Zearalenone and Zeranol

Octylphenol, Nonylphenol

Methoxychlor

Kepone (chlordecone)

Bisphenol A

Selective estrogen receptor

agonists

Idoxifene, Tamoxifen,

Lasofoxifene, Arzoxifene,

FC1271a, Raloxifene

Aromatase inhibitors

Fenarimol – pesticide

Exemestane,, Anastrazole,

Fadrazole, Letrozole

Studies included in the review

Measured multiple endpoints affected related to disruption of the

estrogen or androgen signaling pathways

Primarily, Reproductive, one or multigenerational studies (if any)

Primarily, Oral administration – diet or gavage

Included a broad range of dosage levels from “low” to “high”

Definitions of “Low Dose” used in the review ng/kg for chemicals like EE2 and E2, µg/kg for pesticides and toxic substances, or

A dose below the reported NOEL

Preferred – 6 or more dosage levels, but no less than 4 dose levels

(three treated groups and a control group)

Primarily rodent studies also includes some porcine, primate and

human studies

Review includes about in vivo 200 studies with

>70 of which had 6 or more dose levels

>40 for the Androgen signaling pathway

>30 for the Estrogen signaling pathway

17-Methyltestosterone concentrationin the diet causes sex reversal in Nile talapia.

Phelps and Okoko (2011)

1 10 100 10000

20

40

60

80

100

MT mg/kg diet

Pe

rce

nt

Ma

le

“ROBUST” NMRDC

Effect of Tamoxifen in adult female

rats in the OECD 407 assay

0 5 30 2000

10

20

30

40

Dose g/kg/day

Rela

tiv

e U

teri

us p

lus

cerv

ix w

eig

ht

Not so “ROBUST”

NMRDC

Question

Do all the effects of EDCs display a threshold?

No, it does not appear so.

OECD Hershberger assay validation studies with

Testosterone Propionate and Flutamide given for ten days sc to

castrate immature male rats: No apparent threshold

Testosterone Propionate stimulatesAndrogen-dependent

tissue growth in castrate male rats

0.0 0.2 0.4 0.6 0.8 1.00

20

40

60

80

100 VP

SV

LABC

GLANS

COWS

TP mg/kg/d sc

PE

RC

EN

T O

FM

ax

imu

m

0 1 2 3 4 5 6 7 8 9 100

25

50

75

100 LABC

VP

SV

GP

Flutamide reduces androgen dependent

organ weights in castrate immature,

androgen-treated male rats

mg/kg/dP

erc

en

t o

f c

on

tro

l

•Hormone dependent endpoints

•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

1 10 1000

25

50

75

100 Hypospadias

Ectopic Testis

AGD

Nipples

Areolae

Ventral Prostate

ED50 HillSlope

Areolae 11.91 1.208

Ventral Prostate 36.39 1.125

Nipples 36.72 7.451

AGD 44.46 1.29

Hypospadias 50.28 36.39

Ectopic Testis 197.4 2.28

Vinclozolin mg/kg/d GD 14 to PND 3P

erc

en

t A

ffe

cte

d

Question Do EDCs induce

non-monotonic effects in vitro?

Yes

“Low dose” hypothesis: EDCs produce nonmonotonic responses in vitro and in vivo.

Toxicology testing studies are conducted at high dosage levels at the right side of the

curves whereas the relevant “low dose” studies are conducted on the left side of the

curve and can see the opposite effects from the high dose studies.

Data examined

to date on E and

A in vitro gene

expression

assays clearly

show that

current

toxicology

studies on

EDCS are here

Toxicologists ???

Endocrinologists ???

X

EE2 T47D KBLUC

10-4

10-3

10-2

10-1

100

101

102

103

104

105

106

107

108

0

20

40

60

80

100Serum EE2 level in

rats treated with a

very high oral dose of

EE2 - 1mg/kg

Conc in whole lake

causing fish

population crash

Concentration ppt

Pe

rce

nt

of

0.1

nM

E2

High concs irrelevant in vivo

TrenboloneNMDRC

0.00

10.

01 0.1 1 10 10

0

1000

1000

0

1000

00

1000

000

0

5

10

15

TB conc in tissue associated with infertility in thefathead minnowTB conc in amniotic fluid in female rat fetus resulting inreproductive tract malformations

Cytotoxicity

Concentration (ppb)

Fo

ld I

nd

uc

tio

n

High concs

irrelevant in vivo

What about NMDRCs in vivo?

Do EDCs induce robust, reproducible

non-monotonic effects, if so

• At low dosage levels?

• Is the effect clearly adverse or causally

linked to an adverse effect?

• Would it alter risk assessment?

My classification of in vivo NMDRCs

and an estimate of their prevalance

“Robust”, reproducible, biologically plausible

“Trivial”, frequency of one effect declines as a more

severe one develops

“False positive”, multiple comparisons or invalid

analyses

“Confounded”, NMDRC is reported at dosage levels

well below background levels

“Imaginary”, no group differs significantly from control

but the data are interpreted as displaying a NMDRC

“Oblivious”, interpretation of high dose NMDRC ignores

overt toxicity or adverse effects at much lower doses

Dose-response database with Estrogens:

number of studies reviewed (About 70 E and A studies with six or more dose groups

(studies with 6 or more dose groups/total studies examined)

Estradiol (2 / 9)

Ethinyl estradiol ( 7 / 10)

Genistein(2 /12)

BPA (4 / 14, and more on the way)

DES (1, 7)

Zearalenone (2, 8)

Octylphenol (1, 2)

Nonylphenol (2, 3)

Methoxychlor (3, 7)

Kepone (3, 5)

SERMs (2, 16)

Aromatase Inhibitors (3, 9)

vom saal PNAS 1997 vs Ashby 1999Does an oral low dose of DES increase F1

male mouse prostate weight?

0 2 20 200

2000

2000

0

2000

00 020

0 020

0

0

20

40

60

Ashby 1999

VOM SAAL 1997

Cagen 1999

DES ng/kg

Pro

sta

te w

eig

ht

CF

-1 M

ale

Mic

e

Vom Saal PNAS 1997. Oral DES administrationGD11-17 was reported to induce a NMDRC onF1 male mouse prostate weight. However, the

dose of DES required to reduce prostate weightis clearly not a "low dose" effect

1 10100

1000

10000

10000020

25

30

35

40

45

50

55

60

Dose range that

accelerates

puberty in

female rats

Dose that reduces

early embryo

viability GD4-8

Dose in mg/kg in tablets

prescribed for women

Dose that

reduces F1

male mouse

prostate weight

DES ng/kg

Pro

sta

te w

eig

ht

CF

-1 M

ale

Mic

eNeonatal Doses

reducing fecundity

Dose-response database for Antiandrogens and Androgens

(studies with 6 or more dose groups / total studies)

Flutamide (2 / 9)

Vinclozolin (4 /8)

Procymidone (4 / 6)

DEHP (9 / 19)

DBP (3 / 6)

Finasteride (2 / 2)

Semicarbizide (1 / 3)

Prochloraz (3 / 5)

Linuron (0 / 6)

Testosterone (12 (4 rat, 8 men) / 13)

SARMS (1 – so far, more to come)

Low Dose Studies with Androgens

and SARMS

A few effects in rats display non-monotonic responses, but most do not Testosterone Propionate (sc) to the pregnant rat (GD14-18)

Testosterone propionate (sc) in the adult male rat

None of these studies used the oral route of administration

Dose response data in men do not show NMDRCs

Extensive trenbolone data base unavailable to the public and general scientific community, but lots of low dose studies in many species

Non-monotonic response of the adult male

rat testis to TP implants. Many other effects

showed monotonic responses

“ROBUST” NMDRC

F1 female hydrometrocolpos

with vaginal agenesis

10 100

1000

1000

0

0

25

50

75

100

Testosterone Propionate (sc)ng per pregnant rat GD 14-19

Pe

rce

nt

Aff

ec

ted

“ROBUST” NMRDC

Low Dose Studies with

Antiandrogens

Antiandrogens

Finasteride

Rat studies – no evidence of non-monotonic effects

Flutamide

Rat studies – no evidence of non-monotonic effects

Dibutyl phthalate and DEHP

Rat studies – no reproducible evidence of non-monotonic effects for adverse effects at low dosage levels

Low to high-dose, dose response studies with the “antiandrogen” Finasteride

administered orally to the dam during gestation at doses ranging from 0.0003 to 300

mg/kg/d. There were no nonmonotonic effects on the male offspring.

Threshold effects of in utero flutamide on the incidence of male

rat reproductive tract malformations later in life

F1 male rat malformations after in utero flutamide exposureTen dose levels over 4 orders of magnitude

Data combined from two studies(results of dose of 10 mg

in one study combined with 12.5 mg in the other)

0.1 1 10 100

0

25

50

75

100 hypospadias

epididymal agenesis

prostate agenesis

Sv agenesis

Ectopic testis

labc agenesis

Flutamide mg/kg

Pe

rce

nt

aff

ec

ted

Low to high-dose, dose response studies. Data are from ten one generation

studies with different phthalate esters that all disrupt male rat sexual

differentiation via the same mode of action that also display similar potencies in

short-term in vivo screening assays. Testis/Epididymal malformations and

seminal vesicle weights are shown. None of the effects in these studies

displayed any non-monotonic responses.

Testis-epididymal malformation

data pooled from 10 studies

0.01 0.1 1 10 100 1000

0

20

40

60

80

100 LogEC50

HillSlope

EC50

2.586

3.332

385.3

mg/kg/d PE

Pe

rce

nt

Aff

ec

ted

DEHP DR Low dose study

Andrade et al 2006

No non-monotonic low dose adverse effects

Body Weight

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0

100

200

300

400

500

Dose mg DEHP/kg/d

WE

IGH

T (

g)

TESTES WEIGHT

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0.0

0.5

1.0

1.5

2.0

2.5

Dose mg DEHP/kg/d

WE

IGH

T (

mg

)

Epididymal weight

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0

200

400

600

800

Dose mg DEHP/kg/d

WE

IGH

T (

mg

)

Seminal Vesicle

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0

200

400

600

800

1000

Dose mg DEHP/kg/d

WE

IGH

T (

mg

)

Prostate weight

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0

200

400

600

Dose mg DEHP/kg/d

WE

IGH

T (

mg

)

Percent without Phthalate Syndrome

Reproductive Tract Malformations

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0

50

100

150

Dose mg DEHP/kg/d

Pe

rce

nt

No

rma

l

F1 Male Mating Behavior

and Fertility

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

0

50

100

150

Dose mg DEHP/kg/d

Pe

rce

nt

No

rma

l

Daily testis sperm production

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

His

t Contr

ol0

10

20

30

40

50

DS

P u

ne

dit

ed

da

ta(m

illi

on

s)

DEHP DR Low dose study

Andrade et al 2006

“non-monotonic “low dose effect

Range of phthalates in rodent chows

Reported NMDRC for AGDDietary phthalate levels estimated

from another paper

0.0

0.5

1.0

5.0

21.4

214.

0

500.

0

5000

0.0

5000

00.0

1.0

1.2

1.4

1.6

1.8

2.0

2.2Range of BackgroundDietary Phthalate Levels

DEHP micrograms per kg per day

An

og

en

ita

l D

ista

nc

e (

mm

)“Confounded and

Imaginary” NMRDC ??

PUP BIRTH WEIGHTNO STATISTICALLY SIGNIFICANT EFFECTS

NOTED BY AUTHOR

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

4

5

6

7

Dose DEHP mg/kg/d

Gra

ms

AGE AT VAGINAL OPENING* INDICATES STATISTICALLY SIGNIFICANT

EFFECTS NOTED BY AUTHOR

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

32

34

36

38

40

* ** *

range of

phthalates

in control

diets

Dose DEHP mg/kg/d

Ag

e a

t "P

ub

ert

y"

AGE AT FIRST ESTRUSNO STATISTICALLY SIGNIFICANT

EFFECTS NOTED BY AUTHOR

0.00

0

0.01

5

0.04

5

0.13

5

0.40

5

1.21

5

5.00

0

15.0

00

45.0

00

135.

000

405.

000

36

38

40

42

Dose DEHP mg/kg/d

Ag

e a

t "P

ub

ert

y"

Data from Grande et al., 2006,

cited as examples of NMDRCs

induced in female rat offspring

from in utero exposure to DEHP

Imaginary NMDRC

My current conclusions on the shape of the

dose response curves for EDCs- EDCs appear to induce some effects that do not appear to display a

threshold (apparent Linear No Threshold responses)

NMDRCs for EDCs

Biologically plausible

Occur frequently in vitro, but these are generally not relevant to in vivo effects and do not occur at low concentrations

It appears that NMRDCs are more common

in studies with short-term exposures and

On “upstream” mechanistic events versus “downstream” adverse phenotypic effects.

A few of the effects of androgens given sc are non-monotonic, but other effects in the same study occur at lower dosage levels and they display “normal” (monotonic) dose responses

A number of multigenerational studies of estrogens and antiandrogens have been reviewed. To date, these did not indicate that robust, reproducible NMDRCs were common events at low dosage levels.

Additional data needs to be examined from robust, multigenerational studies using a broad range of dosage levels for other pathways

Impact of NMDRCs and LNT responses on EDC

screening and testing

Impact for EDC screening – NONE • Do the EDC screening assays fail to detect E or A activity? NO

Impact for multigenerational testing • Estimation of shape of the dose response curve in the low dose

region could be enhanced by using more dose groups

• For example, keep total N litters in a study as is, increase number of

dose groups from 3 to 6 with half as many litters per dose group

• Examining more than one animal per litter enhances endpoint

sensitivity by increasing the statistical power to detect low dose

categorical effects like malformations and histopathological lesions

(Blystone et al., 2010; Hotchkiss et al., 2008).

Sensitivity of Endpoints to low dose effects in EDC testing • Recent studies conducted with E and A active chemicals have

identified more sensitive endpoints that should be added on a case-

by-case basis.

• Many of these are not explicitly included in any current protocol