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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