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2009 37: 100 originally published online 15 January 2009 Toxicol Pathol
and L. Earl Gray, Jr
Cynthia V. Rider, Vickie S. Wilson, Kembra L. Howdeshell, Andrew K. Hotchkiss, Johnathan R. Furr, Christy R. Lambright
Cumulative Effects of In Utero Administration of Mixtures of ''Antiandrogens'' on Male Rat Reproductive
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Cumulative Effects of In Utero Administration
of Mixtures of “Antiandrogens” on Male
Rat Reproductive Development
CYNTHIA V. RIDER,1,2VICKIE S. WILSON,1KEMBRA L. HOWDESHELL,1ANDREW K. HOTCHKISS,1,2,3
JOHNATHAN R. FURR,1CHRISTY R. LAMBRIGHT,1AND L. EARL GRAY JR1
1MD-72, Endocrinology Branch, Reproductive Toxicology Division,
NHEERL, ORD, U.S. Environmental Protection Agency, RTP, North Carolina, USA
2North Carolina State University/USEPA Cooperative Training Grant (CT826512010), Raleigh, North Carolina, USA
3Current address: NCEA, ORD, US Environmental Protection Agency, RTP, North Carolina, USA
Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act (FQPA) required the
Environmental Protection Agency (EPA) to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are
conducting studies with mixtures to provide a framework for assessing the cumulative effects of “antiandrogenic” chemicals. Rats were dosed
during pregnancy with antiandrogens singly or in pairs at dosage levels equivalent to about one half of the ED50for hypospadias or epididymal age-
nesis. The pairs include: AR antagonists (vinclozolin plus procymidone), phthalate esters (DBP plus BBP and DEHP plus DBP), a phthalate ester
plus an AR antagonist (DBP plus procymidone), and linuron plus BBP. We predicted that each chemical by itself would induce few malformations;
however, by mixing any two chemicals together, about 50% of the males would be malformed. All binary combinations produced cumulative, dose-
additive effects on the androgen-dependent tissues. We also conducted a mixture study combining seven “antiandrogens” together. These chemicals
elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibition of androgen synthesis). In this
study, the complex mixture behaved in a dose-additive manner. Our results indicate that compounds that act by disparate mechanisms of toxicity dis-
play cumulative, dose-additive effects when present in combination.
reproductive system; male reproduction; endocrine disrupters.
As a result, the field of “mixtures toxicology” is emerging
as an area of increasing scientific and regulatory focus in the
United States and abroad. For example, in 1996 the U.S.
Environmental ProtectionAgency (EPA) began considering the
cumulative risk of chemicals that act via a common mechanism
of toxicity as mandated in the Food Quality Protection Act
(FQPA). The U.S. EPA’s Offices of Water (OW) and Research
and Development (ORD) and the U.S. EPA Superfund, Solid
Waste, and Air Programs also have ongoing programs in this
area. In 2003, the U.S. EPA National Center for Environmental
Assessment (NCEA), ORD, published a “Framework” report
that initiated a long-term effort to develop cumulative risk
.cfm?deid= 54944). The report identifies the basic elements of
the cumulative risk assessment process and provides a flexible
structure for conducting and evaluating cumulative risk assess-
ment, and for addressing scientific issues related to cumulative
risk. It is intended that the NCEA Framework report will serve
as a foundation for developing future guidance. In this regard,
the research from our laboratory, described herein, is intended
to contribute to the development of a guidance framework for
assessing cumulative risks to reproduction and development
from exposure during pregnancy.
There is now widespread awareness that humans (Calafat
et al. 2008; Eskenazi et al. 1999; Landrigan et al. 1999; Silva,
Barr et al. 2004; Silva, Reidy et al. 2004; Wolff, Britton et al.
2008; Wolff, Engel et al. 2007; Wolff, Engel et al. 2008), fish
(Ankley et al. 2007; Jobling et al. 1998; Jobling and Tyler 2006;
Jobling et al. 2006), and wildlife (Hall and Thomas 2007) are
exposed to multiple contaminants on a continuous basis. The
chemicals found in some aquatic systems include not only pes-
ticides (Hela et al. 2005; Jaspers et al. 2006) and industrial
chemicals (Hall and Thomas 2007), but also pharmaceuticals
and hormones (Durhan et al. 2006; Kolpin et al. 2002).
The research described in this article has been reviewed by the National
Health and Environmental Effects Research Laboratory, ORD, U. S.
Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies of
the Agency nor does the mention of trade names or commercial products con-
stitute endorsement or recommendation for use.
Address correspondence to: L. Earl Gray Jr., MD-72, Endocrinology
Environmental Protection Agency, Research Triangle Park, NC 27713, USA;
Division,NHEERL, ORD, U.S.
Toxicologic Pathology, 37: 100-113, 2009
Copyright © 2009 by Society of Toxicologic Pathology
ISSN: 0192-6233 print / 1533-1601 online
Vol. 37, No. 1, 2009CUMULATIVE EFFECT OF ANTIANDROGENS IN UTERO 101
Although many studies have examined the effects of mix-
tures in vitro or in short-term in vivo assays with mature ani-
mals, few studies have examined the effects of mixtures of
chemicals on mammalian reproductive development. Since the
early 1980s, our laboratory has been studying the effects of
pesticides and toxic substances administered in utero on fetal
and postnatal rodent reproductive development. We have con-
ducted dose response studies on the postnatal reproductive
effects of environmental estrogens, androgens, antiandrogens,
dioxins and PCBs, and germ cell toxicants. Along with these
long-term in vivo studies, we also have conducted mechanistic
studies in vitro and in vivo to identify the mechanisms and
modes of action of these different toxicants.
Currently, we are using this information to design mixture
studies to examine how members of one of these classes of tox-
icants, the “antiandrogens,” interact when they are adminis-
tered during sexual differentiation of the laboratory rat.
Toxicants studied include pesticides and phthalates that disrupt
sexual differentiation by acting as androgen receptor (AR)
antagonists and/or inhibitors of fetal testosterone synthesis.
The review that follows will: (1) describe the chronology of
events that led us to initiate a “mixtures” research program with
vinclozolin and procymidone and then phthalates and the pesti-
cides prochloraz and linuron; (2) describe the modes of action
in vitro and in vivo of the individual chemicals that we selected
to study as the program evolved; (3) describe the mathematical
modeling procedures that we now use; (4) present the results of
our completed mixture studies; (5) describe our future research
plans; and (6) present an alternative framework for selecting
chemicals for inclusion in cumulative assessments.
Our First Mixture Study
In the late 1990s the Agency began an examination of
whether some or all members of the dicarboximide class of
fungicides, which includes vinclozolin, iprodione, and pro-
cymidone, shared a common mechanism of toxicity. At this
time, the scientific information on the mechanisms of toxicity
of this class of fungicides was incomplete. For this reason, the
EPA concluded in 2000 that “The Agency does not currently
have a fully developed understanding of whether vinclozolin
shares a common mechanism of toxicity with iprodione and
procymidone because the androgen system is highly complex.
As a result, the Agency has not determined if it would be
appropriate to include them in a cumulative risk assessment.
Therefore, for the purposes of this assessment, the Agency has
assumed that vinclozolin does not share a common mechanism
of toxicity with iprodione and procymidone” (http://www.epa
.gov/opp00001/reregistration/vinclozolin/). In addition, in a
risk assessment on procymidone in 2005, the Agency con-
cluded that “EPA has not made a common mechanism of toxi-
city finding and therefore, has not assumed that procymidone
has a common mechanism of toxicity with other substances
for the purposes of this tolerance action” (http://www.epa.gov/
oppsrrd1/REDs/procymidone_tred.pdf). At the encouragement
of the program office, we initiated studies at EPA’s National
Health and Environmental Effects Laboratory to better eluci-
date the mechanism of toxicity for these antiandrogenic fungi-
cides as well as mixture studies on how they interact. Since then,
several studies from our laboratory and other laboratories have
been completed that address these uncertainties. These studies
demonstrate that vinclozolin and procymidone share a common
mechanism of toxicity and interact in a cumulative manner.
Our Mixture Studies with Phthalates
Following the project with vinclozolin and procymidone, we
1 and 2) to determine how these chemicals interact when present
as mixtures. The effects of mixtures of phthalates are a concern,
since humans are exposed to multiple phthalates at one time
et al. 2007). To address this issue, in 2006 the EPA requested that
the NationalAcademy of Sciences establish a panel to provide the
Agency with recommendations on how to address the cumulative
effects of the phthalates. The data from several of the phthalate
mixture studies presented herein were given to the NAS panel for
di-n-butyl (DBP) plus di-n-ethyl hexyl phthalate (DEHP)
fetal testosterone levels (Howdeshell et al. 2008), and a complex
mixture study that included three phthalates and four pesticides
(Rider et al. 2008) (http://www8.nationalacademies.org/cp/
Concerns about some phthalates in toys led to passage of the
2008 US Consumer Protection Agency Modernization Act
(Public Law No: 110-314, section 108), which prohibits the
sale of certain products containing phthalates (DEHP, DBP,
benzyl butyl-[BBP], di-iso-nonly phthalate [DINP], di-
iso-octyl phthalate DIOP and di-n-octyl phthalate [DNOP]).
Additionally, the law established the Chronic Hazard Advisory
Panel charged with examining “the potential health effects of
each of these phthalates both in isolation and in combination
with other phthalates” and “to consider the cumulative effect of
total exposure to phthalates, both from children’s products and
from other sources, such as personal care products.”
Our Mixture Studies with Pesticides
and Phthalates with Diverse Modes of Toxicity
In addition to toxicants that disrupt sexual differentiation
predominantly via one mechanism of toxicity (i.e., AR antago-
nists or inhibitors of testosterone synthesis), it became evident
that several pesticides, including linuron and prochloraz, act
via dual mechanisms of toxicity. These pesticides display AR
antagonist activity and inhibit testosterone synthesis with
varying potencies. We were interested in exploring how these
chemicals with complex mechanisms of action interact in mix-
tures. Therefore, we conducted two binary mixture studies, one
102 RIDER ET AL.TOXICOLOGIC PATHOLOGY
with linuron with BBP (Hotchkiss et al. 2004) and a second
with procymidone with DBP. Subsequently, we conducted a
study with a mixture of seven chemicals (including vinclozolin,
procymidone, linuron, prochloraz) and three phthalates (DBP,
DEHP, and BBP) (Rider et al. 2008), and we have initiated a
similar complex mixture study with ten chemicals.
TABLE 1.—In vitro and/or ex vivo mechanistic assay results and in vivo effects in male rat offspring
Abbreviations: LDHC, lowest dose producing hypospadias, cryptorchidism, or epididymal agenesis; LDRE, lowest dose producing a reproductive effect.
TABLE 2.—Brief methods and chemical dosage levels used in the chemical mixture studies discussed in this review
examining the effects of mixtures of “antiandrogens” with similar and diverse mechanisms of toxicity.
1. Studies using chemicals with common mechanisms of toxicity
Vinclozolin plus procymidone administered on gestational days 14 (GD0=day of sperm) to 18. Study was a 2 x 2 factorial with a control (vehicle), vinclozolin
(50 mg/kg/d), procymidone (50 mg/kg/d) and combination (50 vinclozolin plus 50 procymidone) groups. All F1 male offspring examined as fully mature adults.
Di-n-butyl phthalate plus benzyl butyl phthalate administered on gestational days 14 (GD0=day of sperm) to 18. Study was a 2 x 2 factorial with a control (vehi-
cle), DBP (500 mg/kg/d), BBP (500 mg/kg/d) and combination (500 DBP plus 500 BBP) groups. All F1 male offspring examined as fully mature adults.
Di-n-ethyl hexyl phthalate plus di-n-butyl phthalate administered on gestational days 14 (GD0=day of sperm) to 18. Study was a 2 x 2 factorial with a control
(vehicle), DBP (500 mg/kg/d), DEHP (500 mg/kg/d) and combination (500 DBP plus 500 DEHP) groups. (Howdeshell et al, 2007). All F1 male offspring exam-
ined as fully mature adults.
Five phthalate mixture study with individual chemicals and the mixture administered on gestational days 8 (GD0=day of sperm) to 18 (Howdeshell et al., 2008).
Dams were dosed via gavage from GD8 to GD18 with either 0 (vehicle control) or seven dose levels of the mixture. The top dose (100%) included at total of 1300
mg of the five phthalates (BBP, DBP, DEHP and DiBP each at 300 mg/kg/day, plus 100 mg/kg DPP/day) and was administered at 100%, 80%, 60%, 40%, 20%,
10% and 5% of the top dose. This dose ratio (3:3:3:3:1) was selected based upon the ED50 values (mg/kg/d) from the dose response studies of each phthalate in
order that each phthalate would contribute equally to the mixture effects on testosterone production if the mixture behaved in a dose additive manner.
2. Studies using chemicals with diverse mechanisms of toxicity
Linuron plus benzyl butyl phthalate administered on gestational days 14 (GD0=day of sperm) to 18. Study was a 2 x 2 factorial with a control (vehicle), Linuron
(75 mg/kg/d), BBP (500 mg/kg/d) and combination (75 Linuron plus 500 BBP) groups.
Di-n-butyl phthalate plus procymidone administered on gestational days 14 (GD0=day of sperm) to 18. Study was a 2 x 2 factorial with a control (vehicle), DBP
(500 mg/kg/d), procymidone (50 mg/kg/d) and combination (500 DBP plus 50 procymidone) groups.
Seven-chemical study using a mixture with each chemical at 1/7thof its ED100 (15 mg/kg/d vinclozolin, 15 mg/kg/d procymidone, 35 mg/kg/d prochloraz, 20
mg/kg/d linuron, and 150 mg/kg/d each of benzyl n-butyl phthalate, dibutyl phthalate and diethylhexyl phthalate in the top dose group. Pregnant rats were dosed
with the vehicle (corn oil, 25%, 50%, 75%, or 100% of the high dose by oral gavage on GD14-18. The two lowest dose groups contain individual chemicals at or
below their no observed effect levels (NOAELs) for inducing male reproductive tract malformations.
Vol. 37, No. 1, 2009CUMULATIVE EFFECT OF ANTIANDROGENS IN UTERO 103
Mechanisms and Modes of Action of the
Individual Chemicals Used in the Mixture Studies
As summarized in Table 1, the research discussed herein
reveals that environmental chemicals can alter the androgen
signaling pathway via several distinct modes of action.
Knowledge of the modes of action of endocrine-disrupting
chemicals (EDCs) allows us to make some predictions about
how individual tissues will be affected when antiandrogens are
combined. The classes of EDCs known to interfere with the
androgen signaling pathway include: dicarboximide fungi-
cides, for example, vinclozolin (Kelce et al. 1994); organochlo-
rine-based insecticides, for example, p,p’-DDT and p,p’-DDE
(Kelce et al. 1995); conazole fungicides, for example, prochlo-
raz (Noriega et al. 2005; Vinggaard et al. 1999); plasticizers,
for example, phthalates; polybrominated diphenyl ethers
(PBDEs) (Gray et al. 2004; Stoker et al. 2005); and urea-based
herbicides, for example, linuron (Lambright et al. 2000;
McIntyre et al. 2000).
Modes of Action of the Individual
Chemicals Used in Our Mixture Studies
AR antagonists: Vinclozolin and Procymidone (fungicides):
Of the dicarboximide fungicides, vinclozolin (Kelce et al.
1994), iprodione (Blystone et al, in preparation), and procymi-
done (Hosokawa et al. 1993; Nellemann et al. 2003; Ostby
et al. 1999; Vinggaard et al. 1999) act as AR antagonists in
vitro and/or in vivo. These pesticides, or their metabolites,
competitively inhibit the binding of androgens to AR, which
leads to inhibition of androgen-dependent gene expression in
vitro and in vivo (Kelce and Wilson 1997). Vinclozolin and
procymidone both act as AR antagonists in the Hershberger
assay (castrated-immature androgen- and pesticide-treated male
rats), an effect replicated in several laboratories (Ashby et al.
2004; Charles et al. 2005; Kang et al. 2004; Kennel et al. 2004;
Owens et al. 2007; Shin et al. 2007;Yamasaki et al. 2003).
of pubertal landmarks and reduce androgen-dependent organ
weights in the young male rat (Monosson et al. 1999). In a
Hershberger assay using castrated immature testosterone-treated
male rats, vinclozolin and procymidone (0, 25, 50, and 100
mg/kg/d) alone or in combination inhibited testosterone-induced
growth of androgen-dependent tissues (ventral prostate, seminal
tive fashion (Gray et al. 2001; Nellemann et al. 2003).
Administration of vinclozolin during sexual differentiation
demasculinizes and feminizes the male rat offspring such that
treated males display female-like AGD at birth, retained nip-
ples, hypospadias, suprainguinal ectopic testes, a blind vaginal
pouch, and small to absent sex accessory glands (Gray et al.
1994). In contrast to the phthalates and linuron, even at high
dosage levels (200 mg/kg/d), epididymal hypoplasia was rare
and gubernacular agenesis was not displayed in vinclozolin-
treated male offspring.
25, 50, or 100 mg/kg/d from gestational day 14 to postnatal day
3) reduces neonatalAGD and increases the incidence of retained
nipples/areolae in infant male rats (Gray, Ostby et al. 1999). In
adult life, ventral prostate weight is permanently reduced (at
6.25, 25, 50, and 100 mg/kg/d) and male offspring display per-
manent female-like nipples. Treatment at 50 and 100 mg/kg/d
induces hypospadias and other reproductive tract malformations
(Gray, Ostby et al. 1999; Hellwig et al. 2000). The most sensi-
tive period of development to the disruptive effects of vinclo-
zolin is GD 16-17, with less severe effects seen in males exposed
to vinclozolin on GD 14-15 and GD 18-19 (Wolf et al. 2000).
When procymidone is administered from day 14 of preg-
nancy to day 3 after birth at 25, 50, 100, or 200 mg/kg/d, AGD
is shortened in male pups, and the males display retained nip-
ples, hypospadias, cleft phallus, a vaginal pouch, and reduced
sex accessory gland size (Ostby et al. 1999). Hypospadias was
displayed by males in the 50 mg/kg/d dose group and above and
ectopic, undescended testes displayed at 200 mg/kg/d.
These two dicarboximide pesticides not only induce reproduc-
tive tract malformations and permanent reductions in androgen-
prostatic and vesicular tissues abnormally such that F1 male off-
spring develop high rates of inflammation in these tissues later in
life. In 1999, we observed that in utero procymidone treatment
induced fibrosis, cellular infiltration, and epithelial hyperplasia in
the dorsolateral and ventral prostatic and seminal vesicular tissues
(Ostby et al. 1999). More recently, similar effects were seen in
males exposed to vinclozolin in utero (Cowin et al. 2008). One
hundred percent of male rats exposed to 100 mg/kg/d during sex-
ual differentiation displayed prostatitis after puberty. The authors
also reported that prostatic inflammation “was not associated with
the emergence of premalignant lesions, such as prostatic intra-
epithelial neoplasia or proliferative inflammatory atrophy, and
hence mimics nonbacterial early-onset prostatitis that commonly
occurs in young men” (Cowin et al. 2008).
after either short-term procymidone or vinclozolin treatments.
In summary, the effects of procymidone and vinclozolin are
identical in vivo and in vitro.
Inhibitors of Fetal Reproductive Development and Testis
Hormone Production: Phthalates: The phthalates represent a
development. This class of chemicals does not appear to act via
estrogen and androgen nuclear receptors. Although a few
studies suggested that some of the phthalates are estrogenic,
DBP injections do not induce a uterotropic response or
estrogen-dependent sex behavior (lordosis) in ovariectomized
adult female rats (Gray and Ostby 1998), and oral DBP
treatment fails to accelerate vaginal opening or induce constant
estrus in intact female rats (Gray, Wolf et al. 1999). The
phthalate diesters and their monoester metabolites also do not
104 RIDER ET AL.TOXICOLOGIC PATHOLOGY
appear to compete significantly with androgens for binding to
AR at environmentally relevant concentrations (Parks et al.
2000). In vivo, the phthalate diesters fail to display consistent
AR antagonist activity. DBP and BBP produce negative results
in a Hershberger assay, whereas DEHP causes equivocal
reductions in androgen-induced tissue growth even at 1000
mg/kg/d (Gray, in preparation; Stroheker et al. 2005).
In utero, some phthalate esters alter the development of the
male rat in an antiandrogenic manner. Prenatal exposure to
DBP, BBP, DINP, and DEHP treatment cause a syndrome of
effects, including underdevelopment and agenesis of the epi-
didymis and other androgen-dependent tissues and testicular
abnormalities (Foster et al. 2001; Gray et al. 2000) character-
ized as the “Phthalate Syndrome.” Prenatal exposure to DBP
from day 10 to day 22 of gestation produces effects nearly
identical to those seen with DEHP, with effects occurring at
dosage levels of 50–100 mg/kg/d (Mylchreest and Foster 2000;
Mylchreest et al. 1999). Among the antiandrogenic EDCs, the
phthalates are unique in their ability to induce agenesis of the
gubernacular cords, a tissue whose development is dependent
upon the peptide hormone insulin-like peptide-3 and critical
for testis descent.
A Pesticide with Dual Modes of Toxicity: Linuron (herbicide):
In vitro, the herbicide linuron is an AR antagonist (Lambright et
al. 2000; McIntyre, Barlow, and Foster 2002; McIntyre, Barlow,
Sar et al. 2002; McIntyre et al. 2000; Turner et al. 2003). In
contrast to some AR antagonists, neither short-term (Lambright
et al. 2000; O’Connor et al. 2002) nor long-term (Gray,Wolf et al.
1999) linuron administration induces elevated serum LH levels.
male rat testosterone synthesis during sexual differentiation,
demonstrating that linuron is antiandrogenic via dual mecha-
nisms of action (Table 1), inhibiting androgen synthesis and as an
AR antagonist (Wilson et al. 2004) (Wilson et al. unpublished).
When administered in utero, linuron exposure causes malfor-
to 100 mg linuron/kg/d (GD14-18) displayed epididymal and tes-
dosage levels as low as 12.5 mg/kg/d (exposed from GD 10–22)
(McIntyre et al. 2000).The testicular effect seen in adult F1 males
results from epididymal lesions rather than a direct effect of lin-
uron on testis morphology. When male rat fetuses or offspring
were necropsied on GD 17, 19, and 21, and postnatal days (PND)
7 and 14, epididymal malformations were not observed in fetuses
from linuron-treated dams but were seen in linuron-exposed male
offspring on PND 7 and 14 (McIntyre, Barlow, Sar et al. 2002).
The testicular lesions are seen only in adults and not in younger
animals. These lesions develop as a consequence of pressure atro-
at any time point during fetal or infant life.
In contrast to the effects of vinclozolin and procymidone,
malformed external genitalia and undescended testes were
rarely displayed by linuron-exposed males. The syndrome of
effects induced by linuron is atypical of an AR antagonist and
more closely resembles the Phthalate Syndrome.
A Pesticide with Dual Modes of Toxicity: Prochloraz
Prochloraz is a fungicide that also disrupts reproductive
development and function by several modes of action (Noriega
et al. 2005; Vinggaard et al. 2005; Vinggaard et al. 2006).
Prochloraz inhibits the steroidogenic enzymes 17, 20 lyase and
aromatase, and it is an AR antagonist (Blystone, Furr et al.
2007; Blystone, Lambright et al. 2007). In a study in which rat
dams were dosed from GD 14 to 18, Wilson et al. (2004) found
that prochloraz reduced fetal testis testosterone and increased
progesterone production tenfold on GD 18 without affecting
Leydig cell ins13 mRNA levels.
In a transgenerational study, prochloraz treatment from GD
14 to 18 at doses of 62.5, 125, 250, and 500 mg/kg/d delayed
parturition and altered reproductive development in the male
offspring in a dose-related manner (Noriega et al. 2005).
Treated males displayed reduced AGD and female-like areolas
(33%, 71%, and 100% in 62.5, 125, and 250 mg/kg groups,
respectively), and males in the 250 mg/kg treatment group dis-
played hypospadias. However, the epididymides and gubernac-
ular ligaments were relatively unaffected.
In male rat offspring, the profile of effects induced by pre-
natal prochloraz appears to more closely resemble that of an
AR antagonist, like vinclozolin, rather than an inhibitor of fetal
testosterone synthesis, like a phthalate or linuron.
Dose-response Analysis of Individual Chemicals
Over the past several decades, research in our laboratory has
focused on defining the effects of individual chemicals on the
reproductivedevelopment of male rats. However, recent advance-
ments in analytical techniques have drawn attention to the preva-
lence of environmental chemical mixtures, which has shifted
focus from individual chemical effects to mixtures effects (CDC
2008; Kolpin et al. 2002; Squillace et al. 2002). Now, individual
chemical data are input into mathematical models of mixture tox-
icity to make predictions about the potential effects of mixtures
on male reproductive tract development. Predicted mixture
responses are compared to observed data generated from mixture
exposures to determine the type of joint action (dose addition,
The mixture toxicity models we use require dose-response
data from individual chemical exposures. These data were
compiled from studies conducted in our laboratory over the
past twenty years. Assumptions were introduced when dose-
response data were incomplete. For example, we previously
evaluated the effects of only one dose of BBP on reproductive
development; the BBP dose coincided with the dose-response
data from DBP, thus we assumed that BBP had a similar dose-
response curve to DBP. Historical data included studies
conducted by different researchers with several rat strains and
Vol. 37, No. 1, 2009 CUMULATIVE EFFECT OF ANTIANDROGENS IN UTERO105
slightly different dosing schedules. However, all studies
included exposure to the chemical during the critical window
of reproductive tract differentiation in utero.
Once the raw data were compiled, we transformed the data
to fit a 0-to-100 scale. For continuous end points (AGD and
organ weights), we converted the data to percentage change
from the control value. For malformation data, we presented
the data as percentage incidence. We then graphed the data on
a log-linear scale and fit the data with a logistic equation (see
example in Figure 1):
FIGURE 1.—Individual chemical dose-response data fit with a logistic model from a mixture study with vinclozolin, procymidone, prochloraz,
linuron, and phthalates. This figure is reproduced from Rider et al. (2008).
where R is the response, D is the chemical dose, ρ is the power
or Hill slope of the curve, and ED50 is the exposure dose
eliciting a 50% response. The parameters (Hill slope and
ED50) generated from the logistic fit to the individual chemi-
cal data were used in models to make predictions of the mix-
Modeled versus Observed Responses
There are three types of joint action models: dose addition,
response addition, and integrated addition. The dose addition
model, first introduced by Loewe and Muischnek (1926), has
been applied to mixtures of chemicals that have the same
mechanism of action (Altenburger et al. 2000; Silva et al.
2002); the response addition model has been associated with
mixtures containing chemicals with different mechanisms of
action (Backhaus et al. 2000), and the integrated addition model
to mixtures containing both same and different mechanism of
action components (Altenburger et al. 2005; Rider and Leblanc
2005). In the dose-addition model, mixture components can be
thought of as dilutions of one another. Once the potencies of
the individual chemicals are accounted for, their doses can sim-
ply be added together to determine the total dose of the mix-
ture. From this total mixture dose, we can then calculate the
predicted response. The dose addition equation that we used to
calculate predicted responses of mixtures is:
where R is the response to the mixture, Diis the concentration
of chemical i in the mixture, ED50iis the concentration of
chemical i that causes a 50% response, and ρ´ is the average
power (Hill slope) associated with the chemicals.
approach. The toxic equivalency approach is often associated with
dioxin-like compounds (Safe 1990). For this approach, a reference
chemical was selected for each end point based on the strength of
the individual chemical dose response data for that end point. For
dias in one mixture study because it had the most complete dose-
response data of the chemicals in the mixture (Rider et al. 2008).
Relative potency factors were calculated by dividing the ED50 of
the reference compound by the ED50 of each of the other mixture
doses were added to get the total mixture dose in terms of the ref-
erence chemical, which could then be inserted into the logistic
equation for the reference chemical to calculate the predicted mix-
The response-addition model, also referred to as independ-
ent-action model, was first introduced by Bliss (1939) and has
been used to describe mixtures of chemicals with different
mechanisms of action. However, there has been discussion of
whether it is possible for chemicals to have completely inde-
pendent action at a common target tissue given the complexity
of biological systems (Hermens and Leeuwangh 1982). The
equation for response addition is based on probability theory
and is expressed as:
The integrated addition model, introduced relatively
recently by several different groups (Altenburger et al. 2005;
Rider and Leblanc 2005; Teuschler et al. 2004), combines the
dose and response addition models. In this approach, chemicals
with the same mechanism of action are grouped, and the total
dose associated with each group is calculated using dose addi-
tion. The groups are then combined using response addition.
The integrated addition model is expressed mathematically as:
Results of Our Mixture Studies
Brief Methods and Dosage Levels Used in Our Mixture
Studies (Table 2): In our binary studies, rats were dosed singly
or in pairs during pregnancy with antiandrogens at dosage
levels equivalent to about one half of the ED50for hypospadias
or epididymal agenesis. In these studies, we focused dose
selection on the induction of malformations and permanent
effects (higher-dose effects, rather than low-dose effects) for
two reasons. First, there is no unanimity among risk assessors
about using anogenital distance (AGD) at birth in male rats or
the induction of female-like nipples in infant male rats as
adverse endpoints, whereas there is no question that
hypospadias, epididymal agenesis, and undescended testes are
adverse effects. Second, the dose-response curves for these
malformations are generally nonlinear and quite steep (Rider
et al. 2008), which enables us to easily distinguish dose-
addition model predictions from response or integrated addition-
model predictions. In contrast to the malformation data,
anogenital distance (AGD) and several other low-dose effects
appear linear in the low dose range and one cannot easily
distinguish the manner of interaction (dose versus response-
addition model predictions) of the chemicals in the mixture.
Mixtures with Chemicals That Have the Same Mechanism
of Toxicity: Our first research goal was to confirm that
antiandrogenic chemicals with the same mechanism of toxicity
106 RIDER ET AL.TOXICOLOGIC PATHOLOGY
conformed to a model of dose addition. We began this work by
assessing simple binary mixtures. The first binary mixture was
composed of the AR antagonists vinclozolin and procymidone
(Gray et al. 2001); a second consisted of two phthalate esters
with a common active metabolite (DBP and BBP); and the third
was made up of two phthalate esters with different active
metabolites (DEHP and DBP) (Howdeshell et al. 2007). The
results from our binary studies with chemicals that disrupt
androgen signaling via a similar mechanism of action were
generally consistent with predictions based on a dose-addition
model for inducing malformations in male rats exposed in utero.
Vinclozolin plus Procymidone Mixtures
In ourAR antagonist binary study (Gray et al. 2001), in utero
exposure to vinclozolin alone resulted in a 10% incidence of
hypospadias and a 0% incidence of vaginal pouch development
in male rats, whereas procymidone exposure resulted in a 0%
incidence of either malformation. The combination exposure,
however, resulted in a 96% incidence of hypospadias and 54%
incidence of vaginal pouch in treated animals (Figure 2).
Similar effects have been seen in studies of F1 male rats
exposed to mixtures of vinclozolin and procymidone during
sexual differentiation (Christiansen et al. 2008; Gray et al. 2001;
Gray et al. 2006; Hass et al. 2007; Metzdorff et al. 2007; Rosen
et al. 2005; Wilson et al. 2008). Short-term studies using cas-
trated, immature male androgen-treated rats (Gray et al. 2001;
Nellemann et al. 2003) also demonstrate that vinclozolin and
procymidone induce cumulative effects when coadministered.
Phthalate Mixture Studies
In both binary phthalate mixture studies, exposure to the
individual chemicals resulted in no malformations or low
incidences of malformations, and the combination exposures
typically resulted in 50% or greater incidences of malforma-
tions (Figures 3 and 4) (Howdeshell et al. 2007).
In a more complex mixture study, we assessed the cumulative
effects on fetal testosterone production following in utero expo-
sure to a mixture of five phthalates: DBP, di-iso-butyl phthalate
(DiBP), BBP, DEHP, and DPP (Howdeshell et al. 2008). First,
we characterized the individual chemical dose-response relation-
ships as described above (Figure 5). We then dosed animals on
GD 8–18 with a mixture of the five phthalates. The mixture was
designed such that every chemical would contribute equally to
the reduction in fetal testosterone production. Finally, observed
mixture responses were compared to responses predicted based
on a model of dose addition (Figure 6).
From these mixture studies, we conclude that chemicals that
target the androgen signaling pathway via the same mechanism
of action are dose additive when present in a mixture. Currently,
cumulative risk assessments have not been performed on the
antiandrogenic chemicals. This work indicates that these chem-
icals would be good candidates for cumulative risk assessment
and supports the use of the dose-addition model for determining
the effects of these mixtures.
Mixtures with Chemicals That Have
Different Mechanisms of Toxicity
We hypothesized that chemicals with different mechanisms
of toxicity that target the same signaling pathway would
exhibit cumulative effects that conform to a model of dose
addition, not response addition. To test this hypothesis, we first
assessed the joint effects of binary mixtures of chemicals with
different mechanisms of action.
The first binary mixture consisted of a fetal testosterone
inhibitor (BBP) and an antiandrogen with multiple mechanisms
FIGURE 2.—Male rat reproductive tract malformations following in
utero exposure to vinclozolin and procymidone alone or in combination
(Hotchkiss et al. unpublished data).
FIGURE 3.—Male rat reproductive tract malformations following in
utero exposure to BBP and DBP alone or in combination (Hotchkiss,
et al. unpublished data).
Vol. 37, No. 1, 2009 CUMULATIVE EFFECT OF ANTIANDROGENS IN UTERO107
of action (linuron) (Hotchkiss et al. 2004). The second binary
mixture consisted of DBP and the AR antagonist procymidone.
In these studies, pregnant rats were dosed on GD 14–18 with
either the individual compounds or the binary mixture at a dose
level equivalent to approximately one half of the ED50 value for
malformations (Table 2).
BBP plus Linuron: In the BBP and linuron study, in utero
exposure to BBP alone elicited a 0% incidence of hypospadias
and vaginal pouch formation and a 12% incidence of
epididymal agenesis in male rats. In utero exposure to linuron
alone resulted in a 0% incidence of hypospadias and vaginal
pouch development and a 63% incidence of epididymal
agenesis. However, exposure to the combination resulted in
cumulative effects, with males displaying 56%, 40%, and 97%
FIGURE 4.—Male rat reproductive tract malformations following in
utero exposure to DEHP and DBP alone or in combination. Results
were originally presented in Howdeshell et al. (2007).
FIGURE 6.—Comparison of fetal testosterone reduction following indi-
vidual phthalate exposure to a mixture of five phthalates (BBP, DPP,
DEHP, DBP, and DiBP) and predictions based on a dose-addition
model. Data were originally presented in Howdeshell et al. (2008).
FIGURE 7.—Male rat reproductive tract malformations following in
utero exposure to BBP and linuron alone or in combination. Data were
originally presented in Hotchkiss et al. (2004).
FIGURE 5.—Individual chemical dose-response data fit with a logistic
model from six individual phthalate dose-response studies with BBP,
DPP, DEHP, DBP, DiBP, and DEP. Results were originally presented
in Howdeshell et al. (2008).
incidence of hypospadias, vaginal pouch, and epididymal
agenesis, respectively (Hotchkiss et al. 2004) (Figure 7).
Di-n-butyl Phthalate plus Procymidone: In the procymidone
plus DBP study, procymidone, or DBP alone induced low
incidences of hypospadias (1.5% and 0%, respectively) and
vaginal pouch (0% and 0%, respectively), whereas the males
treated with the combination of procymidone and DBP displayed
49% and 27% incidences of hypospadias and vaginal pouch,
respectively, indicating that the interaction was at least dose
108 RIDER ET AL.TOXICOLOGIC PATHOLOGY
additive (Figure 8). We are currently conducting an expanded
binary study including multiple doses of a fixed-ratio mixture of
DBP and procymidone. Initial results demonstrate that responses
to the binary mixture conform to a model of dose addition, not
response addition (Hotchkiss et al. unpublished data).
Vinclozolin, Procymidone, Prochloraz, Linuron, and Three
Phthalates (DBP, BBP, DEHP): To further test our hypothesis,
we designed a study with seven antiandrogenic chemicals with
diverse mechanisms of action including AR antagonists
(vinclozolin and procymidone), mixed-mechanism chemicals
and prochloraz), and testosterone synthesis–inhibiting phthalates
(BBP, DBP, and DEHP) (Rider et al. 2008). According to the
current mixtures paradigm, this seven-antiandrogenic-chemical
mixture should conform to a model of integrated addition.
However, we found that models of integrated addition or
response addition consistently underestimated the effects of our
model and the related toxic equivalency approach provided
estimates of mixture responses that approximated the observed
responses (Figure 9). For example, hypospadias was seen in
100% of the high-dose animals, and whereas dose addition and
toxic equivalency models predicted 70% affected, integrated and
response-addition models predicted 0% affected.
Our future studies will be designed to answer questions
concerning the characteristics of chemicals that contribute to
making them dose additive. Currently, the focus of cumulative-
risk assessments is on chemicals with the same mechanism of
action. However, we have demonstrated that chemicals with
different mechanisms of action that target a common signaling
pathway can also display dose additivity. Our future studies
will be aimed at refining our experimental designs for mixture
assessments and testing the boundaries of dose additivity with
chemicals that target male reproductive tract development. In
future studies, we will continue testing mixtures of chemicals
that target androgen signaling in studies designed to provide
clear distinctions among predictions based on each of the mix-
ture models. For example, we are currently building on our pre-
vious study with antiandrogens by increasing the number of
chemicals included in the mixture to ten. The goal of increas-
ing the number of chemicals is to have all mixture components
present in the mixture at doses clearly below their individual no
observable adverse effect (NOAEL) levels. This scenario
allows for a greater distinction between models of response
addition (where chemicals below their NOAELs do not con-
tribute to the mixture toxicity) and dose addition (where chem-
icals below their NOAELs do contribute to the overall mixture
To date, the chemicals tested in our mixtures studies target
male reproductive tissues through interference with androgen
signaling. Other chemicals disrupt male reproductive tissue
development through mechanisms that are not fully under-
stood. For example, in utero exposure to TCDD results in epi-
didymal malformations in male rats that do not involve AR
antagonism or testosterone synthesis inhibition. Currently, we
are assessing a binary mixture of TCDD and DBP to ascertain
whether these chemicals act in a dose-additive manner to elicit
Our binary mixture studies were designed to combine pairs
of chemicals at doses where each chemical would produce few
if any malformations, but doubling the dose of one would
induce malformations in about 50% of the males. If the chem-
icals behaved in a dose-additive, cumulative fashion, then the
mixture would produce malformations in 50% of the males, but
if they interacted independently, then few males would be mal-
formed. Our results clearly show that all binary combinations
produced cumulative, dose-additive effects on the androgen-
We also conducted a complex mixture study combining
seven “antiandrogens” together. These chemicals elicit antian-
drogenic effects at two different sites in the androgen signaling
pathway (i.e., AR antagonist or inhibition of androgen synthe-
sis). In this study, the complex mixture also behaved in a dose-
Our results indicate that compounds that act by disparate
mechanisms of toxicity display cumulative, dose-additive
effects when present in combination. The results also suggest
that a modification of the approach for cumulative risk assess-
ments from one based upon “common mechanism of toxicity”
to one that includes the cumulative assessment of chemicals that
FIGURE 8.—Male rat reproductive tract malformations following in
utero exposure to procymidone and DBP alone or in combination
(Hotchkiss et al. unpublished data).
Vol. 37, No. 1, 2009 CUMULATIVE EFFECT OF ANTIANDROGENS IN UTERO109
disrupt development of the same reproductive tissues during
sexual differentiation would result in target organ- and timing-
based approach rather than on a narrow mechanism of toxicity.
We propose that the primary focus should be on the biological
system (e.g., androgen signaling pathway) rather than the mech-
anism of toxicity and that a cumulative risk assessment could
potentially include all chemicals that target that system during
the same critical developmental period.
FIGURE 9.—Comparison of observed and predicted responses to a mixture of seven antiandrogens including: vinclozolin, procymidone, linuron,
prochloraz, and three phthalates (BBP, DBP, and DEHP). Originally presented in Rider et al. (2008).
110 RIDER ET AL.TOXICOLOGIC PATHOLOGY
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