Critical Reviews in Toxicology, 35:713719, 2005

Copyright
c Taylor and Francis Inc.

ISSN: 1040-8444 print / 1547-6898 online
DOI: 10.1080/10408440591007395

Mode of Action: Impaired Fetal Leydig Cell

FunctionEffects on Male Reproductive Development
Produced by Certain Phthalate Esters
Paul M. D. Foster
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
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Certain phthalate esters (di-2-ethylhexyl; di-n-butyl and butyl benzyl) have profound effects on
the developing male reproductive system when administered orally to pregnant experimental
animals during a critical window of development. These esters produce a syndrome of adverse
effects that are characteristic of a disturbance in androgen-mediated development and include
a variety of reproductive tract malformations and effects on developmental phenotypic mark-
ers. A testicular dysgenesis syndrome has been proposed to explain the secular increases in a
number of human male reproductive deficits, including decreased semen parameters, increased
incidence of cryptorchidism and hypospadias (two of the most common human birth defects),
and increased incidence of testicular (germ cell derived) cancer. The rodent phthalate data
lend support to the hypothesis. This example illustrates a number of points in the use of the
Human Relevance Framework. First, chemical agents may have more than one mode of action
(MOA): for example, phthalate-induced peroxisome proliferation leading to hepatocarcinogen-
For personal use only.

sis, compared with the induction of developmental effects via effects on androgen signaling.
Second, the case demonstrates the life-stage sensitivity of the response to these compounds.
Third, because humans may be exposed to multiple phthalate esters producing adverse effects
on reproductive development, these compounds may be useful in testing the utility of the Human
Relevance Framework (HRF) approach for evaluating cumulative and aggregate risk.

Keywords Di-n-Butyl Phthalate, Endocrine Disruptors, Human Relevance, Mechanisms

Certain phthalate esters (di-2-ethylhexyl, DEHP; di-n-butyl,
DBP; and butyl benzyl, BBP) have profound effects on the de-
veloping male reproductive system when administered orally to
experimental animals during a critical window of development
(gestation days 1221 for the rat) (Mylchreest et al., 1998, 1999a,
1999b; Gray et al., 2000, 2003). These results are of interest to
risk assessors because of the potential for human exposure from
a wide variety of sources. Although several esters have effects,
studies with DBP and, to a more limited extent, DEHP provide
the basis for this article.
These esters produce a syndrome of adverse effects in male
rat offspring after in utero exposure that is characteristic of
a disturbance in androgen-mediated development. The syn-
drome is characterized by malformations of the epididymis,
The views presented in this article do not necessarily reflect those
of the National Institute of Environmental Health Sciences.
Address correspondence to Dr. Paul M. D. Foster, National Institute
of Environmental Health Sciences, P.O. Box 12233, Mail Code E-
106, Research Triangle Park, NC 27709 USA. E-mail: foster2@niehs.
nih.gov
vas deferens, seminal vesicles, prostate, external genitalia (hy-
pospadias), cryptorchidism, and testicular injury, together with
permanent changes (feminization) in the retention of nipples/
areolae (sexually dimorphic structures in rodents) and demas-
culinization of the growth of the perineum resulting in a reduced
anogenital distance (AGD). These effects constitute a continuum
of response, with the most severe manifestations and highest in-
cidence of reproductive-tract malformations observed at higher
dose levels (500 mg DBP/kg/day), whereas changes in AGD
and areolae retention are frequently seen at lower dose levels
(100 mg DBP/kg/day). These may be permanent phenotypic
changes in affected individual animals. The general potency of
response on reproductive development is DEHP > DBP > BBP.
Interestingly, most of these adverse responses have not been ob-
served in classical (exposure on gestation days 615) teratology
studies in rats. In the case of DBP, in utero exposure has also re-
sulted in testicular Leydig cell adenoma in 90-day-old offspring
(Mylchreest et al., 1999a). This tumor type is usually found at
around 35% in controls of the rat strain employed in the study
just described and is generally considered to be a tumor of old
age (most frequently noted at the end of a 2-year bioassay).

713
 714 P. M. D. FOSTER

A testicular dysgenesis syndrome (i.e., a failure of normal in
utero development of the testis) has been proposed to explain
the secular increases in a number of human male reproductive
deficits, including decreased semen parameters, increased inci-
dence of cryptorchidism and hypospadias (two of the most com-
mon human birth defects), and increased incidence of testicular
(germ-cell-derived) cancer (Sharpe, 2001, 2003; Skakkebaek,
2002). Thus far, no cause-and-effect relationship has been es-
tablished between any environmental agent and these human
deficits. However, the rodent data lend support to the hypothesis
(Sharpe, 2001).
This example illustrates a number of interesting points in
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Wolffian duct system to develop normally into the vas deferens,
epididymis, and seminal vesicles (Barlow and Foster, 2003).
Lower testosterone levels also impact the dihydrotestosterone
(DHT)-induced development of the prostate and external geni-
talia (testosterone is converted to DHT by 5-reductase). DHT
is also responsible for the normal apoptosis of nipple anlagen in
males, resulting in the lack of nipple development, and also
for the growth of the perineum to produce the normal male
anogenital distance (approximately twice that of the female).
Testicular descent into the scrotum also requires normal andro-
gen levels and a failure of descent results in cryptorchidism.
However, the initial stages of testicular descent involve the ac-

the use of the human relevance framework. First, chemical
agents may have more than one MOA: in this case, phthalate-
induced peroxisome proliferation leading to hepatocarcinogen-
sis, as well as induction of developmental effects. Second,
the case demonstrates the life-stage sensitivity of the response
to these compounds. Specifically, phthalate esters have been
known for decades to produce testicular effects in rodents
(Foster et al., 1980) with important stage-related differences:
Sertoli cells as the probable testicular target in pubertal and adult
animals (Foster et al., 1982; Creasy et al., 1987), but a different
MOA and different initial testicular target (fetal Leydig cells) in
fetal animals. Third, because humans may be exposed to mul-
For personal use only.
tion of another Leydig cell product, insl3 (insulin-like factor 3),
the message of which is also downregulated by DEHP, DBP,
and BBP (Wilson et al., 2004). Female rat fetuses are predom-
inantly unaffected after treatment with these specific phthalate
esters.
Key Events
Table 1 describes the key events in the MOA of phthalate
esters (based primarily on data from DBP and DEHP) in animals.
A critical first step in the toxicity of phthalate diesters is the
conversion, by hydrolysis, to the monoester (Lake et al., 1977).
This can occur in the gut, in the liver, or in the blood. There is

tiple phthalate esters producing adverse effects on reproductive
development in test animals, these compounds may be useful in
testing the utility of the Human Relevance Framework (HRF)
approach for evaluating cumulative and aggregate risk.
I. IS THE WEIGHT OF EVIDENCE SUFFICIENT
TO ESTABLISH THE MOA IN ANIMALS?
A significant decrease in the testicular production of testos-
terone by rat fetal Leydig cells (Parks et al., 2000; Shultz
et al., 2001; Mylchreest et al., 2002) at time points critical in
male reproductive system development results in failure of the
some evidence that monoesters are preferentially absorbed from
the gut. Once converted to the monoester, the entity believed
responsible for toxicity, it then readily crosses the placenta to
transfer to the fetus (Singh et al., 1975; Saillenfait et al., 1998).
One of the earliest phthalate-related fetal effects observed
in rats was disturbance of normal fetal testicular Leydig cell
function and/or development (Parks et al., 2000; Shultz et al.,
2001; Mylchreest et al., 2002; Barlow et al., 2003). This can
result in fetal Leydig cell hyperplasia or large aggregates
of fetal Leydig cells (at gestation day 21) in the developing
testis. Downregulation of genes critical for normal steroid

TABLE 1
Key events in the animal MOA for phthalate

Key event Evidence in animals References

1. Hydrolysis to produce monoester
and transport to target
2. Impaired Leydig cell function
a. Decrease in fetal testicular
androgen production
b. Decreased androgen mediated
gene expression and insl3
expression in fetal testes
3. Androgen insufficiency syndrome/
testicular dysgenesis
YES. Hydrolysis measured in a number of
animal species; passage of monoester to the
fetus measured in rats
YES. Levels of testosterone measured in fetal
testes; increased levels of precursors
indicate functional change at CYP 17
YES. Measured in fetal rat testes. insl3
knock-out mouse results in cryptorchidism
YES. Noted in rats (and rabbits). Effects noted
on Wolffian duct development, nipples,
prostate, hypospadias, cryptorchidism
Lake et al., 1977; Singh et al., 1975;
Saillenfait et al., 1998
Parks et al., 2000; Mylchreest et al.,
2002; Shultz et al., 2001
Barlow et al., 2003; Adham et al.,
2000
Gray et al., 2000; Barlow and Foster,
2003; Higuchi et al., 2003
 IMPAIRED FETAL LEYDIG-CELL FUNCTION
production and transport of critical intermediates is noted prior
to observation of increased cell aggregates (Shultz et al., 2001;
Barlow et al., 2003).
Recent data indicate a decrease in the expression of the
Leydig cell product insl3 in rat fetal testes after DEHP (750
mg/kg/day), DBP (1 g/kg/day), or BBP (1 g/kg/day) exposure
in utero on GD 1418 (Wilson et al., 2004). This may be related
to an increased incidence of cryptorchidism after fetal exposure
to phthalates. Knockouts of this gene in mice show complete
cryptorchidism (Nef and Parada, 1999; Adham et al., 2000; Nef
et al., 2000). The insl3 gene is associated with Leydig cell dif-
ferentiation and overall levels of testosterone, with high levels
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in fetal rat testes that decrease after birth, rise again at puberty,
but decrease again in old animals (Paust et al., 2002). These au-
thors also review the relationship of the SF-1 promotor found
on the insl3 gene with those found on many of the genes con-
trolling the steroidogenic machinery in Leydig cells. Although
human polymorphisms for insl3 have not been reported, they
have been noted for the insl3 receptor (LGR8), which has been
shown recently to be related to cryptorchidism in humans (Ivell
and Hartung, 2003).
In addition to morphological changes in Leydig cells in
the interstitium of the fetal rat testis, the seminiferous cords
also show a marked disturbance of normal gonocyte prolifera-
For personal use only.
tion, resulting in multinucleated cells (Mylchreest et al., 2002;
Barlow and Foster, 2003) and subsequent changes in the de-
veloping germ-cell population. In the human fetus, impaired
gonocyte development is an early change in the induction of
germ-cell-derived testicular cancer (Rajpert-De Meyts et al.,
1998). A marked decrease in testosterone production by fetal
Leydig cells is critical to the subsequent reproductive tract mal-
formations in rats exposed to specific phthalate esters (Parks
et al., 2000; Mylchreest et al., 2002). This reduction in an-
drogen leads to abnormal reproductive tract development. For
example, lower AR expression in epithelial cells of fetal epi-
didymis is followed by Wolffian duct failure to coil and de-
velop normally into the epididymis (Mylchreest et al., 2002;
Barlow and Foster, 2003). These androgen-dependent adverse
outcomes affecting the Wolffian duct and prostatetogether
with hypospadias, cryptorchidism, retention of nipples, and a
permanently reduced anogenital distancedescribe a syndrome
of effects characteristic of specific phthalate esters. The interre-
lationship of key factors in the animal MOA is displayed dia-
grammatically in Figure 1. This syndrome has parallels with the
reported human testicular dysgenesis syndrome (Sharpe, 2001;
Fisher et al., 2003) and also shows similarities to other known
human genetic syndromes involving impaired androgen respon-
siveness in sexual differentiation of the reproductive tract (for
review, see Hughes, 2001).
Almost all the mechanistic studies on gene expression and
MOA with phthalates noted above have utilized dose lev-
els that ensure significant prevalence of fetal reproductive
abnormalitiesfor example, 500 mg/kg/day for DBP; however,
a recent paper (Lehmann et al., 2004) reports significant de-
715
creases in testosterone levels and significant changes in gene ex-
pression associated with Leydig cell differentiation in fetal testes
at dose levels below the 50-mg/kg/day no-observed-adverse-
effect level (NOAEL) previously reported for DBP.
Other Information
Studies on the reproductive effects of specific phthalate es-
ters have been conducted predominantly in rats, but some sim-
ilar events have also been noted in the rabbit (Higuchi et al.,
2003). No specific studies have been conducted evaluating re-
productive development in mice exposed to phthalates during
gestation. However, several phthalate esters produce reproduc-
tive toxicity in adult male and female mice and are teratogenic
to fetuses exposed in classical teratology studies when treatment
is from implantation to closure of the hard palate, with fetuses
examined just prior to term (see Center for the Evaluation of
Risk to Human Reproduction [CERHR] reviews). Phthalate es-
ter monoesters (and not the parent diesters) can impair Leydig
cell function in adult rat cells in vitro (Jones et al., 1993), but
at dose levels significantly higher (1 mM) than those affecting
Sertoli cell function (10100 nM) in culture (Lloyd and Foster,
1988). Moreover, in vivo effects on the adult rat testis occur at
significantly higher dose levels than in fetal rats (100 mg/kg/day,
versus 2 g/kg/day for DBP).
In contrast to its role in the hepatic effects of phthalate es-
ters, PPAR is unlikely to be involved in these testicular and
developmental toxicities. This conclusion is based in part on
the presence of testicular and developmental toxicity in PPAR
knockout mice (Peters et al., 1997; Ward et al., 1998), and in part
on data showing that the guinea pig, a nonresponder to PPAR
ligands, is as sensitive to the testicular effects of phthalates as
are rats (Gray et al., 1982). While all the manifestations of spe-
cific phthalate esters on reproductive development are similar
to those seen with classical androgen receptor (AR) antagonists
such as flutamide, phthalate diesters and monoesters have not
been shown to interact with either rat or human AR using binding
or transcriptional activation assays (Foster et al., 2001).
II. ARE KEY EVENTS IN THE ANIMAL MOA PLAUSIBLE
IN HUMANS?
The action of certain phthalate esters on male reproductive
development has been evaluated primarily in rats, with more lim-
ited evidence obtained in rabbits (DEHP; Higuchi et al., 2003)
and one report on DPB effects on gonadal development in frogs
(Ohtani et al., 2000). Although there are no human data relating
exposure to phthalate esters with adverse outcomes in terms of
reproductive development, the critical metabolites for reproduc-
tive and developmental toxicity, the phthalate monoesters, are
produced in humans (Anderson et al., 2001; Barr et al., 2003)
and are commonly found in urine samples from the general
population (Blount et al., 2000; Barr et al., 2003) and in children
(Brock et al., 2002).
The critical enzymes involved in steroidogenesis are identi-
cal in rats and humans, and all mammals are believed to have
 716 P. M. D. FOSTER
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FIG. 1. Diagrammatic representation of the interaction between the fetal seminiferous cords and Leydig cells and critical gene
involvement following in utero phthalate exposure in rats. Deficits in androgen production result in a syndrome of malformations
(shown in yellow) that includes malformations of the epididymis, seminal vesicles, and prostate together with an increased incidence
of cryptorchidism and hypospadias. Male offspring also show a feminized phenotype with a permanently reduced anogenital distance
(AGD) and retention of nipples. Genes significantly down regulated by di-n-butyl phthalate include stem-cell factor and its receptor
c-kit, the high-density lipoprotein (HDL) receptor (SRB-1), steroid acute regulatory protein (StAR), and the genes coding for the
enzymes involved in the conversion of cholesterol to pregnenlone (side-chain cleavage enzyme), pregnenolone to progesterone (3
hydroxysteroid dehydrogenase), and progesterone to androstenedione (CYP 17), resulting in a lowered production of testosterone
necessary for normal reproductive development (Barlow et al., 2003). Others have shown a decrease in insl3 gene expression
following in utero exposure to DEHP (Wilson et al., 2004).

parallel activation mechanisms for androgen dependent pro-
cesses. Based on our current knowledge of comparative physi-
ology and endocrinology, it is thus possible for fetuses that have
been sufficiently exposed to specific phthalate monoesters to
experience antiandrogenic effects. Table 2 illustrates the plau-
sibility of the biological process occurring in humans, although
there is no direct evidence that this MOA occurs.
The testicular tumors observed in rats are Leydig cell ade-
nomas that have a developmental origin (Mylchreest et al.,
1999a; Barlow et al., 2004), not the germ-cell tumors most fre-
quently noted in humans. However, abnormal fetal gonocyte
development is a precursor event to germ-cell cancer in humans
(Skakkebaek et al., 1987; Rajpert-De Meyts et al., 1998), and
this has been seen in fetal testes from rats treated with DBP
(Barlow and Foster, 2003) and DEHP (Parks et al., 2000). Inter-
estingly, Leydig cell adenomas (using the rodent pathological
definition) appear to be fairly common in humans, but they are
termed micronodules, rather than tumors, according to a recent
publication (Holm et al., 2003). Adverse reproductive conse-
quences have been reported in some studies that purport to re-
late specific phthalate exposure with adverse human outcomes
including sperm measures (Duty et al., 2003) and premature
breast development (Colon et al., 2000), but these limited case
reports should be treated with caution. Several NTP Center for
the Evaluation of Risk to Human Reproduction (CERHR) mono-
graphs on specific phthalate esters, including DEHP (Kavlock
 IMPAIRED FETAL LEYDIG-CELL FUNCTION 717

TABLE 2
Concordance analysis of key events in phthalate MOA

Key event Evidence in animals Evidence in humans References

1. Hydrolysis to produce
monoester and transport to
target
2. Impaired Leydig cell
Function
a. Decrease in fetal
testicular androgen
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YES. Hydrolysis measured in
a number of animal species;
passage of monoester to the
fetus measured in rats.
YES. Levels of testosterone
measured in fetal testes;
increased levels of
precursors indicate
YES. Phthalate monoesters Schmid and Schlatter, 1985;
measured in human urine Anderson et al., 2001
No. data. However, steroid Hughes, 2001
synthetic enzymes and
pathways similar/identical
in humans; androgen

production functional change at CYP critical for normal human

b. Decreased androgen
mediated gene
expression and insl3
expression in fetal testes
3. Androgen insufficiency
syndrome/ testicular
dysgenesis
For personal use only.
17
YES. Measured in fetal rat
testes; insl3 knock-out
mouse results in
cryptorchidism
YES. Noted in rats (and
rabbits); effects noted on
Wolffian duct development,
male development, with
syndromes reported for
genetic defects
No. data. Insl3 exists in Ivell and Hartung, 2003
humans. Polymorphisms of
the insl3 receptor (LGR8)
have been related to
cryptorchidism.
No. data. Human syndromes Barthold et al., 2000; Nitsche
exist related to Low DHT and Hiort, 2000;
levels, or impaired Imperato-McGinley, 2002;

nipples, prostate, androgen receptor function; Yong et al., 2003; Toppari

hypospadias, cryptorchidism and et al., 2001
cryptorchidism hypospadias are the most
common human birth
defects

et al., 2002a) and DBP (Kavlock et al., 2002b), address some of
these issues in more detail.
III. TAKING INTO ACCOUNT KINETIC AND DYNAMIC
FACTORS, IS THE ANIMAL MOA PLAUSIBLE
IN HUMANS?
Toxicodynamic factors critical to adverse effects on repro-
ductive outcomes from phthalate exposure during development
are similar in rats and humans. In addition, humans can indeed
produce the metabolites responsible for reproductive and devel-
opmental toxicity in animals. For these reasons, key events in the
animal MOA are plausible in humans if the exposure level is high
enough. For DEHP, humans produce more of the inactive mo-
noester glucuronide in urine than rodents do for a given dose,
but the placenta and fetus have active glucuronidase enzymes
that could release active monoester at critical targets. More-
over, humans and primates appear to have saturated metabolism
in terms of producing free phthalate monoester, with nonlinear
production of the monoester from diesters administered at dose
levels above approximately 200250 mg/kg/day (for DEHP).
However, adverse effects on reproductive development in rats
have been reported below this saturation level, where the re-
sponse is linear for rodents and humans. Actualization of the
MOA in humans depends, then, on the likelihood of human ex-
posure levels becoming sufficient to induce these key events in
the MOA.
IV. STATEMENT OF CONFIDENCE AND IMPLICATIONS
Confidence is high that the evidence presented supports the
described MOA of specific phthalate esters in rats. The com-
ponent features of the MOA are also plausible in humans.
Elements of the phthalate syndrome on reproductive develop-
ment can be noted in animals (for DEHP) at dose levels below
30 mg/kg/day, whereas real-world human exposure in the gen-
eral population to individual phthalates has been reported in the
50100 g/kg/day range. Whether such exposure could trigger
the key events just noted is unknown. Clearly, better information
is required in humans relating the known exposure to adverse
health outcomes, particularly in specific groups such as pregnant
women and infant boys.
ACKNOWLEDGMENTS
The ILSI Risk Science Institute and the Human Relevance
Phase II Workgroup greatly appreciate the work of the June 2004
 718 P. M. D. FOSTER
Peer Review Panel and Dr. John Moores skilled facilitation as
chairman. This ILSI Risk Science Institute project was supported
by funding from the Office of Pesticide Programs and the Office
of Pollution Prevention and Toxics of the U.S. Environmental
Protection Agency and by the Existing Substances Division of
Health Canada.
In addition to the authors of the overview for this special
issue, members of the Phase II Workgroup included Dr. Elaine
Faustman, Dr. Robert Snyder, Dr. Vicki Dellarco, and Dr. Karl
Baetcke. Dr. James Klaunig chaired the workgroup.
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