In vivo effects of bisphenol A in laboratory rodent studies

Article (PDF Available)inReproductive Toxicology 24(2):199-224 · August 2007with73 Reads
DOI: 10.1016/j.reprotox.2007.06.004 · Source: PubMed
Abstract
Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/(kg day), in laboratory exposures with mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol and ethinylestradiol. For most effects, the potency of BPA is approximately 10-1000-fold less than that of diethylstilbestrol or ethinylestradiol. Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long lasting, organizational effects in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely, but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that developmental effects occur in the female reproductive system.

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Reproductive Toxicology 24 (2007) 199–224
Review
In vivo effects of bisphenol A in laboratory rodent studies
Catherine A. Richter
a,
, Linda S. Birnbaum
b
, Francesca Farabollini
c
, Retha R. Newbold
d
,
Beverly S. Rubin
e
, Chris E. Talsness
f
, John G. Vandenbergh
g
,
Debby R. Walser-Kuntz
h
, Frederick S. vom Saal
i
a
U.S. Geological Survey, Columbia Environmental Research Center, Columbia, MO 65201, United States
b
U.S. Environmental Protection Agency, Research Triangle Park, NC, United States
c
Department of Physiology, University of Siena, Siena, Italy
d
National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC, United States
e
Department of Anatomy and Cell Biology, Tufts University School of Medicine, Boston, MA, United States
f
Institute of Clinical Pharmacology and Toxicology, Charit´e Universit¨atsmedizin Berlin, Berlin, Germany
g
Department of Zoology, North Carolina State University, Raleigh, NC, United States
h
Department of Biology, Carleton College, Northfield, MN, United States
i
Division of Biological Sciences, University of Missouri, Columbia, MO, United States
Received 2 March 2007; received in revised form 6 June 2007; accepted 11 June 2007
Available online 26 June 2007
Abstract
Concern is mounting regarding the human health and environmental effects of bisphenol A (BPA), a high-production-volume chemical used in
synthesis of plastics. We have reviewed the growing literature on effects of low doses of BPA, below 50 mg/(kg day), in laboratory exposures with
mammalian model organisms. Many, but not all, effects of BPA are similar to effects seen in response to the model estrogens diethylstilbestrol
and ethinylestradiol. For most effects, the potency of BPA is approximately 10–1000-fold less than that of diethylstilbestrol or ethinylestradiol.
Based on our review of the literature, a consensus was reached regarding our level of confidence that particular outcomes occur in response to low
dose BPA exposure. We are confident that adult exposure to BPA affects the male reproductive tract, and that long lasting, organizational effects
in response to developmental exposure to BPA occur in the brain, the male reproductive system, and metabolic processes. We consider it likely,
but requiring further confirmation, that adult exposure to BPA affects the brain, the female reproductive system, and the immune system, and that
developmental effects occur in the female reproductive system.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Behavior; Neuroendocrine; Endocrine disruptors; Immune system; Metabolism; Mouse; Rat; Reproduction
Abbreviations: 2F, located in utero between female siblings; 2M, located in utero between male siblings; ADI, acceptable daily intake; AhR, aryl hydrocarbon
receptor; ARC, arcuate nucleus; AVPV, anteroventral periventricular preoptic area; BPA, bisphenol A; Crl:CD(SD), Charles River Laboratories Sprague–Dawley rat
strain; DES, diethylstilbestrol; DRN, dorsal raphe nucleus; EDC, endocrine disrupting chemical; E2, 17-estradiol; ER, estrogen receptor protein ;ER, estrogen
receptor protein ; Esr1, estrogen receptor gene 1 (); Esr2, estrogen receptor gene 2 (); GD, gestation day; HRT, hormone replacement therapy; IFN, interferon; IgE,
immunoglobulin E; i.m., intramuscular; i.p., intraperitoneal; LD50, lethal dose, 50% of population; LH, luteinizing hormone; LoEC, lowest effective concentration;
LOAEL, lowest observed adverse effect level; MPA, medial preoptic area; NMU, N-nitroso-N-methylurea; NTP, National Toxicology Program; PND, postnatal day;
PIN, prostate interepithelial neoplasia; p.o., oral adminisration; RAR, retinoic acid receptor; RXR, retinoid X receptor; SERM, selective estrogen receptor modulator;
s.c., subcutaneous; Th1, T helper lymphocyte 1; Th2, T helper lymphocyte 2; TH, tyrosine hydroxylase; VMH, ventromedial nucleus
The information in this document has been subjected to review by the National Health and Environmental Effects Research Laboratory, U.S. Environmental
Protection Agency, and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or
commercial products constitute endorsement or recommendation for use.
Corresponding author at: 4200 New Haven Road, Columbia Environmental Research Center, USGS, Columbia, MO 65201, United States.
Tel.: +1 573 876 1841; fax: +1 573 876 1896.
E-mail address: Crichter@usgs.gov (C.A. Richter).
0890-6238/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.reprotox.2007.06.004
200 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
Contents
1. Introduction ............................................................................................................ 200
1.1. Rationale for experimental design, analysis and reporting criteria ...................................................... 201
1.1.1. Animal model ............................................................................................ 202
1.1.2. Feed, water, and housing ................................................................................... 202
1.1.3. Method of dosing ......................................................................................... 202
1.1.4. Design and analysis issues ................................................................................. 202
1.2. Definition of “low dose” ........................................................................................... 202
1.3. Timing of exposure ............................................................................................... 203
2. Developmental effects of BPA due to exposure during gestation through puberty............................................... 203
2.1. Effects on neurotransmitters and their receptors, neuroendocrine function and hormone receptors in the brain in
males and females ................................................................................................ 203
2.1.1. Receptors ................................................................................................ 203
2.1.2. Neuroendocrine effects .................................................................................... 203
2.1.3. Rapid signaling effects .................................................................................... 210
2.1.4. Brain structure ............................................................................................ 210
2.1.5. Behavioral effects ......................................................................................... 210
2.1.6. Sex differences ........................................................................................... 211
2.2. Developmental effects on the female reproductive tract ............................................................... 211
2.2.1. Timing of puberty ......................................................................................... 211
2.2.2. Mammary gland .......................................................................................... 211
2.2.3. Uterus and vagina ......................................................................................... 212
2.2.4. Ovary, oocytes and fertility ................................................................................ 212
2.3. Developmental effects on the male reproductive tract ................................................................. 212
2.3.1. Serum testosterone levels .................................................................................. 212
2.3.2. Prostate .................................................................................................. 213
2.3.3. Testes, epididymis, sperm and seminal vesicles .............................................................. 213
2.4. Developmental effects on metabolism ............................................................................... 214
2.5. Developmental effects on the immune system ........................................................................ 214
3. Effects of BPA exposure during adulthood ................................................................................. 214
3.1. Adult effects on the brain and behavior .............................................................................. 214
3.2. Adult effects on the female reproductive tract ........................................................................ 215
3.3. Adult effects on the male reproductive tract .......................................................................... 215
3.4. Adult effects on metabolism ....................................................................................... 215
3.5. Adult effects on the immune system ................................................................................ 216
4. Comparison of findings of significant effects and no-significant effects in low dose BPA studies ................................. 216
4.1. Strain differences ................................................................................................. 216
4.2. BPA as a SERM: responses in different tissues and in different animal models .......................................... 217
4.3. Batch-to-batch variability in feed: impact on low dose endocrine disruptor research ...................................... 217
4.4. The use of positive controls ........................................................................................ 217
4.5. The importance of dose ............................................................................................ 218
5. Conclusions and levels of confidence for different outcomes ................................................................. 218
5.1. Based on existing evidence, we are confident of the following ......................................................... 218
5.1.1. Developmental effects on the brain and behavior ............................................................. 218
5.1.2. Developmental effects on the male reproductive tract ......................................................... 218
5.1.3. Developmental effects on enzyme activity, growth and metabolism ............................................. 218
5.1.4. Adult effects on the male reproductive tract .................................................................. 219
5.2. We consider the following to be likely but requiring confirmation ...................................................... 219
5.2.1. Developmental effects on the female reproductive tract ....................................................... 219
5.2.2. Adult effects on the brain and behavior ...................................................................... 219
5.2.3. Adult effects on the female reproductive tract ................................................................ 219
5.2.4. Adult effects on the immune system ........................................................................ 219
Acknowledgements ..................................................................................................... 219
References ............................................................................................................. 219
1. Introduction
Bisphenol A (BPA) is used as the monomer to manufacture
polycarbonate plastic, the resin that lines most food and bev-
erage cans, dental sealants, and as an additive in other plastics
(Fig. 1, Table 1) [1]. BPA is one of the highest volume chemicals
produced worldwide; global BPA production capacity in 2003
was 2.2 million metric tonnes (over 6.4 billion pounds), with a
6–10% growth in demand expected per year [2]. Heat and either
acidic or basic conditions accelerate hydrolysis of the ester bond
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 201
Fig. 1. Chemical structure of bisphenol A (BPA).
linking BPA monomers, leading to release of BPA and the poten-
tial for human and environmental exposure. Studies conducted
in Japan [3] and in the USA [4] have shown that BPA accounts
for the majority of estrogenic activity that leaches from landfills
into the surrounding ecosystem.
BPA has been demonstrated in both in vivo and in vitro
experiments to act as an endocrine disrupting chemical (EDC)
(reviewed in [5,6]). There is extensive evidence that BPA is
an estrogen-mimicking chemical, although recent findings have
revealed that BPA is a selective estrogen receptor modulator
(SERM), since BPA and the potent endogenous estrogen 17-
estradiol (E2) do not always show identical effects, and in some
studies BPA has been shown to antagonize the activity of E2 [7].
There is evidence that, similar to other estrogens, BPA can bind
to androgen receptors and inhibit the action of androgen [8].In
addition, there is evidence for an anti-thyroid hormone effect of
BPA [9]. However, effects of BPA mediated by binding to andro-
gen and thyroid hormone receptors appear to require higher
doses than those required to elicit estrogenic or antiestrogenic
responses [7].
Chemicals classified as endocrine disruptors include not only
hormone-mimics or antagonists that act via binding to receptors,
but also chemicals that can interfere with hormone synthesis and
clearance, as well as other aspects of tissue metabolism. Experi-
ments have shown that BPA influences enzyme activity and thus
metabolism in various tissues. Another mechanism of endocrine
disruption is the alteration of hormone receptor expression, and
experiments described below have shown that BPA alters hor-
mone receptor numbers and hormone receptor gene activity in
target tissues.
In this review, we summarize the recent literature on low dose
effects of BPA in laboratory animals. The majority of the studies
used rats and mice; only a few used other mammalian species.
We conclude with a series of statements expressing our level
of confidence concerning various effects of BPA in laboratory
animals at low doses.
Table 1
Physical, chemical, and acute toxicological properties of bisphenol A (BPA)
[1,199]
Molecular formula (CH
3
)
2
C(C
6
H
4
OH)
2
Molecular weight 228.29
Water solubility 120–300 mg/l
log k
ow
3.40
Melting point 150–155
C
Boiling point 220
C (4 mmHg)
LD50
a
, rat, oral 3300–4240 mg/kg [199]
LD50, mouse, oral 2500–5200 mg/kg [1]
LD50, fish (Pimephales promelas), 96 h 4.6 mg/l [1]
a
Acute lethal dose, 50% of population.
1.1. Rationale for experimental design, analysis and
reporting criteria
Endocrinology experiments with laboratory animals are
particularly vulnerable to confounding effects. Among other dif-
ficulties, treatment effects can be masked by hormonally active
components of feed, water, or caging; and species and strains
differ greatly in their sensitivity to different hormonally active
compounds. Therefore, proper reporting of experimental design
is critical to evaluation of studies in the literature. Below, we
discuss several of the most important aspects of experimen-
tal design, which we considered when evaluating the studies
discussed in this review.
1.1.1. Animal model
Published results must identify precisely the animal model
and supplier being used. For example, Sprague–Dawley rats
from different commercial breeders cannot be assumed to be
the same, since it is an outbred stock. In particular, the out-
bred Sprague–Dawley CD rat from Charles River Laboratories
[Crl:CD(SD)] has very low sensitivity to exogenous estrogens,
and after more than 50 years of selective breeding by Charles
River for large body size and litter size, it would be inappropri-
ate to identify these rats as just Sprague–Dawley (Table 2). In
contrast to the Crl:CD(SD) rat, male and female CD-1 (ICR)
mice are highly sensitive to exposure to low doses of BPA
during development as revealed by over 20 published studies
(reviewed below) reporting significant effects of low doses of
BPA in this outbred stock. This high sensitivity of the CD-1
mouse to BPA is predicted by the high responsiveness to posi-
tive control estrogens: E2, ethinylestradiol and diethylstilbestrol
(DES), as revealed by both the in vivo studies discussed here
and other studies of CD-1 cells and organs in primary culture
[10–12].
The marked difference in sensitivity of different animal mod-
els used in toxicological, pharmacological and endocrinological
research is just one of many reasons why it is essential that
experiments include appropriate positive controls, which is dis-
cussed in more detail in following sections. With regard to
examination of the in vivo estrogenic activity of BPA, which
is the subject of this review, the sensitivity of the endpoint of
interest in the chosen animal model should be characterized
with a positive control such as E2 (appropriate if adminis-
tration is by injection or subcutaneous capsule, due to very
limited oral absorption of E2) or either DES or ethinylestra-
diol (appropriate if chemicals are administered orally, since they
are orally active at very low doses). For example, in the CD-1
mouse, which is the animal model used by the U.S. National
Toxicology Program, an appropriate positive control dose of
ethinylestradiol or DES to detect a response to BPA within
Table 2
Lack of effects reported in low dose in vivo bisphenol A (BPA) research using
the Charles River Laboratories Sprague–Dawley (Crl:CD(SD)) rat strain
Effect 0 (0%)
No effect 13 (100%)
202 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
the low dose range (below 50 mg/(kg day)) would be an oral
dose not greater than 5 g/(kg day). This suggestion is based
on numerous reports that for responses mediated by nuclear
estrogen receptors, estimates of BPA potency in CD-1 mice
range between 10 and 1000-fold less than either ethinylestra-
diol or DES, depending on the specific response being measured
[10,12–16]. Also, doses of DES above 5 g/(kg day) can result
in opposite effects relative to lower doses; this has been shown
following developmental exposure for the prostate [16,17] and
uterus [18]. High doses of these positive control estrogenic
chemicals are thus not appropriate as a positive control for low
dose effects of BPA or other estrogenic endocrine disrupting
chemicals.
1.1.2. Feed, water, and housing
The exact feed used must be identified. Ideally, the estro-
genicity of the feed should be characterized, since estrogenic
components have been demonstrated to occur in both soy-based
and non-soy-based animal feeds. There is also the possibility
of variation in estrogenic activity between different lots of feed
commonly used in toxicological research [19]. If possible, the
same lot of feed (same mill date) should be used throughout an
experiment.
The type of caging should be carefully selected to avoid
estrogenic contamination of experimental animals. In particu-
lar, polycarbonate cages and water bottles should not be used,
since they will leach uncontrolled concentrations of BPA to the
experimental animals [20]. Polypropylene cages have been used
successfully in studies of estrogens in mice, and polysulfone
cages are available to replace polycarbonate; polysulfone is a
co-polymer containing BPA and sulfone, but it is reported to
be more resistant to degradation at high temperatures relative
to polycarbonate [20]. Similarly, the source of drinking water
must be free of BPA and other estrogens; reverse osmosis and
carbon filtration is often necessary to achieve this requirement.
In addition, if BPA is delivered in drinking water, the water
must be free of chlorine or other reactive ions. Adding BPA to
chlorinated water results in formation of tetrachlorobisphenol
A.
1.1.3. Method of dosing
In all cases the precise method of dosing the animals and
the time of dosing should be identified. Methods of adminis-
tration of BPA include—(1) oral (p.o.): by gavage, by adding
BPA to feed or drinking water, or by feeding the chemi-
cal in oil; (2) injection: subcutaneous (s.c.), intraperitoneal
(i.p.), intracisternal, or intramuscular (i.m.) routes; and (3)
implantation of Silastic
®
capsules or Alzet
®
minipumps that
lead to steady-state exposures. The rationale for the dos-
ing method must also be stated. For example, minipump
implants model continuous exposure and avoid the first-pass
metabolism of BPA in the liver that results from oral expo-
sure.
The positive control chosen must be compatible with the
selected route of exposure. For example, E2 has very low activity
when administered orally. For studies in which BPA is admin-
istered orally, DES or ethinylestradiol are appropriate positive
controls. When BPA is administered in constant release cap-
sules, E2 would be an appropriate positive control, while an
estrogen agonist with a long half-life would be inappropriate.
Route of administration influences the rate of metabolism of
BPA, at least in adults [21], and there is some evidence for a
higher contribution from ingestion of BPA in humans relative
to inhalation or absorption through the skin [22]. However, the
report that BPA levels in plasma collected from pregnant women
in Germany show a range encompassing two orders of magni-
tude, between 0.1 and 10 parts per billion [23], suggests the
likely possibility of variable exposure to multiple sources of
BPA. Thus, although oral delivery appears to be most relevant
for extrapolation to humans, all delivery methods may reveal
effects of BPA.
1.1.4. Design and analysis issues
When animals are assigned to groups, the litter must be taken
into account. It is well established that for both outbred stocks
and inbred strains, the litter is a significant source of variation
and needs to be accounted for in assigning animals to groups.
Ideally, one animal per litter should be used. In cases when this is
not possible, there are statistical methods (such as including litter
as a main effect variable and dividing the F value for treatment
effects by the F value for litter effects) that can be used when
more than one animal per litter is used, or litter can be used as a
covariate in ANCOVA. The method used to avoid confounding
litter effects must be reported.
For all experiments, the positive and negative controls must
be clearly identified. Experiments using a replicate block design
should include a positive and a negative control in each block,
if possible. Experiments showing no effect and lacking positive
control data cannot be interpreted.
When taking measurements from adult animals, care must
be taken to normalize the reproductive state of the experimental
animals. Males should be singly housed for 2–4 weeks before
collection, in order to avoid physiological differences arising
from differences in dominance status of the males. Normal-
ization of adult female reproductive status can be achieved by
assaying their estrous cycles and collecting females at the same
stage, or by using ovariectomized females with or without hor-
mone replacement [24].
1.2. Definition of “low dose”
Low dose effects of environmental endocrine disrupting
chemicals generally refer to effects being reported at doses
lower than those used in traditional toxicological studies for
risk assessment purposes. “Low dose” is also commonly used
to refer to environmentally relevant doses, i.e., doses resulting
in serum levels close to those observed in human serum. For
BPA, prior to 1997, the lowest dose studied for risk assessment
purposes was 50 mg/(kg day), which in the USA remains the cur-
rently accepted lowest observed adverse effect level (LOAEL)
that was used to calculate the current EPA reference dose (and
FDA acceptable daily intake or ADI dose) of 50 g/(kg day); this
presumed “safe” dose is estimated by dividing the LOAEL by
three 10-fold safety factors (i.e. by 1000) [25]. Thus, we included
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 203
Table 3
Published studies since 1997 (reviewed in detail in Table 4) that reported low
dose effects of bisphenol A (BPA) in mice and rats resulting from exposure
during development or during adulthood
Number of studies (total number
of studies, including citations in
other sections)
Observed adverse effects
51 (51) Altered brain physiology, structure,
and behavior
22 (27) Altered male reproductive organs,
including lower sperm production
17 (19) Altered female reproductive organs,
including accelerated puberty
8 (22) Altered metabolism
7 (7) Altered immune system
in our analysis studies dosing with less than 50 mg/(kg day) BPA
(Tables 3 and 4).
1.3. Timing of exposure
Exposures to endocrine disruptors have different effects
depending on the life stage of the exposed animals. Effects
resulting from adult exposure are generally reversible and are
termed “activational”. Effects resulting from exposure during
organ development (beginning during prenatal development and
continuing in postnatal life through puberty) may result in per-
sistent alterations of the affected systems, even in the absence of
subsequent exposure; these effects are termed “organizational”
[26]. Some organizational effects are measurable immediately
upon exposure and persist throughout the life of the animal [16].
Other organizational effects are undetectable at the time of expo-
sure, but they become apparent in subsequent adulthood [26,27].
Windows of vulnerability, also known as critical periods, dur-
ing which the developing system is most sensitive to exposure,
are common features of organizational effects [28]. Exposures
occurring outside the critical periods will not elicit organiza-
tional effects. There is evidence that organizational effects of
estrogenic endocrine disruptors such as BPA are mediated by
epigenetic alterations in DNA [29]. Organizational and activa-
tional effects on the same tissue often differ qualitatively as
well as in duration and in the dose required to elicit effects. In
this review, we will first discuss organizational effects of pre-
natal through pubertal BPA exposure, termed “developmental
effects” and then activational effects of adult BPA exposure,
termed “adult effects”.
2. Developmental effects of BPA due to exposure during
gestation through puberty
Many studies have examined the effects of prenatal, neona-
tal (shortly after birth) and lactational (birth through weaning)
exposure to low doses of BPA. These experiments involved
examining effects of exposure to low doses of BPA during
“critical periods” in the development of different tissues. These
critical periods continue through puberty, the period of physio-
logical transition to fertility.
2.1. Effects on neurotransmitters and their receptors,
neuroendocrine function and hormone receptors in the
brain in males and females
A large number of studies have involved examination of the
effects of exposure to low doses of BPA during critical periods in
brain development on subsequent brain structure, function and
behavior.
2.1.1. Receptors
Increases in estrogen receptors (ER) and (ER)have
been observed in diverse areas of the brain in response to devel-
opmental BPA exposure. BPA was administered to Wistar rats
via minipumps at doses of 25 and 250 g/(kg day) from gesta-
tion day (GD) 8 through parturition [30]. The male offspring of
these mothers were examined, and BPA was found to perma-
nently up-regulate estrogen receptor 2 () (Esr2) mRNA levels
in the preoptic area at 25 g/(kg day) [30]. Female offspring
were not examined in this study [30]. Neonatal [postnatal days
(PND) 1–5] injection of Fischer 344 rat pups with a dose of
approximately 15 mg BPA/(kg day) resulted in increased estro-
gen receptor 1 () (Esr1) mRNA expression in the medial basal
hypothalamus of females, and increased both Esr1 and Esr2
mRNA expression in the anterior pituitary in males [31].In
Sprague–Dawley rats (Harlan), fed 40 g BPA/(kg day) at early
puberty, from PND 23–30, an increased number of ER-labelled
neurons was found at puberty (PND 37) in the arcuate nucleus
(ARC) in males and females, and in the ventromedial nucleus
(VMH) in females only. At maturity, more ER-labelled neu-
rons were found in the medial preoptic area (MPA) in females
[32]. Esr1 and Esr2 mRNA expression were also increased in the
dorsal raphe nucleus (DRN) of male ICR (CD-1) mice prena-
tally exposed to 2 g BPA/(kg day) via oral administration to the
pregnant female on GD 11–17 [33]. In this study, serotonin, sero-
tonin transporter, and serum testosterone were not altered [33].
BPA induced changes in somatostatin receptors in the brains
of offspring born to Sprague–Dawley rats that received an oral
dose of 400 g BPA/(kg day) during pregnancy and lactation.
Exposed offspring were examined on PND 10 and 23 [34].BPA
also induces increased mRNA expression of the ligand-activated
transcription factors aryl hydrocarbon receptor (AhR), retinoic
acid receptor (RAR) , and retinoid X receptor (RXR) in
embryonic brain [35,36].
2.1.2. Neuroendocrine effects
BPA-induced changes in function of the hypothalamus–
pituitary–gonad axis have been observed in both males and
females. In females, disruption of LH and estrous cyclicity
have been reported. The female offspring of Sprague–Dawley
rats exposed during gestation and lactation to approximately
1.2 mg/(kg day) of BPA in their drinking water exhibited dis-
rupted, prolonged estrous cycles and decreased hypersecretion
of LH in response to ovariectomy, suggesting lasting neuroen-
docrine effects of early BPA treatment [37]. Disruption of adult
estrous cycles seen as an increase in the length of the estrus
phase was also reported to occur in female offspring of preg-
nant Sprague–Dawley rats administered BPA orally at a dose of
204 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
Table 4
Published papers reporting biological effects in animal studies (excluding aquatic animals) for bisphenol A (BPA) in the low dose exposure range (<50,000 g/kg BW/day), sorted by tissue or endpoint examined
Reference In other sections Animal Sex Exposure, vehicle Time of exposure Doses tested,
g/kg BW/day
(*P < 0.05)
Endpoints Age at collection Other chemicals tested
Effects on brain physiology
Akingbemi [41] Sex difference,
testosterone,
metabolism
Long Evans rat,
CRL
M Oral, oil oral
gavage
PND 21–PND
35; GD 12–PND
21
2.4*, 10(*), 100,000,
200,000; 2.4*, 10*,
100,000*, 200,000;
2.4*
Serum LH, testosterone
suppression, serum estradiol
suppression, pituitary LH
and ER mRNA expression;
body weight; seminal vesicle
weight (see also in vitro)
PND 35; PND 90
Aloisi [120] Rat F Oral, oil Adult 40,000* ER Adult
Ceccarelli [32] Sprague–Dawley
rat, Harlan, Italy
F, M Oral PND 23–30 40* Hypothalamic ER PND 27, PND 90 Ethinylestradiol
Evans [39] Sheep, Poll
Dorset
F Injection, oil 4 weeks
(prepuberty)–11
weeks
3500* Tonic LH secretion; LH pulse
frequency and amplitude
11 weeks DES, octylphenol
Facciolo [34] Sprague–Dawley
rat (CRL, Italy)
F Oral, oil GD 0–PND 23 40, 400* GABA
A
receptors PND 10, PND 23
Funabashi [117] Rat F Injection, s.c. Adult 50,000* Hypothalamic preoptic area
progesterone receptors
Estradiol, butyl benzyl
phthalate
Funabashi [119] Rat F Injection s.c. Adult 40,000* Frontal and temporal cortex
change in progesterone
receptor mRNA
Adult Octylphenol,
nonylphenol
Funabashi [118] Rat F Oral, oil Adult 4, 40, 400*, 4000* Hypothalamic progesterone
receptors, POA and VMH
Adult Estradiol
Kawai [33] ICR mice M Oral GD 11–17 2* Change in ER, and ER
immnoactivity in dorsal raphe
nucleus
PND 35, PND 63,
PND 91
Kawato [42] Wistar rat M Oral, water GD 11–PND 21 0.1*, 1, 10, 50 mg/l
in drinking water
Brain steroidogenesis 4 weeks
Khurana [31] Fischer 344 rat F, M Injection, oil PND 1–5 15,000*, 75,000*;
15,000*
Hyperprolactinemia; pituitary
ER,ER expression
PND 10–PND 30 DES, octylphenol
MacLusky [44] Rat F Injection, oil Adult 40*, 120*, 400*; 45* Hippocampal pyramidal
neuron synaptogenesis
Adult 17-Estradiol,
17-estradiol
Nikaido [15] Puberty,
metabolism
Crj:CD-1 (ICR)
mouse
F Injection, DMSO GD 15–GD 18 500*, 10,000*; 500,
10,000*
Cycle length, diestrus,
reduced corpora lutea,
mammary gland
development; age at vaginal
opening
4, 8, 12, 16 weeks
and weeks 9–11
examined for
estrous cycles
DES, genistein,
zearalenone,
resveratrol
Ramos [30] Prostate Wistar-derived
rat
M Osmotic
minipump
GD 7–22 25*, 250* Ventral prostate, HPG axis,
ER
PND 15, 30, 120
Rubin [37] Metabolism Sprague–Dawley
rat, Taconic
F Oral, water GD 6–PND 21 100*, 1200* Body weight, cyclicity, LH BW from PND
2–110, cycles 4
and 6 months
Savabieasfahani
[40]
Suffolk Ewes F s.c. injection in
cottonseed oil into
pregnant dams
GD 30–GD 90 5000* Low birth weight, increased
LH levels during first 2
months of postnatal life,
extended breeding season,
dampened magnitude of LH
surge
6 weeks–40+
weeks of age
Methoxychlor
Steinmetz [121] Rat F Implant Adult 300* Serum prolactin; PRF activity
(see also in vitro)
Adult Estradiol
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 205
Talsness [38] Prostate,
metabolism
Sprague–Dawley
rat,
Harlan–Winkelmann
F, M Oral, oil GD 6–GD 21 100*, 50,000*; 100*,
50,000
Reproductive system effects,
body weight; estrus cycle;
AGD in males
Adult, 3 months,
PND 3, 15, 21
Ethinylestradiol
Zoeller [9] Sprague–Dawley
rat, Zivic Miller
F, M Oral, feed, via
treated wafer
GD 6-duration
not defined
1000*, 10,000*,
50,000*
Serum thyroxin; dentate
gyrus RC3/neurogranin
expression
PND 4, 8, 15, and
35
Zsarnovszky
[43]
Sprague–Dawley
rats
F, M Injections into
cerebellum
PND 4–PND 19
and adults
250–300 g
Concentrations of
10
12
to 10
6
M
BPA were injected
into the cerebellum
(3 l in PND 4–10,
5 l into older
animals)
Rapidly increased pERK
beginning on PND 8;
co-administration with E2
inhibited rapid E2 induced
ERK1/2 activation. Dose
dependent
PND 4–PND 19
and adults
Cyclodextrin
encapsulated estradiol
Effects on brain structure
Kubo [45] Sex difference Wistar rat F, M Oral, water Mating to
weaning
1500* Loss of sexual dimorphism in
behavior and in size of locus
coeruleus
6–12 weeks
Kubo [46] Sex difference,
prostate
Rat F, M Oral, water Mating to
weaning
30*, 300* Reversal in the sexually
dimorphic volume of the
locus coeruleus, behavioral
changes
Postnatally, 6,
11–12 and 14
weeks
DES, resveratrol
Masuo [49] Behavior Wistar rat M Intra-cisternal
injection, oil
10 l into pups
PND 5 Approximately 2,
20*, 200*, 2000*;
2000*
Motor hyperactivity
(spontaneous motor activity);
midbrain gene expression
PND 56 Nonylphenol,
octylphenol, DEHP
Nakamura [50] ICR/Jcl mice F, M s.c. injection, oil GD 0-collection 20* Altered neuronal migration
and gene expression
GD 10.5–GD 16.5
Patisaul [48] Sex difference Sprague–Dawley
rat, CRL
F, M s.c. injection in
sesame oil into
pups
PND 1-2 Approximately
75,000*
Altered AVPV development,
altered sexual dimorphism of
dopamine (TH) neuron
number and number of
TH/ER expressing neurons
PND 19
Rubin [47] CD-1 mouse F, M Osmotic
minipump
GD 8–PND 21 0.025, 0.250* Dopamine neuron number in
the AVPV reduced/loss of
expected sexual dimorphism
PND 24–27
Effects on behavior
Adriani [64] Sprague–Dawley
rat (Italy)
F, M Oral, oil GD 0–PND 26 40* Behavioral tests,M&F;
amphetamine response, M
PND 30–35, PND
70
Aloisi [56] Sprague–Dawley
rats, CRL
F, M Oral, oil, trained
to suck BPA from
pipette
Dams exposed
thru pregnancy
and lactation.
Two groups:
placental exp
suckling exp
40* Pain behavior;
formalin-induced nociception
22 weeks of age
Della Seta [61] Metabolism Rat F Oral, oil Adult 40* Maternal behavior
Della Seta [67] Testosterone Sprague–Dawley
(Italy)
M Oral, oil PND 23–PND 30 40* Decreased serum testosterone PND 37, 45, >90 Ethinylestradiol
Dessi-Fulgheri
[62]
Sprague–Dawley
rat (Italy)
F, M Oral, Oil 40, GD 0–PND
21; 400, GD
14–PND 6
40*, 400*, 40*, 400,
40, 400*
Play behaviors PND 35, 45, 55
Farabollini [51] Sprague–Dawley
rat (Italy)
F, M Oral, oil GD 0- GD 22;
PND 1–PND 21
40* Aggression, Sexual Behavior PND 100
Ishido [54] Metabolism Wistar rat M Intracisternal
injection, oil 10 l
PND 5 3, 30, 300*,
3000*; 3, 30,
300, 3000*
(87 fmol–87 nmol
BPA/rat)
0.02–20 g/rat
Hyperactivity; reduced gene
expression of dopamine
receptor and dopamine
transporter; reduded midbrain
TH-IR
4–8 weeks
206 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
Table 4 (Continued )
Reference In other sections Animal Sex Exposure, vehicle Time of exposure Doses tested,
g/kg BW/day
(*P < 0.05)
Endpoints Age at collection Other chemicals tested
Kawai [52] Testosterone CD-1 mouse M Oral, oil GD 11–17 2*, 20* Aggression, testis weight 8, 12, 16 weeks
Laviola [66] Sex difference CD-1 mouse
CRL
F, M Oral, oil, drink
from syringe
GD 11–18 10* Amphetamine-induced
conditioned place preference
60 days Methoxychlor
Masuo [53] Rat Intracisternal
injection
Neonatal 20 g/kg* Motor hyperactivity 4–5 weeks
Mizuo [55] ddY mice Oral, feed GD 0–PND 14 0.002, 0.5*, 2* mg/g
in food
Enhanced reward effect and
hyperlocomotion in response
to morphine
Adult
Negishi [59] F344/N rat M Oral, oil (corn)
gavage
GD 3–PND 20 100* Behavioral alterations active
avoidance test
8–24 weeks Nonylphenol
Nishizawa
[35,36]
Mouse F, M Oral, oil GD 6–13, GD
6–17
0.02*, 2*, 200*,
2000*
Embryonic brain and gonad
AhR expression; RAR,
RXR expression
Palanza [60] Metabolism CD-1 mouse,
CRL
F Oral, oil GD 14–18 10* Maternal behaviors In adulthood
Porrini [63] Sprague–Dawley
rat, CRL, Italy
F, M Oral, peanut oil,
micro-pipette
Daily from
mating to
parturition
40* Socio-sexual behaviors 35, 45, 55 days
Razzoli [122] Gerbil F Oral, oil Adult 2*, 20; 2*, 20*; and
2, 20*
Social investigative behavior,
free exploratory tests
Ethinylestradiol
Ryan [57] Puberty,
metabolism
C57Bl6 mice Ovx F Oral, gavage
needle into back
of mouth
GD 3–PND 21 2, 200* Accelerated puberty, altered
anxiety related behaviors
Adult, 6 weeks + Ethinylestradiol
Suzuki [65] ddY mouse M Oral, in feed GD 0–PND 21 0.002, 0.5, 2 mg/g
feed, 300*,
75,000*,
300,000*
Dopamine D1
receptor-mediated enhanced
induced abuse state
Not specified
Effects on sex differences in the brain
Carr [68] Fischer 344 rat F, M Oral, oil gavage in
pups
PND 1–PND 14 100, 250*; 100*, 250 Morris water maze;
elimination of sex difference
Tests begin PND
34
Estradiol
Farabollini [58] Sprague–Dawley
rat (Italy)
F, M Oral, oil GD 0–PND 21 40*, 400* Exploratory behavior, activity PND 85
Fujimoto [73] Wistar rat F, M Oral, drinking
water
GD 0–PND 1 15* Sex differences in open field
and forced swimming
behavioral tests
7–9 weeks
Funabashi [72] Wistar rat F, M Oral, drinking
water
Gestation and
lactation, no GD
identified
2500*, (2500) Sex differences in CRH
neurons in bed nucleus of the
stria terminalis
4–7 months
Imanishi [70] ICR mouse F, M Oral, oil GD 6–GD 17 2* Placental nuclear receptor
gene expression, 9 of 20
examined; six non-nuclear
receptor genes
GD 18
Nishizawa [71] ICR mouse F, M Oral, oil GD 6-collection 2* Embryonic brain and gonad
retinoid receptor expression
RAR, RXR
GD 12, 14, 16, or
18
Shibata [69] Metabolism Wistar rat M Oral 2 or 4 weeks,
adulthood
1500* BPA and sex steroid
metabolism in adult male
Adult DES
Effects on puberty in females
Honma [13] Metabolism ICR/Jcl mouse F, M s.c. injection, oil GD 11–GD 17 2*, 20*; 2*, 20; 2,
20*
BW, estrous cycle length,
male AGD, vaginal cytology;
female AGD; age of vaginal
opening, first estrus
Up to PND 120 DES
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 207
Howdeshell [78] Metabolism CF-1 mouse F, M Oral, oil GD 11–GD 17 2.4* Puberty, BW Puberty
Vandenberg [79] Mammary gland CD-1 mouse F Alzeet osmotic
pump
GD 8–18 0.25* Mammary gland morphology GD 18
Effects on the mammary gland
Colerangle [85] Rat F Osmotic
minipump
4 weeks old,
administered 11
days
100*, 54,000* Mammary gland
differentiation
5.5 weeks DES
Durando [82] Wistar rat F Osmotic
minipump
GD 7–22 25* Mammary gland, cancer PND 30, 50, 110,
180
Markey [80] CD-1 mouse F Osmotic
minipump
GD 8–GD 19 25*, 250* Mammary gland PND 10, 1 month,
6 months
Munoz-de-Toro
[81]
Mouse F Osmotic
minipump
GD 8 lactation 0.025*, 0.250* Mammary gland
morphogenesis, terminal end
bud density
PND 30, 120
Murray [83] Wistar rat F Osmotic
minipump
2.5*, 25*, 250*,
1000*
Mammary gland hyperplasia
(carcinoma precursor)
PND 55, 95
Effects on the uterus and vagina
Markey [88] Mouse F Osmotic
minipump
GD 9-lactation 0.025*, 0.250* Genital tract alterations; ER
and progesterone receptor
up-regulation
developmentally
Adult
Nagel [173] Mouse F Injection, oil Adult 25 (P < 0.06), 791*,
25,000*
Uterus, gene expression DES
Sch
¨
onfelder [87] Rat F Oral GD 6–21 100*, 50,000* Vaginal ER expression,
vaginal histology
Adult Ethinylestradiol
Sch
¨
onfelder [86] Sprague–Dawley
rat
F Oral GD 6–21 100*, 50,000*;
50,000*; 100*,
50,000*
Uterine epithelial histology;
uterine ER expression;
uterine ER expression
Adult Ethinylestradiol
Suzuki [14] Mouse F Injection, oil GD 10–18 10,000*, 100,000* Uterine and vaginal mitotic
indices following prenatal
exposure
PND 30 DES
Effects on the ovary, oocytes and fertility
Al-Hiyasat [90] Mouse F Oral, water Adult for 28 days 5, 25*, 100*; 5, 25,
100*
Increased resorptions, uterine
weight; ovarian weight
Adult
Berger [92]
Mouse F Injection s.c. GD 1–4 3, 375*; 10, 125* Implantation rate; fecundity GD 5, PND 1
Hunt [89] C57BL/6, mouse F Oral, oil Peripubertal for
6–8 days
20*, 40*, 100* Disruption of meiosis;
aneuploidy
30 days old
Susiarjo [91] C57BL/6, mouse F Oral, injection GD 11–18 20* Disruption of chromosomes GD 18
Effects on testosterone in males
Takao [133] Mouse M Oral, drinking
water
Adult 120, 12,000* Plasma free testosterone Adult
Effects on the Prostate
Gupta [10] Mouse M Oral, oil GD 14–18 50* Prostate weight, prostate AR
(see also in vitro)
PND 3, 20, 60 DES, aroclor
Ho [29] Sprague–Dawley
rat
M Injection s.c. PND 1–5 10* Prostate cancer (PIN lesions) Adult Estradiol
Nagel [94] Metabolism CF-1 mouse M Oral, oil GD 11–GD 17 2*, 20* Prostate weight 6 months Octylphenol
Ramos [97] Wistar rat M Osmotic pump,
DMSO
GD 7–GD 22 25*, 250* Ventral prostate PND 30
Stoker [100] Wistar rat M s.c. injection, oil PND 22–PND 32 50,000* Serum prolactin, lateral
prostate weight
PND 29, PND
120
Estradiol, Pimozide
Timms [16] Mouse M Oral, oil GD 14–18 10* Fetal development of prostate
and urethra
GD 19 Ethinylestradiol, DES
Effects on the testes, epididymis, sperm and seminal vesicles
Aikawa [102] SHN mouse M s.c. injection, oil PND 1–PND 5 175*, 17,500*;
175, 17,500*
Abnormal sperm; decreased
motility
10 weeks Estradiol
208 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
Table 4 (Continued )
Reference In other sections Animal Sex Exposure, vehicle Time of exposure Doses tested,
g/kg BW/day
(*P < 0.05)
Endpoints Age at collection Other chemicals tested
Al-Hiyasat [129] Metabolism Swiss mouse M Oral Adult, for 1
month
5*, 25* 100* Reduced fertility,
epididymidal sperm, body
weight
Adult
Chitra [131,132] Metabolism Wistar rat M Oral, oil Adult, for 45
days
0.2*, 2*, 20*; 0.2*,
2*, 20*; 0.2*, 2*,
20*; 0.2, 2*, 20*;
0.2*, 2*, 20*; 0.2*,
2*, 20*
Decreased testis, epididymis
weight; increased ventral
prostate weight; reduced
sperm motility; sperm count;
oxidative stress enzymes;
increased H
2
O
2
Chitra [132] Wistar rat M Oral Adult 0.2*, 2*, 20* Decrease in sperm motility,
decrease in oxidative stress
enzymes
Adult
Fisher [104] Wistar rat M s.c. injection, oil PND 2–PND 12 37,000* Efferent duct epithelial height PND 10, 18, 25,
35, and 75
DES, ethinylestradiol,
genistein, octylphenol,
parabens
Sakaue [127] Rat M Oral, oil Adult 0.2, 2, 20*, 200*,
2000*, 200,000*
Sperm production
Takahashi [136] Rat, mouse M Injection,
propylene glycol
adult 2000, 20,000* BPA low dose challenge after
extended high-dose exposure
Takao [134] Mouse M Oral, drinking
water
Adult 200, 20,000* Testicular ER,ER
Thuillier [188] Sprague–Dawley
rat, CRL
M Gavage (BPA);
s.c. injection
(DES)
GD 14–GD 22 100, 1000*, 10,000*,
200,000*
Neonatal testicular gonocyte
PDGF, PDGF
GD 21–PND 3 DES, genistein,
coumestrol
Tohei [135] Rat M Injection Adult 3000* Testes and serum hormones
Toyama [103] ICR mouse,
Wistar rat
M s.c. injection, oil PND 2–PND 12 71, 714*, 3600*,
7100*; 180, 1800*,
18,000*
Spermatogenesis, mouse; rat 2–10 weeks Estradiol, estradiol
benzoate
Toyama [130] Mice M Injection Adult, for 6 days 20*, 200*; Abnormal sperm
morphology;
Adult
vom Saal [101] CF-1 mouse M Oral, oil GD 11–GD 17 2*, 20*; 2, 20* Reproductive organs; sperm
production
6 months Octylphenol
Wang [105] Sprague–Dawley
rat, CRL
M Oral, oil GD 14–GD 22 1000, 10,000*,
200,000*; 1000*,
10,000, 200,000
Estrogen receptor-associated
protein expr; Hsp90, p23
GD 21, PND 3,
PND 21
DES, genistein,
coumestrol
Wistuba [106] Sprague–Dawley
rat (Germany)
M Oral gavage,
Cornstarch
suspension
GD 6–GD 21 100*, 50,000* Sertoli cell number per testis PND 290–370 Ethinylestradiol
Effects on metabolism
Alonso-
Magdalena
[140]
OF1 mice M Injection, oil Adult 10* Insulin release, insulin
sensitivity
Estradiol
Kabuto [141] ICR mice M Injection i.p. for 5
days
Adult 25,000*, 50,000* Antioxidant scavenging
enzymes
Kabuto
[114] Mouse M Oral, drinking
water
GD 1-lactation 2.5–5* Testis weight, brain weight,
kidney weight, various
oxidation markers
PND 28
Lemmen [189] Mouse F, M Injection, oil GD 13.5 100, 1000*, 10,000* Transgenic embryonic gene
expression in whole embryo
lysates
DES, estradiol
propionate
Markey [110] Mouse F Osmotic
minipump
GD 8–19 25*, 250* Estrus cycle alterations,
blood-filled ovarian bursae,
mammary gland budding
Adult
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 209
Nunez [139] Sprague–Dawley
rat
F Osmotic, pump Adult 5000, 18,000*,
23,000*
Body weight, blood and
tissue levels of BPA
Adult
Takai [107] B6C3F1 mouse F, M Ex vivo in culture
medium
GD 2–4 0.1, 0.3, 1*, 3*, 10,
100,000* nM (0.023,
0.068, 0.23*, 0.68*,
2.3, 23,000* ppb)
Increased rate of development
at 1 and 3 nM, decreased rate
of development at
100,000 nM
Blastocyst
Takai [108] B6C3F1 mouse F, M During IVF in
culture medium
GD 0–2 1 nM (0.228 ppb)* Postnatal weight gain
increase
Weaning
Effects on the immune system
Lee [145] Mouse, BALB/c F Injection (i.p.) Adult 25,000* Increased IL-4 production,
increased serum
antigen-specific
immunoglobulin E (IgE)
levels
Adult Nonylphenol
Sawai [142] Mouse,
C57BL/6,
NZB/WF1
F, M Oral, in feed PND 35–42 2.5* Decreased IFN and IL-10
secretion by splenic
mononuclear cells; decreased
IgG2a production; protection
in lupus-prone mice (see also
In vitro)
Adult
Sugita-Konishi
[147]
Mouse, BALB/c F Injection (s.c.), oil Adult 5000* Immune cells and functions,
reduced IL-6 production and
neutrophil phagocytic activity
against bacterial infection
Adult
Tian [144] Mouse,
BALB/cA
M Oral, oil Adult 370, 1800*, 9000*;
370, 1800, 9000*
IL-10 production; IL-4
production
Adult
Yoshino [143] Mouse, DBA/1J F, M Oral, oil Adult 3, 30, 300*, 3000*;
3, 30*, 300*, 3000*
Increased serum anti-HEL
IgG2a; augmentation of
cytokine response
Adult Estradiol
Yoshino [116] Mouse, DBA/1J F, M Oral, oil GD 0–GD 17 3, 30*, 300*, 3000*;
3, 30, 300*, 3000*
Increased serum anti-HEL
IgG2a; T-helper cytokines
(both Th1 and Th2) increased
Adult
Yurino [148] BWF1 mice Silastic capsule 4 weeks 25 ng/ml in blood* IgM antibodies 5 months Estradiol DES
The information indicated in the column headings provides information in published studies. If information is not reported, this is indicated by “NR”. Day plug observed: GD 0. Mice GD 19: PND 1. Rats GD 22:
PND 1. F: female; M: male; DES: diethylstilbestrol.
210 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
100 g/(kg day) on GD 6–21 [38], and prolonged estrous cycles
were observed in female offspring of CD-1 mice injected with a
dose of 500 g/(kg day) BPA on GD 15–18 [15]. Exposure via
i.m. injection of prepubertal female Poll Dorset lambs to DES
(0.005 mg/(kg day)) or BPA (1 mg/(kg day)) for 7 weeks begin-
ning at 4 weeks of age (prior to puberty) had significant effects on
LH secretion (a decrease in LH pulse frequency) that were sim-
ilar, with the dose of DES being 200-fold lower than BPA [39].
Treatment of pregnant Suffolk Ewes with 5 mg/(kg day) of BPA
dissolved in cottonseed oil via daily subcutaneous injections
from GD 30–GD 60 resulted in marked effects in the female off-
spring. Levels of LH were increased during the first 2 months of
postnatal life, breeding season was extended, and the magnitude
of the LH surge was dampened [40].
In males, effects on LH, prolactin, and brain aromatase
activity have been observed. Exposure of weanling males to
2.4 g/(kg day) BPA for 15 days resulted in decreased serum
LH and testosterone levels due to alterations in LH synthesis
and secretion at the pituitary level; neuroendocrine effects in
this study were observed following exposure of the weanling
mice to BPA, but not following gestational and lactational expo-
sure [41]. Injecting approximately 15 mg/(kg day) of BPA into
neonatal Fisher 344 rat pups from postnatal days 1–5 resulted
in an increase in serum prolactin levels (hyperprolactinemia)
in both males and females on PND 30 [31]. Male offspring
of pregnant and lactating rats exposed to 20 g/(kg day) BPA
in drinking water showed an increase in estrogen synthesis in
hippocampal neurons due to an increase in aromatase activity
[42].
BPA exposure also induces alterations in the hypothalamus–
pituitary–thyroid axis. Dietary exposure to doses of 1, 10 or
50 mg/(kg day) of BPA in Sprague–Dawley rats from GD 6
through weaning caused a significant increase in serum total
T4 in pups on PND 15, but serum thyroid-stimulating hormone
was not different from controls. The expression of the thyroid
hormone-responsive gene RC3/Neurogranin, measured by in
situ hybridization, was significantly up-regulated by all BPA
doses in the dentate gyrus. These findings suggest that BPA acts
as an antagonist of TR, which mediates the negative feedback
effect of thyroid hormone on the pituitary gland, but that BPA
is less effective at antagonizing TR, leaving TR-mediated
events to respond to elevated plasma thyroid hormone levels
[9].
2.1.3. Rapid signaling effects
BPA has effects similar to E2 (similar potency and efficacy)
in stimulating rapid-response ERK1/2 activity in the cerebel-
lar cortex of Sprague–Dawley rat pups at PND 5–19; treatment
consisted of a 6-min intracerebellar injection followed by rapid
fixation for immunohistochemistical analysis for phosphoryla-
tion of ERK1/2. Significant effects of both BPA and E2 were
observed at the lowest dose examined of 1 pM (0.228 ppt).
Interestingly, in addition to acting as an estrogen, when co-
administered in conjunction with E2, BPA was able to inhibit
the rapid E2-induced ERK activity in the developing cerebellum,
thus disrupting the action of E2 in a dose-dependent manner [43].
There is another report that BPA can act as an estrogen antago-
nist in the hippocampus when BPA is co-administered with E2
[44].
2.1.4. Brain structure
Developmental exposure to BPA resulted in a significant
change in the locus coeruleus, where BPA at oral doses of
30 g/(kg day) (in drinking water) and above reversed the nor-
mal sex differences in this brain structure and eliminated sex
differences in behavior [45,46]. Pregnant and lactating CD-
1 mice were exposed to very low doses of BPA, 0.025 and
0.250 g/(kg day) BPA, via Alzet minipumps, and the brain
of the offspring were examined at PND 24–27. The expected
robust sex difference in the number of tyrosine hydroxylase
(TH) positive neurons in the sexually dimorphic anteroventral
periventricular preoptic area (AVPV) was apparent in the con-
trol offspring. However, in the offspring exposed to BPA, the
sex difference in TH neuron number was undetectable due to
a decline in TH neuron number in BPA exposed females. No
significant difference in TH neuron number was noted in the
males [47]. It is interesting to note that injection of much higher
doses of BPA (approximately 300,000-fold higher then the high-
est daily dose administered to pregnant and lactating females in
the study discussed above, 500 g/(day pup) or approximately
75 mg/(kg day)) directly into rat pups on PND 1–2 also altered
TH neuron number in the AVPV; however, the alteration was
in a different direction from that reported with very low doses
of BPA. With this much higher dose of BPA, the male off-
spring showed differences from controls, and the male AVPV
resembled the female AVPV, whereas the females showed no
significant effect of the BPA injection. These data suggest that
at this high dose, BPA may have acted as an antiestrogen and
interfered with the masculinization of TH neuron number [48].
The importance of dose is discussed in more detail below.
In a study with male Wistar rats that received a single intracis-
ternal injection of approximately 20 g BPA/kg dose on PND 5,
BPA reduced the number of dopamine-containing neurons and
resulted in changes in gene expression [49].A20g/(kg day)
injection of BPA into pregnant ICR (CD-1) mice disrupted nor-
mal neocortical development in fetuses by accelerating neuronal
differentiation/migration [50].
2.1.5. Behavioral effects
2.1.5.1. Aggression. BPA resulted in an increase in defensive
aggression in male Sprague–Dawley rat offspring prenatally
exposed to BPA (administered orally to mothers throughout ges-
tation) at a dose of 40 g/(kg day); no effect was found in the
offspring of mothers treated during lactation [51]. In addition,
increased aggressiveness (using a composite score of aggres-
sion) in male CD-1 mouse offspring occurred as a result of oral
administration of 2 g/(kg day) of BPA to pregnant females on
GD 11–17
[52].
2.1.5.2. Activity level and reactivity to stimuli. Treatment of
male Wistar rat pups at PND 5 with an intracisternal injection
of 0.87 nmol (approximately 20 g/kg) BPA resulted in hyper-
activity at 4 weeks of age [49,53]. Ishido et al. reported that
single intracisternal injections of approximately 15 g BPA/kg
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 211
or 150 g BPA/kg at PND 5 increased spontaneous motor activ-
ity at 4 weeks of age [54]. Prenatal and lactational exposure
to BPA in the feed (approximately 3–300 mg/(kg day)) also
resulted in an increase in activity with dose and altered the
response to morphine [55]. BPA increased reactivity to painful
or fear-provoking stimuli in the offspring of Sprague–Dawley
rats fed 40 g/(kg day) throughout pregnancy and lactation: a
hyperalgesic effect was found following prenatal, but not post-
natal exposure [56]. When pregnant and lactating C57BL/6 mice
were administered an oral BPA dose of 2 or 200 g/(kg day),
BPA increased anxious behavior in a dose-dependent fashion
when females were examined in adulthood. Significant effects
were observed in animals exposed to 200 g/(kg day) of BPA
and to 5 g/(kg day) of ethinylestradiol, which was used as a
positive control for the study [57]. In contrast, Farabollini et
al. reported that maternal exposure to 40 g/(kg day) of BPA
throughout gestation and lactation resulted in reduced anxiety
in adult male offspring, and lowered motivation to explore in
both male and female adult offspring [58].
2.1.5.3. Learning. BPA impaired learning of both passive and
active avoidance tasks in offspring of Fisher 344 rats fed
100 g/(kg day) of BPA during pregnancy and lactation [59].
2.1.5.4. Maternal behavior. Feeding pregnant CD-1 mice
10 g/(kg day) decreased subsequent maternal behavior in
female offspring, similar to behavioral effects seen with adult
exposure [60,61].
2.1.5.5. Social interactions and response to addictive drugs.
Prenatal and lactational oral exposure of Sprague–Dawley rats to
40 g/(kg day) altered adult play and other socio-sexual behav-
iors in both males and females [62,63]. Prenatal and lactational
exposure to BPA in the feed (approximately 3–300 mg/(kg day))
resulted in an altered response to morphine [55]. The behav-
ioral response to amphetamine was enhanced in rats and mice
at 40–300 g/(kg day) [64,65]. However, at a prenatal dose of
10 g/(kg day) of BPA, exposed adult female CD-1 mice lost
their responsiveness to the reward pathways normally stimu-
lated by amphetamine, possibly through effects on the dopamine
system; in this study there was no effect on male amphetamine
responsiveness, and there was no effect on activity levels [66].
2.1.5.6. Sexual behavior. An impairment in the timing of cop-
ulatory sequence was found in Sprague–Dawley male rats,
perinatally exposed to BPA (40 g/(kg day) fed to the moth-
ers through gestation or lactation) [51] or treated during early
puberty [67]. In animals exposed via oral administration perina-
tally, a reduced performance in terms of latency and frequency
of intromission was observed [51]. Effects in the same direc-
tion were found with prepubertal exposure to BPA; this was
in agreement with results obtained with the positive control
ethinylestradiol [67].
2.1.6. Sex differences
One of the most interesting findings concerning organiza-
tional effects of BPA on the brain and behavior and other
aspects of physiology is the loss of sex differences that
are typically observed in control males and females. Also
intriguing is the effect of BPA on one sex but not the other
[45,46,48,58,66,68–73]. The mechanisms responsible for dif-
ferent effects of BPA in males and females are not clear,
although it is known that BPA metabolism is influenced by
testosterone [74,75], and BPA modifies the metabolism of testos-
terone [41,76,77]. Thus, one possibility is that the sex-specific
effects of BPA in the brain and other tissues are due to an inter-
action with background levels of gonadal steroids. The effects of
BPA on metabolism are discussed in more detail in a companion
paper in this issue (see: L. Vandenberg et al.).
2.2. Developmental effects on the female reproductive tract
2.2.1. Timing of puberty
Early onset of sexual maturation in females occurred at mater-
nal doses between 2.4 and 500 g/(kg day) in a number of
different mouse strains [13,15,57,78]. Puberty in female rodents
can be assayed by age at vaginal opening or by age at first ovula-
tion, signaling the onset of fertility. The age at first ovulation can
be detected by assessing the age at which the vaginal epithelium
is first cornified, indicating that the female is in estrus, or by
pairing prepubertal females with experienced males and moni-
toring the females’ age at first parturition. Timing of puberty is
linked to postnatal growth, since puberty is dependent on age,
body size, and energy stores. A study with CF-1 mice showed
that the response to prenatal exposure (via oral dosing of the
mother) to a very low dose of BPA (2.4 g/(kg day)) from GD
11–17 was influenced by the relative position of the fetus with
respect to its female and male siblings in the uterus. The response
to BPA was greatest in the females that were located in utero
between female siblings (2F females) in terms of stimulating
an increase in postnatal growth and accelerating the age at first
vaginal estrus; postnatal growth was also stimulated in males
that were located in utero between female siblings (2F males)
but not in male offspring that were located in utero between
male siblings (2M males). This finding is consistent with the
observation that a very low maternal dose of BPA stimulated
a change in the developing mammary gland in 2F female but
not 2M female CD-1 mouse fetuses [79]. Taken together, these
findings suggest that the endogenous concentrations of E2 and/or
testosterone are significant factors is determining the response
to BPA during fetal life in mice.
2.2.2. Mammary gland
Stimulation of mammary gland development in CD-1 mice
was observed in the offspring of dams exposed to the very low
maternal dose of 0.025 and 0.250 g/(kg day) delivered tonically
by an Alzet pump [80]. A significant increase in the percent-
age of ducts, terminal ducts, terminal end buds, and alveolar
buds was observed in female offspring of BPA-treated dams at
6 months of age, collected during proestrus. At 6 months of
age, the authors identified terminal end buds as appearing much
less bulbous than terminal end buds observed during puberty.
The treatment-induced changes in histo-architecture, coupled
with an increased presence of secretory product within alve-
212 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
oli observed in the BPA-exposed offspring, resembled those of
early pregnancy. Examination of BPA-exposed females (using
the same paradigm) at 30 days of age [81] revealed an increase in
mammary gland area and number of terminal end buds, as well
as an increase of progesterone receptor-positive ductal epithelial
cells that were localized in clusters, suggesting future branch-
ing points. These sites may be involved in the increase in lateral
branching noted in the mammary glands of offspring born to
BPA-exposed dams [80].
The female offspring of pregnant Wistar rats implanted
throughout pregnancy with an Alzet minipump that released
25 g/(kg day) of BPA were examined at various times up
to 6 months of age [82]. At puberty, animals exposed
prenatally to BPA showed an increased mammary gland pro-
liferation/apoptosis ratio both in the epithelial and stromal
compartments. BPA exposed animals showed an increased num-
ber of hyperplastic ducts with signs of desmoplasia, suggesting
a heightened risk of neoplastic transformation. Administration
of a sub-carcinogenic dose of N-nitroso-N-methylurea (NMU)
to the female rats exposed prenatally to BPA increased the
percentage of hyperplastic ducts and induced the develop-
ment of neoplastic lesions. In a related report Wistar-Furth
offspring born to mothers implanted with osmotic minipumps
that released BPA at a dose of 2.5 g/(kg day) during gesta-
tion and lactation revealed evidence of ductal hyperplasias at
PND 50 and 95 that included increased expression of Ki67 and
Esr1. Some of the ductal lesions were identified as carcinoma
in situ [83].
The effect of exposure to 0.25 g/(kg day) delivered to CD-1
mice by Alzet minipump from the evening of gestational day 8
throughout pregnancy was examined in female fetuses on GD
18 [79]. In unexposed fetuses, the mammary gland ductal tree
was more developed in 2M females than in 2F females [79]; this
result is counterintuitive, since prior research has shown that
2M female fetuses have elevated serum testosterone and lower
serum E2 relative to the 2F female siblings [84]. BPA expo-
sure increased mammary gland ductal area and ductal extension
and eliminated intrauterine position differences; this was due
to BPA stimulating a significant change in 2F females but not
2M females [79]. In the stroma, BPA exposure promoted mat-
uration of the fat pad and altered the localization of collagen.
Within the epithelium, BPA exposure led to a decrease in cell
size and delayed lumen formation [79]. Pubertal exposure to
100 g BPA/(kg day) also increased differentiation of mammary
gland structures and increased proliferation of epithelial cells
[85].
2.2.3. Uterus and vagina
Oral administration of 100 g/(kg day) or 50 mg/(kg day) of
BPA to pregnant Sprague–Dawley rats resulted in a significantly
decreased uterine ER protein expression in BPA-treated ani-
mals at both doses (relative to controls) when measured during
the estrous phase of the cycle. Interestingly, a high dose of
200 g/(kg day) ethinylestradiol caused a similar effect [86].
These same doses of BPA and ethinylestradiol caused strik-
ing changes in vaginal morphology during estrus, including a
decrease in the thickness of the epithelial layer [87]. In addi-
tion, Western Blot analysis indicated that following exposure to
either dose of BPA, the full-length variant (64 kDa) of Esr1 was
not expressed in the vagina of female offspring during estrus,
whereas during the diestrus stage, ER protein expression did
not differ from the control group [87].
Studies in the CD-1 mouse have also revealed alterations
in the uterus and vagina in offspring born to mothers treated
with 0.025 and 0.25 g BPA/(kg day) via Alzet minipumps
[88]. These alterations include decreased vaginal wet weight,
decreased volume of the endometrial lamina propria and
increased protein expression of both ER and progesterone
receptor in the luminal epithelium and subepithelial stroma of
the uterus. Consistent with these effects of BPA, studies have
shown that exposure during early development (via injection on
PND 1–5) to very low doses of DES (0.01 g/(kg day)) alters
ER receptor levels and morphology of the uterus in CD-1 mice
[18].
2.2.4. Ovary, oocytes and fertility
Significant disruption of the alignment of chromosomes dur-
ing meiosis was observed in developing oocytes due to leaching
of BPA from polycarbonate drinking bottles at doses between
15 and 70 g/(kg day); this finding leads to the prediction that
exposure to BPA during the time that meiosis is reinitiated by
the mid-cycle surge in luteinizing hormone (LH) can result in an
increase in aneuploidy, which is one of the major causes of spon-
taneous abortion in humans [89]. As predicted by this finding,
there was an increase in mortality of embryos that occurred at a
maternal dose of 25 g/(kg day) [90]. Subsequently, a defect in
meiosis was induced in C57BL/6 mouse embryos by maternal
oral or injected BPA at 20 g/(kg day) by Hunt et al., and the
effect was similar to the defect seen in untreated Esr2-knockout
mice [91]. The impact of this effect would not be directly on the
in utero exposed female, but on the embryos produced from her
oocytes (F2 generation effect). Implantation of embryos is not
affected at low BPA maternal doses (as low as 10 g/(kg day)),
and is significantly decreased only at a maternal dose of approx-
imately 70 mg/(kg day), which is just above the low dose range
[92].
2.3. Developmental effects on the male reproductive tract
2.3.1. Serum testosterone levels
Male Long-Evans rats born to mothers exposed to
2.4 g/(kg day) of BPA via feeding, from GD 12 thorough lac-
tation had decreased levels of testicular testosterone, which was
not due to a decline in serum LH levels but was consistent with a
decline in the steroidogenic capacity of Leydig cells in the BPA
exposed males [41]. Decreased serum testosterone in male CD-1
mice also occurred at a maternal dose of 2 g/(kg day) given on
GD 11–17 [52]. In Sprague–Dawley (Harlan) male rats, alter-
ations in circulating testosterone levels were observed after BPA
treatment with 40 g/(kg day) at early puberty (PND 23–30): in
this study testosterone was reduced in the juveniles (PND 37),
and this decrement persisted in the adults [67].
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 213
2.3.2. Prostate
Development of the prostate gland from the urogenital sinus
is dependent on systemic testosterone, which is metabolized
to 5-dihydrotestosterone in the urogenital sinus mesenchyme.
The mesenchyme directs the development of the epithelium
into the glandular structure of the prostate [93]. An increase
in adult prostate size in male offspring occurred when preg-
nant females were fed BPA at 2 or 20 g/(kg day) on GD 11–17
in CF-1 mice [94],at10g/(kg day) on GD 14–18 in CD-1
mice [16], and 50 g/(kg day) on GD 16–18 in CD-1 mice [10];
in the experiments conducted by Timms et al. [16] and Gupta
[10], DES caused the same effects as BPA at a maternal oral
dose of 0.1 g DES/kg/day. Gupta [10] also showed that the
effect of BPA on the prostate was directly on prostate tissue
by removing the prostatic region of the fetal urogenital sinus
and examining the effect of BPA in organ culture; the low-
est effective concentration (LoEC) of BPA that stimulated an
increase in prostate growth was 50 pg/ml (ppt). DES also stim-
ulated prostate growth in primary culture at a dose of 0.5 pg/ml
(ppt) [11]. Richter et al. removed the prostatic region of the fetal
urogenital sinus and examined the prostatic mesenchyme cells,
which contain the androgen and estrogen receptors, in primary
culture. With a constant physiological concentration of 5-
dihydrotestosterone, E2 stimulated androgen receptor mRNA
levels at 1 pM (0.27 pg/ml), while BPA caused the same effect
at 1 nM (0.23 ng/ml), which is within the range of BPA detected
in human fetal umbilical cord blood [23]. Maternal exposure to
DES (0.1 g/(kg day), p.o.), an increase in free serum E2 (of
0.1 pg/ml, via Silastic implant) and BPA (50 g/(kg day), p.o.)
all caused a permanent increase in prostatic androgen receptors
in mice in addition to an increase in adult prosate weight, relative
to negative controls [10,16,17].
In addition to effects on prostate size and androgen respon-
siveness, prenatal exposure to BPA may affect the development
of prostate cancer in later life. Timms et al. [16] observed in
GD 19 male CD-1 mouse fetuses that a maternal oral dose of
10 g BPA/(kg day) on GD 14–18 stimulated an increase in the
number of primary prostatic ducts as well as proliferation of
basal cells (the progenitor cells thought to be responsible for
the development of prostate cancer) in the dorsolateral, but not
ventral, primary ducts. This is of interest in that a similar dose
of BPA administered via injection to neonatal rats resulted in
100% of the subsequent adult males exhibiting prostate interep-
ithelial neoplasia (PIN) lesions, which are pre-tumorous prostate
cancer lesions [29]. Epigenetic changes occurring during devel-
opment may be the basis for the adult altered susceptibility to
disease [95]. BPA is also implicated as a factor in the disruption
of therapy for human prostate cancer, since BPA can bind and
activate a mutant form of the androgen receptor, T877A, found
in some human prostate cancers, and thus stimulate prolifera-
tion of human prostate cancer cells in the absence of androgen
[96].
The prostate in rodents is composed of distinct lobes that
respond differently to endocrine disruptors. Differences in cell
proliferation and differentiation markers in stromal cells were
observed in ventral prostate following prenatal exposure to
25 g/(kg day) of BPA in prepubertal Wistar rats [30,97]. This
finding is different from those reported by Timms et al. [98],in
which male Sprague–Dawley rats exposed to the highest natural
serum levels of E2 (due to developing between female fetuses
[99]) showed enlargement of the dorsolateral prostate but not
the ventral prostate. In another study, prepubertal male Wis-
tar rats were injected with 50 mg BPA/(kg day) between PND
22–32. BPA induced a transient surge in prolactin (implicated
in the regulation of prostate growth) and a subsequent increase
in adult prostate size in later adulthood [100]. On PND 70,
prostate weight was significantly increased and daily sperm pro-
duction was decreased in Sprague–Dawley rats exposed during
gestation via a maternal oral dose of 100 g/(kg day) of BPA
[38]. In contrast to these findings, DES (6.5 g/(kg day)) or
BPA (30 and 300 g/(kg day)) administered via drinking water
to pregnant and lactating Wistar rats did not induce signifi-
cant effects on the ventral prostate (the only region examined)
[46].
2.3.3. Testes, epididymis, sperm and seminal vesicles
In contrast to development of the prostate from the uro-
genital sinus, development of the epididymis, vas deferens
and seminal vesicles from the Wolffian ducts is dependent on
diffusion of testosterone from the testis rather than systemic
testosterone, and does not involve metabolism of testosterone to
5-dihydrotestosterone. Thus, drugs and environmental chem-
icals can have quite different impacts on differentiation of
the Wolffian duct vs. the urogenital sinus [93]. Adult CF-1
male offspring that had been exposed prenatally to a mater-
nal oral BPA dose of 2 g/(kg day) showed decreased weights
of the epididymis and seminal vesicles, but increased weights
of the prostate and preputial glands; a 20 g/(kg day) dose
of BPA resulted in a decrease in daily sperm production per
gram testis [101]. Injection (s.c.) of BPA (50 g/animal, about
15–20 mg/(kg day)) for the first 5 days after birth resulted in
a decrease in the percentage of moving sperm, an increase
in the incidence of malformed sperm, and an increase in
the number of ER-positive cells in the epididymides of
SHN strain mice at 10 weeks of age [102]. Abnormalities
in the acrosomal granule and nucleus of step 2–3 spermatids
were also observed in neonatal ICR (CD-1) mice and Wis-
tar rats injected with 300 g BPA/(kg day) or 1 g E2/(kg day)
[103]. Ectoplasmic specialization between the Sertoli cell and
spermatids was also affected, and some specializations were
partially or totally deleted [103]. Decreased epithelial height
in the efferent ducts of the testes was observed in prepu-
bertal Wistar rats exposed prenatally to 37 mg BPA/(kg day)
[104].
Alterations in heat shock proteins in response to BPA
were examined in testicular germ cells, since they are
involved in the response to environmental stress and have
also been implicated in developmental events. Hsp90 pro-
tein levels were significantly increased at maternal oral
doses of 1 mg/(kg day) of BPA and 0.01 g/(kg day) of
DES [105]. A finding that contrasts with those described
above occurred when pregnant Sprague–Dawley rats were
fed BPA at 0.1 or 50 mg/(kg day). Spermatogenesis was
qualitatively normal in all groups, but both doses of BPA
214 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
increased testicular weight and Sertoli cell number per organ
[106].
2.4. Developmental effects on metabolism
The earliest reported exposure regime is direct dosing of
pre-implantation mouse embryos at the two-cell stage [107].
In this experiment, development to the eight-cell stage and to
the blastocyst stage was accelerated by exposure to 1–3 nM
(0.23–0.69 ng/ml (ppb)) BPA, and was delayed by exposure to
100 M (23 g/ml) BPA. Both the acceleration and the delay of
development to blastocyst were inhibited by co-treatment with
100 nM tamoxifen, which seems unexpected since that dose is
1000-fold less than the high BPA concentration. Also, the effects
on embryo development were not seen with E2 over a dose range
of 10 fM to 1 M [107]. When embryos treated with 1 nM BPA
were implanted in a female and allowed to develop, the resulting
pup weights at weaning were significantly increased compared
to control pups treated with solvent only (0.1% ethanol) as
embryos [108].
Increased postnatal growth in both male and female
rats and mice occurred at maternal doses between 2.4 and
500 g/(kg day) [15,37,41,78,107,109,110]. Evidence is accu-
mulating that during critical periods in development, estrogenic
chemicals can have unexpected effects on the differentiation of
adipocytes as well as postnatal growth [111]. Newbold et al.
reported that neonatal exposure to a low dose (1 g/(kg day))
of DES stimulated a subsequent increase in body weight and
an increase in body fat in mice [18]. In a related study a high
100 M dose of BPA stimulated an increase in the glucose trans-
porter GLUT4 and glucose uptake into 3T3-F442A adipocytes
in cell culture [112]. In a separate study of transgenic mice
over expressing the GLUT4 gene, increased basal and insulin-
induced glucose uptake was observed in whole body and in
isolated adipocytes [113]. Whether the mouse 3T3 cell lines,
which are relatively insensitive to estrogen, are an appropri-
ate model to study the effects of BPA in vitro remains to be
determined.
Some studies have found decreased body weight in response
to developmental BPA exposures [13,38,94], and some have
found no effects on body weight [54,57,114]. Recent research
on the effect of the type of animal feed used in an experiment
on postnatal growth suggests that whether or not an increase
or decrease in body weight occurs may be related to the type
of feed used [115]. In addition, the impact of exposure to BPA
during pregnancy and/or lactation on the maternal behavior and
lactational efficiency of mothers cannot be ignored [60,61].For
example, an experiment by Howdeshell et al., which reported
increased body weight in response to prenatal BPA exposure
[78], differed in design from an experiment conducted by Nagel
et al., which reported decreased body weight [94]. Howdeshell
cesarean delivered the pups and fostered them to untreated dams.
In contrast, Nagel allowed prenatally treated mice to nurse their
own offspring, leading to an opposite effect on adult body
weight. These two studies used the same low (2 g/(kg day))
maternal dose of BPA, the same CF-1 strain of mice, and the
same type of feed.
2.5. Developmental effects on the immune system
There has been only one study of the developmental effects
of BPA on immune function. Prenatal BPA exposure appeared
to increase all tested immune responses to soluble antigen in
exposed offspring [116]. Following feeding of DBA/1J mice a
30 g BPA/(kg day) dose from day 0 through day 17 of gestation,
adult male offspring produced increased antigen-specific IgG2a
antibody. A higher prenatal dose of 300 g/(kg day) increased
adult production of both T helper 1 interferon gamma (IFN-
) and T helper 2 IL-4 cytokines in exposed male and female
offspring [116]. The two cytokines tested have wide-ranging and
distinct effects on immune function; dysregulation of cytokine
production could have implications for inflammation or allergic
responses.
3. Effects of BPA exposure during adulthood
BPA exposure at low doses has diverse activational effects.
Some of these effects are predicted due to the affinity of BPA for
ER and ER, while other effects diverge from those observed
in response to activation of estrogen receptors.
3.1. Adult effects on the brain and behavior
Funabashi et al. [117–119] reported that a single injec-
tion of adult ovariectomized female Wistar rats with BPA at
400 g/kg–40 mg/kg increased progesterone receptor protein in
the preoptic area, ventromedial hypothalamus and frontal cor-
tex, as well as progesterone receptor mRNA in the preoptic area.
Specifically, estrogen acts to induce progesterone receptor in the
hypothalamus, and Northern blot analysis revealed an increase
in progesterone receptor mRNA in the preoptic area and anterior
pituitary of adult female rats acutely dosed with approximately
35 mg BPA/kg [117]. In this study, BPA caused responses simi-
lar to estrogen in the preoptic area and anterior pituitary, but not
in the mediobasal hypothalamus [117]. A single dose of approx-
imately 35 mg BPA/kg in adult female rats induced progesterone
receptors in the frontal cortex, but repressed progesterone recep-
tor expression in the temporal cortex [119].
Activational effects of BPA in the brain include an increase in
ER-expressing cells in the medial preoptic area of pregnant and
lactating adult females and non-pregnant, cycling adult female
Sprague–Dawley rats at a BPA dose of 40 mg/(kg day) admin-
istered orally for a period of 42 days, corresponding to the time
from mating to weaning in the pregnant and lactating group.
In contrast, a decrease in ER-expressing cells was observed
in the arcuate nucleus of pregnant and lactating females but
not non-pregnant, cycling females in response to this treatment
[120].
Injection of ovariectomized E2-treated female Sprague–
Dawley rats with 40–300 g BPA/(kg day) resulted in a mono-
tonic dose-related inhibition of E2-stimulated increase in
hippocampal synapses, with all doses producing significant
effects [44]. Thus, in the hippocampus, BPA acted to antagonize
the action of E2 on synaptogenesis.
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 215
BPA induced a significant increase in serum prolactin levels
in 8–10 week old ovariectomized Fisher 344 rats when adminis-
tered at a low dose of 40–45 g/(kg day) of BPA for a period of
3 days via Silastic capsules. In contrast, Sprague–Dawley rats
were relatively insensitive to both BPA and E2 in this experi-
ment, which was the first report that the model animal was an
important factor in BPA studies [121].
Exposure of adult female Sprague–Dawley rats from the day
of mating through lactation to 40 g/(kg day) of BPA via feed-
ing resulted in alterations in maternal behavior towards their
young; specifically, grooming was reduced in BPA-exposed
dams [61]. In Mongolian gerbils (Charles River, Italy), expo-
sure to pairs of adult males and females via feeding (in oil) 2
or 20 g BPA/(kg day) for 3 weeks increased their social inter-
actions and reduced their exploratory behavior; similar effects
were observed in response to 0.04 g/(kg day) of ethinylestra-
diol [122].
3.2. Adult effects on the female reproductive tract
Acute exposure of C57BL/6 female mice to 20 through
100 g/(kg day) of BPA resulted in a significant increase in mei-
otic abnormalities in the oocytes when exposure occurred during
the peri-pubertal period, suggesting that BPA exposure would
lead to aneuploidy; this abnormality was also observed in mice
that were housed in polycarbonate cages and that were provided
water in polycarbonate bottles that had been damaged by expo-
sure to a harsh detergent during washing [89]. The effect of BPA
on aneuploidy has also been examined in cell culture [123–126].
3.3. Adult effects on the male reproductive tract
A decrease in daily sperm production and fertility in male
Sprague–Dawley rats was reported at oral doses between 20
and 200,000 g/(kg day) due to adult exposure, and maximum
suppression of sperm production occurred at 20 g/(kg day). At
doses below 20 g/(kg day), daily sperm production was not
significantly different from controls [127]. This suggests that
there is a sub-population of cells that are impacted by BPA,
and an approximately 40% decrease in daily sperm production
is the maximum that occurs, regardless of the dose of BPA
administered above 20 g/(kg day). This finding is similar to
data reported for prenatal exposure to ethinylestradiol, where
maximum suppression of daily sperm production occurred at
2 ng/(kg day), with no further suppression occurring at higher
doses [128].
Similar to the findings in rats, oral exposure of adult Swiss
mice for 1 month to 25 and 100 g/(kg day) resulted in a decrease
in daily sperm production and epididymidal sperm concen-
tration, which was associated with a decrease in fertility. A
dose of 5 g/(kg day) also resulted in a decrease in the weight
of the testes and seminal vesicles [129] (Note that the doses
were incorrectly reported in the paper as ng/(kg day) instead
of g/(kg day).) A dose of 20 g/kg body weight of BPA was
injected (s.c.) to adult ICR (CD-1) mice and Wistar rats for
6 days, and abnormalities were observed in the spermatids:
acrosomal vesicles, acrosomal caps, acrosomes and nuclei of
the spermatids were severely deformed. The ectoplasmic spe-
cialization between the Sertoli cell and spermatids was also
affected: incomplete specialization, redundant ectopic special-
ization, and aplasia were observed [130]. A significant decrease
in testis and epididymidal weight was also reported to occur
in adult male Wistar rats exposed orally for 45 days to 0.2, 2
and 20 g/(kg day), while an increase in ventral prostate weight
occurred at all doses [131,132].
Successive i.p. administration of BPA to adolescent male
C57BL/6 mice at a dose of 20 mg/(kg day) for 4 weeks decreased
the prostate and seminal vesicle weights (but not testis or epi-
didymis weights) and also decreased serum testosterone and
both liver and kidney weights [133].
BPA at concentrations of approximately 0.2 and
20 mg/(kg day) was administered in the drinking water to
young male C57BL/6 mice for 8 weeks beginning at 3 weeks
of age (before the onset of puberty), and the number of ER-
containing cells and Esr2 mRNA per testis were significantly
decreased in the 20 mg BPA/(kg day) treated group compared
with controls. In contrast, ER-immunopositive cells and Esr1
mRNA per testis were markedly increased in these males
relative to controls [134].
An s.c. injection for 2 weeks of 3 mg/(kg day) dose of BPA
significantly reduced testicular testosterone content, and serum
testosterone, while plasma LH showed an increase in adult
male Wistar rats [135]. Injection (i.p.) of BPA at a dose of
20 mg/(kg day) for 4 weeks decreased the prostate and seminal
vesicle weights but not the testis or epididymis weights, and also
decreased serum testosterone and both liver and kidney weight
in Wistar rats [136]. Plasma free testosterone levels were dra-
matically decreased following 8 weeks of 12 mg/(kg day) of BPA
treatment of adult male C57BL/6 male mice compared with con-
trol group, and morphologically abnormal multinucleated giant
cells having greater than three nuclei were found in seminif-
erous tubules in the testis following 8-week BPA treatment at
120 g/(kg day), while no controls showed this [133].
3.4. Adult effects on metabolism
There is considerable experimental evidence that in adult
mice E2 acts via ER to have an inhibitory effect on adipocyte
number and lipogenesis, and removal of estrogen by ovariec-
tomy or ER via a genetic mutation also causes impaired glucose
tolerance and insulin resistance in addition to increased fat mass
[137,138]. Estrogen has central effects on food consumption and
energy expenditure that also contribute to its overall inhibitory
effects on adipose deposition in adults. BPA has also been
reported to decrease body weight in adults [129,139]. These
findings thus contrast numerous reports that low dose BPA or
DES exposure during early development stimulates postnatal
growth.
Very low doses of BPA stimulated rapid secretion of insulin
in mouse pancreatic cells in primary culture through a non-
classical, non-genomic rapid estrogen-response system. In the
same study prolonged exposure to a low oral dose of BPA
(10 g/(kg day)) resulted in stimulation of insulin secretion in
adult mice that was mediated by the classical nuclear estrogen
216 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
receptors; the prolonged hypersecretion of insulin was followed
by insulin resistance [140].
Other effects on metabolic pathways have also been observed
in response to BPA. A decrease in antioxidant enzymes occurred
at the very low dose of 0.2 g/(kg day) of BPA in adult Wistar
rat males [131], and at a higher dose of 50 mg/(kg day) in ICR
(CD-1) male mice [141]. In adult Wistar rats, oral administration
of 1 mg/(kg day) of BPA reduced in adult males but not females
the expression of UGT2B1 and other UGT isoforms that mediate
the glucuronidation of BPA and sex hormones [69].
3.5. Adult effects on the immune system
BPA has been reported to modulate immune function at
doses between 2.5 and 30 g/(kg day) [142,143], including
patterns of cytokine and antibody production, response to
infection, and autoimmune disease progression. T helper lym-
phocytes are a source of cytokine families that stimulate
inflammatory responses and resistance to intracellular infec-
tions (Th1 cytokines), or that shift the response to antibody
production, resistance to extracellular organisms, and allergy
(Th2 cytokines). Antigen-specific IFN- (Th1) secretion was
increased in adult DBA1/J mice given 30 g/(kg day) BPA p.o.
for 20 days and sacrificed 24 h later [143]. In contrast, a shorter
term oral dose of 2.5 g/(kg day) BPA given PND 35–42 to
C57BL/6 and autoimmune-prone NZB/WF1 mice significantly
decreased the production of IFN- by splenocytes stimulated
with 4 g/ml of the T lymphocyte mitogen ConA, up to 5
weeks post treatment [142]. A single oral dose of approximately
9 mg BPA/kg just prior to infection with the parasite Trichinella
spiralis increased production of both IL-4 and IL-10 by lymph
node cells stimulated with parasite antigen [144]. Addition of
BPA at doses ranging from 0.1 to 30 M to cultures of lym-
phocytes from Leishmania major-infected BALB/c or C57BL/6
mice did not increase IFN- production [144]. Other studies
also report an increase in the Th2-associated cytokine, IL-4, fol-
lowing BPA exposure. Injection (i.p.) of 25 mg/(kg day) BPA
every other day for 1 week in adult KLH-primed BALB/c mice
resulted in significant increase of IL-4 production in CD4+ T
cells and of serum antigen-specific immunoglobulin E (IgE) lev-
els [145]. Antigen specific spleen cells of adult BPA-exposed
DBA1/J mice (300 g/(kg day) p.o. for 20 days) produced an
elevated IL-4 response [143]. Thus, most models indicate that
BPA may enhance or shift the pattern of cytokine production fol-
lowing antigen stimulation. Skewing of the Th1/Th2 cytokine
profile by EDCs has been associated with allergy and asthma
[146].
Exposure to BPA has also been associated with modulation
of innate immune system cell function. For example, adminis-
tration of 5 mg/(kg day) s.c. to adult BALB/c mice for 5 days
decreased innate host defense to bacterial infection. Upon chal-
lenge with i.p. E. coli injection, both neutrophil phagocytosis
and IL-6 production were significantly reduced [147].
Autoimmune diseases, particularly those mediated by an anti-
body response to host tissues, are more common in females;
estrogens and prolactin are believed to act as drivers for differ-
ential disease development and progression in individuals with
an underlying genetic susceptibility to autoimmunity. However,
BPA exposure modulates the course of glomerulonephritis in
the NZB/WF1 mouse model of systemic lupus erythematosus.
An oral dose of 2.5 g/(kg day) given on PND 35–42 to female
NZB/WF1 mice decreased the production of IgG2a antibody by
splenocytes and delayed the onset of glomerulonephritis as mea-
sured by albuminuria [142]. Yurino et al. used Silastic implants
to administer BPA, E2, or DES to ovariectomized, 4 week old,
female NZB/WF1 mice [148]. Serum concentrations of 30 ng/ml
BPA were measured 4 months after implantation. At 5 months of
age, both DES- and E2-treated mice showed an increase in IgG
anti-DNA antibody and prominent deposition of immune com-
plexes in the glomeruli, both indicators of disease. In contrast,
although BPA, DES, and E2 increased IgM-class autoantibodies,
there was no evidence of glomerular immune complex deposi-
tion and no increase in serum anti-DNA IgG antibody in the
BPA-exposed mice.
In summary, while it is well accepted that estrogen alters
immune function, the interaction is complex [149]. Estrogen
has been reported to both decrease and increase immune func-
tion, depending on dose, including conflicting reports on the
production of the T helper 1 cytokine, IFN- [150,151]
. The
effects of BPA exposure on the immune system may be crit-
ically dependent on the timing of BPA exposure. Estrogen
receptor expression by lymphocytes is dependent upon the age
and strain of the animal; in addition, recent evidence sug-
gests the spleen undergoes significant molecular remodeling
during puberty, resulting in both age and gender-dependent
differences in immune gene expression [152]. Nevertheless,
studies conducted by Yoshino et al. [116,143] indicate simi-
lar dose-associated, gender-independent immune system effects
in 8-week-old offspring of BPA-exposed dams and animals
exposed as adults. These results suggest quantitative, rather than
qualitative, differences in lifestage-dependent immune system
sensitivity to BPA.
4. Comparison of findings of significant effects and
no-significant effects in low dose BPA studies
As of the end of October 2006 we are aware of 27 in vivo
studies reporting no significant effects in response to low doses
of BPA. The variables that account for most of the studies that
find no significant effects have recently been reviewed [5,153].
Below we discuss a number of these variables.
4.1. Strain differences
The major factor that accounts for 13 studies that draw
the conclusion of no effect of low doses of BPA is the use
of a strain of rat sold by Charles River Laboratories, referred
to as the CD-SD rat [Crl:CD(SD)] (Table 2). According to
Charles River [154], rats were purchased by Charles River
from Sprague–Dawley in 1950. This colony was continuously
subjected to selective breeding for rapid postnatal growth and
large litter size for over 40 years. The Crl:CD(SD) rat strain
is insensitive to BPA, and 0 of 13 studies report low dose
effects [155–167]. In addition, this rat strain is insensitive to
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 217
the drug ethinylestradiol at doses (0.5 g/(kg day)) used in
oral contraceptives [163,167]. As shown above, a large number
of studies show that significant low dose effects of BPA have
been observed in the original Sprague–Dawley rat. However, a
number of studies have reported that the inbred Fisher 344 rat is
more sensitive to low dose effects of BPA relative to the original
Sprague–Dawley rat [121,168,169]. Wistar rats also appear to
be more sensitive than Sprague–Dawley rats to the uterotrophic
effects of BPA [168,170]. One assessment of the uterotrophic
response of immature Sprague–Dawley rats revealed a response
following 3 days of exposure to 300 mg/(kg day) of BPA. In the
same study, a significant response to ethinylestradiol was noted
at 0.3 ug/(kg day) [171]. Why the Sprague–Dawley rat does not
respond to low doses of BPA in the uterotrophic bioassay or in
the vaginal stimulation bioassay has been investigated, and the
lack of responsiveness appears to be due to factors after binding
to estrogen receptors [169].
The importance of the strain of animal used in low dose
research was acknowledged in the 2001 Low Dose National Tox-
icology Program (NTP) report. The NTP panel emphasized the
need to test for the sensitivity of any animal model by including a
positive control, such as the well characterized estrogenic drugs
DES or ethinylestradiol, and stated [172, p. vii]: “Because of
clear species and strain differences in sensitivity, animal model
selection should be based on responsiveness to endocrine active
agents of concern (i.e., responsive to positive controls), not on
convenience and familiarity.
4.2. BPA as a SERM: responses in different tissues and in
different animal models
A difference in the response of tissues to low doses of BPA
is shown in articles that have been published reporting that low
doses of BPA do not stimulate a typical uterotrophic (increase in
uterine weight) response in mice and rats, while low dose effects
are observed in many other endpoints. For example, effects on
uterine weight have been observed at relatively high doses of
BPA (25–40 mg/(kg day)), while changes in uterine gene activ-
ity occurred at lower doses of 10 mg/(kg day) in B6C3F1 mice
and 0.8 mg/kg in transgenic ERIN mice [173–175]. In prepu-
bertal CD-1 mice, a dose of 100 mg/(kg day) injected s.c. for
3 days was required to stimulate an increase in uterine weight
[176]; however, 5 mg/(kg day) was sufficient to cause a signif-
icant increase in the height of uterine epithelial cells in this
strain, Similar findings regarding the inability of low doses of
BPA to stimulate a uterotrophic response in prepubertal CD-1
mice were reported in another study [177], which, in compar-
ison to the B6C3F1 mouse data, provides further evidence for
strain differences in sensitivity to BPA.
The prepubertal uterotrophic assay is thus a relatively insen-
sitive endpoint for studies of BPA, since the dose required to
stimulate a uterotrophic response (one of the most commonly
used bioassays in toxicology to assess estrogenic activity), is
markedly higher relative to doses that cause significant effects in
CD-1 mice in the prostate [10,16], testes [52], mammary glands
[79–81,88], and the brain and behavior [47,52,60,66]. All of
these studies report significant effects in response to develop-
mental exposure of CD-1 mice to doses of BPA at and far below
the reference dose of 50 g/(kg day) (there are over 20 low dose
BPA studies published reporting significant effects of BPA in
CD-1 mice). As a specific example, when the magnitude of the
response to BPA in mammary gland and uterus are compared,
the mammary gland appears to be a more sensitive target for
BPA action, but clearly, in utero exposure to low doses of BPA
does have lasting effects on the uterus in CD-1 mice [88].
4.3. Batch-to-batch variability in feed: impact on low dose
endocrine disruptor research
The other major variable that has been implicated in con-
tributing to discrepancies in the outcome of experiments on low
dose effects of BPA is the type of feed used. Two workshops on
this issue were held in 2005 and 2006 to address this issue, and a
manuscript reviewing the conclusions from these workshops is
being prepared. The consensus was that there is a critical need
for researchers to better understand the potential for components
of commercial feed to impact research outcomes. Importantly,
no one type of feed was deemed appropriate for the many differ-
ent types of research conducted with laboratory rats and mice,
and both soy-based and non-soy-based feeds were reported to
contain variable amounts of estrogenic contaminants that were
not soy phytoestrogens [178]. The major concern was that feed
manufacturers should make every effort to reduce the batch-to-
batch variability in feed components that can lead to phenotypic
variation in control animals.
Findings also presented at the workshops emphasized the
critical importance of including appropriate positive controls in
experimental studies. With regard to BPA for example, the inclu-
sion of an appropriate positive control (based on experimental
objectives and techniques, such as route of administration)
allows one to determine whether or not the test system has been
rendered insensitive to any estrogenic chemical or drug when
drawing conclusions concerning the potential safety of estro-
genic chemicals such as BPA. A specific example was provided
by the finding of Thigpen et al. that a commonly used feed in tox-
icological studies, PMI 5002, showed significant batch-to-batch
variability in phytoestrogens that related to an inability to detect
significant effects of the potent estrogenic drug diethylstilbe-
strol (DES) with some batches of this feed [19]. Some studies
that reported finding no significant effects at low doses of BPA
[166,179] used PMI 5002 feed, raising concerns that effects of
DES would not have been found with the batches of this feed that
were used. In fact, 0.1 g/(kg day) DES (administered orally to
pregnant CF-1 mice) was used as a positive control in one study
[179], and DES-exposed animals did not differ from negative
controls on any outcome.
4.4. The use of positive controls
The NTP panel commented in its 2001 report on the issue
regarding: “a study in which the positive control does not pro-
duce the expected positive response. The prudent course of
action in such cases may be to declare the study inadequate
and repeat it, regardless of the experimental outcome in the test
218 C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224
groups” [172, pp. 5–10]. The NTP panel went on to note that:
“For those studies that included DES exposure groups, those that
showed an effect with BPA showed a similar low dose effect with
DES (e.g. prostate and uterus enlargement in mice), while those
that showed no effect with BPA also found no effect with DES.
In many studies in which no statistically significant effect of low
doses of BPA were reported, no positive control was included
[155,156,159,160,166,180–185]. In particular, a positive control
is the only way to distinguish whether an experiment has been
compromised by the presence of an estrogenic contaminant in
animal feed, water, or housing.
Three studies that concluded that there was no effect of low
doses of BPA on the male [179,186] or on both the male and
female [109] reproductive system in mice suffered from a lack
of statistically significant effect of the positive control, DES. One
study did report some effects at a relatively high (15 g/(kg day))
dose of DES and no significant effects at any dose of BPA [161].
As discussed previously, this high dose of DES is not a valid pos-
itive control in experiments that fail to show effects of low doses
of BPA. In summary, experiments reporting only the absence of
statistically significant effects of low doses of BPA that do not
include a positive control, or that fail to show significant effects
of the positive control, cannot be interpreted. Without positive
control data obtained concurrently with the BPA data, there is
no way to determine the reason that significant effects were not
observed for any test chemical.
There are now numerous studies that have been published
showing similar effects of low doses of BPA and DES, the most
commonly used positive control for examining estrogenic effects
of BPA. For many outcomes, BPA has been reported to have the
same efficacy but a 100–1000-fold lower potency relative to DES
[10,13–16,46,69,105,148,187–189].
There are some studies showing that, particularly for rapid
responses mediated by receptors associated with the cell mem-
brane [190–192], BPA and DES (as well as E2) have equal
potency. This subject is covered in detail in the report from
the panel concerned with molecular mechanisms. As discussed
above, there is evidence that BPA is a SERM, and thus, while
there are studies showing similar effects of BPA and endoge-
nous E2 or estrogenic drugs such as DES, there are also studies
showing qualitative differences in the responses of BPA and
other estrogens.
4.5. The importance of dose
There have been many published in vivo and in vitro mech-
anistic studies providing examples of effects of BPA that are
observed at a low dose, but are not observed at higher doses.
This type of dose–response relationship is known as an inverted-
U-shaped curve. In toxicology, it was long assumed that the
only valid dose-related effect is a monotonic dose–response
relationship. However, as toxicologists have investigated more
sub-lethal endpoints and more extensive dose ranges, non-
monotonic dose–response relationships have been encountered
with increasing frequency. The molecular basis for such find-
ings is beginning to be understood in terms of the stimulation
and inhibition of unique sets of genes as one moves across the
dose–response curve [193,194]. These effects are likely to be
mediated by different receptors [for estrogens, ER,ER, ERR
and membrane-associated receptors [43,195,196], and both pos-
itive and negative feedback effects on estrogen receptors and
other receptors [6,7]]. Several examples of unique effects of
low doses of BPA have now been reported and are available for
review [45,46].
5. Conclusions and levels of confidence for different
outcomes
5.1. Based on existing evidence, we are confident of the
following
The criterion for an outcome being assessed as achieving this
level (we are confident) is that multiple independent studies have
shown the same or similar outcomes.
5.1.1. Developmental effects on the brain and behavior
There is extensive evidence for effects of exposure dur-
ing critical periods to low doses of BPA on subsequent brain
structure and chemistry, receptors for hormones and neurotrans-
mitters, and behavior. We can thus state with confidence that
low doses of BPA during development have persistent effects on
brain structure, function and behavior in rats and mice.
It is likely that due to species and strain differences, effects
of BPA on specific brain structures, functions and behaviors
may show differences. However, some effects appear consis-
tent. For example, a number of studies have shown that expected
differences between males and females are not observed in ani-
mals exposed to BPA during development. In some experiments
this outcome has been due to males showing a more feminine
phenotype and females showing a more masculine phenotype
[47,73], while in other experiments, the loss of the sex difference
was due to a measurable change in only one sex [197]. Further
research may reveal whether estrogenic effects dominate when
low doses of BPA are administered and whether other effects of
BPA such as antiestrogenic or antiandrogenic effects are likely
to be observed in the presence of higher doses of BPA.
5.1.2. Developmental effects on the male reproductive tract
There is extensive evidence that BPA impacts the reproduc-
tive system in male rats and mice, although there appear to be
species and strain differences in the sensitivity of specific out-
comes to BPA. The evidence supports an effect on the testes,
with subsequent changes in testosterone secretion and sperm
production. Impacts on other reproductive structures have been
reported in a number of independent studies, including the epi-
didymis and epididymal sperm, prostate, and seminal vesicles.
These findings are consistent with effects of low doses of positive
control chemicals, such as DES and ethinylestradiol.
5.1.3. Developmental effects on enzyme activity, growth
and metabolism
There is extensive evidence for “programming” effects of
BPA on subsequent activity of enzymes in tissues and thus
metabolic processes. An increase in postnatal growth rate due
C.A. Richter et al. / Reproductive Toxicology 24 (2007) 199–224 219
to developmental exposure to low doses of BPA has been shown
in many studies, and this finding is consistent with the effect of
developmental exposure to a low, but not high, dose of DES.
5.1.4. Adult effects on the male reproductive tract
There is a significant amount of evidence that adult exposure
to BPA has adverse consequences for testicular function in male
rats and mice. This is not surprising as estrogen, while essen-
tial for normal epididymis function, has inhibitory effects on
the brain–pituitary–gonadal axis in males, and it is well docu-
mented that elevated E2 inhibits spermatogenesis and testicular
testosterone secretion [1].
5.2. We consider the following to be likely but requiring
confirmation
The criterion for achieving this level is that significant effects
have been reported, but the number of independent replications
is limited. However, confidence in the findings is increased by
the plausibility of the results, based on mechanistic information
available from other related studies.
5.2.1. Developmental effects on the female reproductive
tract
There is extensive evidence for effects of BPA on devel-
opment of the mammary gland. Studies in both rats and mice
have shown effects of developmental exposure to BPA on mam-
mary gland morphology that may predispose animals to develop
cancer. These findings have not yet been repeated in multiple
independent laboratories.
5.2.2. Adult effects on the brain and behavior
There are a number of studies that have found a variety of
significant outcomes of adult exposure to BPA. Given the more
extensive literature showing developmental effects, there is no
reason to expect that adult effects will not also occur. However,
effects in the adult may require higher doses of BPA exposure
or longer periods of BPA exposure.
An important unanswered question and research need is
whether long-term adult exposure to BPA may have adverse
consequences, as has been recently found to be the case for
hormone replacement therapy (HRT), which is now thought to
increase the risk for a number of diseases [198]. The finding
that BPA can stimulate proliferation of human cancer cells in
the absence of androgen is also cause for concern.
5.2.3. Adult effects on the female reproductive tract
While a number of studies have found significant effects of
BPA on the female reproductive system, not as much research
has been conducted as for the male reproductive system. For
example, effects on meiosis in oocytes need to be confirmed by
additional studies. The recent report that maternal exposure to
a very low dose of BPA also disrupts meiosis in the embryonic
oocyte during formation of the primary follicles adds to our
concern.
5.2.4. Adult effects on the immune system
There is extensive evidence that BPA modulates both T helper
1 and T helper 2 cytokine production and alters antibody pro-
duction.
Acknowledgements
Financial support: This review was prepared in conjunc-
tion with the Bisphenol A Conference, Chapel Hill, NC, 28–29
November 2006. Support was provided by the National Institute
of Environmental Health Sciences and the National Institute of
Dental and Craniofacial Research, NIH, DHHS, the W.M. Keck
Center for Behavioral Biology at NC State University, and from
Commonweal.
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