Monograph on the Potential Human Reproductive and ... - OEHHA

270 CHAPIN ET AL.
doses of potent estrogens induce gross abnormalities in
the male reproductive system; and that only agents that
suppress androgen receptor while increasing ERa and
progesterone receptor are likely to cause gross developmental
abnormalities in the male reproductive system.
Strengths/Weaknesses: Strengths include expertise of
the group coupled to well-performed experiments, data
recording, and interpretation. Bisphenol A was not a
primary target in this study but was one of a series of
estrogenic compounds, allowing comparison with other
similar compounds. However, a significant weakness are
the s.c. route of administration, only a single varying
dose level of bisphenol A was used, and there was no
accounting for litter effects within the context of
individual animal treatments within litters.
Utility (Adequacy) for CERHR Evaluation Process:
This work is inadequate for the evaluation process, based
on lack of clarity for experimental or statistical control for
litter effects.
Rivas et al. (2002), supported by the European Union
and the Spanish Ministry of Education, examined the
effects of bisphenol A exposure on reproductive tract
development of male rats. The main focus of the
study was determining the effects of decreased androgen
production in combination with a low dose of
diethylstilbestrol. Effects of flutamide were also examined
but will not be discussed. Wistar rats were fed a soyfree
diet (rat and mouse soya-free breeding diet; SDS,
Dundee, Scotland). [No information was provided about
caging and bedding materials.] Litters of 8–12 male pups
were assembled by cross-fostering on PND 1 (day of
birth). Male rats were s.c. injected with the corn oil vehicle
or 0.1 mg bisphenol A [purity not indicated] on PND 2, 4,
6, 8, 10, and 12 with and without co-administration of
10 mg/kg GnRH antagonist (a suppressor of androgen
production). [Assuming a 5–25 g body weight during
this interval, the bisphenol A dose would be B20 mg/kg
bw/day at the beginning of the interval and B4 mg/kg
bw/day at the end of the interval.] Additional rats were
s.c. injected with diethylstilbestrol at doses of 0.1 or 10 mg
on PND 2, 4, 6, 8, 10, and 12 with and without
administration of GnRH antagonist. Rats were killed on
PND 15. The testis was fixed in Bouin solution and
testicular structures were measured. Plasma testosterone
levels were measured using an ELISA technique. From 3–
10 animals/group were examined for each endpoint.
Data were analyzed by ANOVA.
Treatment with bisphenol A alone did not affect
plasma testosterone levels but treatment with GnRH
antagonist alone and in combination with bisphenol A
significantly lowered plasma testosterone levels. Treatment
of rats with bisphenol A alone or in combination
with GnRH antagonist had no significant effect on rete
testis luminal area, efferent duct luminal area, efferent
duct epithelial cell height, or vas deferens epithelial cell
height. Exposure to the high diethylstilbestrol dose
increased rete area, and both doses of diethylstilbestrol
decreased plasma testosterone levels, increased efferent
duct luminal area, and decreased epithelial cell height in
efferent duct and vas deferens. The study authors
concluded that the estrogenicity of bisphenol A when
injected at a moderately high-dose was insufficient for
disrupting the estrogen-androgen balance in rats.
Strengths/Weaknesses: This study was performed
carefully and well-documented. Weaknesses include:
the dose of bisphenol A was high and only a single dose
level administered subcutaneously was examined; and
litter effects were not addressed in the context within
litter dosing of cross-fostered litters.
Utility (Adequacy) for CERHR Evaluation Process:
This work is inadequate for the evaluation process, based
on lack of clarity on control for litter effects.
Sharpe et al. (2003), supported in part by the European
Union and the Spanish Ministry of Education, examined
the effects of neonatal exposure of rats to bisphenol A on
Leydig cell development and function. Wistar rat dams
were fed a standard soy-containing feed (rat and mouse
breeding diet; SDS). [No information was provided on
feed given to male offspring following weaning or
bedding and caging materials.] Litters of 9–12 male
pups were created by cross fostering pups on PND 1 (day
of birth). Male pups were s.c. injected with the corn oil
vehicle or 0.5 mg/day bisphenol A [purity not reported]
on PND 2–12. [Assuming 5–25 g body weight during
this interval, the dose would be B100 mg/kg bw/day at
the beginning of the interval and B20 mg/kg bw/day at
the end of the interval.] Other groups of rats received
diethylstilbestrol at 0.1–10 mg/day on PND 2, 4, 6, 8, 10,
and 12. Additional rats were treated with GnRH
antagonist Antarelix or 4-tert-octylphenol, but those
results will not be discussed. Rats were killed on PND
18, 25, 35, or 90. Testes were weighed and fixed in Bouin
solution. Sections of testes were immunostained with the
Leydig cell marker 3b-hydroxysteroid dehydrogenase to
evaluate Leydig cell development in 5–7 animals/group.
Plasma testosterone levels were measured by ELISA.
Group sizes for evaluation of testes weight and plasma
testosterone were 2–23, with most groups containing at
least 8 animals. Data were analyzed by ANOVA.
The only significant effect on plasma testosterone level
following exposure to bisphenol A was an increase on
PND 18 (n 5 4). In rats of the bisphenol A group
examined at each time period, there were no significant
effects on testis weight, percent Leydig cell nuclear
volume/testis, Leydig cell nuclear volume/testis, or total
Leydig cell volume (nuclear1cytoplasmic volume/testis).
Significant results in rats exposed to diethylstilbestrol
included decreased Leydig nuclear cell volume at the
mid- or high-dose on or before PND 35 and reduced
plasma testosterone level and testis weight at all doses
and most time points of evaluation. The study authors
concluded that there were no consistent changes in
Leydig cell development following exposure to bisphenol
A.
Strengths/Weaknesses: A strength is that bisphenol A
was one of a number of compounds examined enabling
internal comparison with other similar molecules. Limitations
include use of a single high but variable dose of
bisphenol A and small sample sizes for critical
endpoints.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate due to small or uncertain
sample sizes for key endpoints.
Khurana et al. (2000), supported by NIH, March of
Dimes, and Pardee Foundation, examined the effects of
neonatal bisphenol A exposure on prolactin levels in rats.
[The type of chow used and composition of bedding
and caging materials were not reported.] On PND 1–5
(day of birth 5 PND 0), 8–10 Fischer 344 rat pups/sex/
group (litter relationships are unclear) were s.c.
Birth Defects Research (Part B) 83:157–395, 2008