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

246 CHAPIN ET AL.
group was a reduction in plasma testosterone level [by
B28%]. No effect was observed on cholesterol level. In
the ex vivo study, prenatal bisphenol A exposure
increased activities of 17a -hydroxysteroid dehydrogenase
[by B140%] and 17b-hydroxysteroid dehydrogenase
[by B70%]. Observations in the 17b-estradiol compared
to the control group included decreased numbers of
offspring delivered, higher body weight of male offspring
at 13 weeks of age, reduced plasma testosterone
level, and increased testicular 17a-hydroxysteroid dehydrogenase
activity. The study authors concluded that
bisphenol A had an estrogenic effect on the testis but did
not decrease activities of enzymes involved in testosterone
production.
Strengths/Weaknesses: A strength of this study is the
examination of testosterone levels at 13 weeks of age.
This strength is negated by the sample size (n 5 2–4),
which is too small to draw any firm conclusions.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate based on insufficient sample size.
Murray et al. (2007), supported by NIH, examined the
effect of prenatal bisphenol A exposure on in situ
induction of mammary tumors. Wistar-Furth rats were
fed Harlan Teklad 2018, which was reported to contain 20
fmol/g estrogen equivalents. Water was supplied in glass
bottles. Caging and bedding materials were not reported,
but they were stated that to test negative in the E­
SCREEN. From GD 9 (GD 1 5 day of vaginal sperm)
through PND 1 [The day of birth was PND 0 (A. Soto,
personal communication, March 2, 2007)], rats received
the 50% DMSO vehicle or bisphenol A [purity not
reported] at 0.0025, 0.025, 0.250, or 1 mg/kg bw/day.
Dosing solutions were delivered by implanted [assumed
s.c.] osmotic pumps. [Number of dams treated was not
reported. Based on a limited amount of information
provided on the number of offspring examined, it
appears that r6 dams/group were treated.] Pup
viability was assessed on PND 1. On PND 2 pups were
sexed and litters were culled to 8 pups. Anogenital
distance was measured on PND 4. Litters were weighed
during the lactation period. Female offspring were
monitored for body weight and vaginal opening in the
post weaning period. Female offspring were killed on
PND 50 or 95. Mammary glands were collected and
whole-mounted or sectioned for histopathological examination.
Morphometric analyses were conducted to
examine possible presence of preneoplastic lesions.
Mammary glands were examined for ERa and Ki-67
protein by an immunohistochemistry technique. Maximal
numbers of ‘‘maternal units’’ were represented in
each dose group. One female/litter was included in
histological examinations. [Apparently r6 offspring/
group were examined in histopathological examinations.
Number of offspring examined for other endpoints
was not reported in the manuscript. According
to an author, n 5 7–21 for the other endpoints (A. Soto,
personal communication, March 2, 2007).] Statistical
analyses included ANOVA followed by post-hoc tests
(Bonferroni or t-test) when significant effects were
observed by ANOVA. [It was not clear if dams or
offspring were considered the statistical unit.]
Bisphenol A exposure did not affect offspring viability,
sex ratio, age at vaginal opening, or female anogenital
distance. Anogenital distance was reduced on PND 4 in
males from the 0.250 mg/kg bw/day group. Percent
hyperplastic ducts was increased in all dose groups on
PND 50 and in the 0.0025 mg/kg bw/day group on PND
95; the study authors noted that the effect on PND 50 was
quantitatively similar in all dose groups (i.e. 3–4-fold
increase). Cribriform structures were observed in the 0.25
and 1 mg/kg bw/day groups. [Incidence was not
reported for the control and lower dose groups.] The
structures were classified as carcinomas-in-situ and were
characterized by increased ductal size resulting from
luminal epithelium proliferation, enlarged luminal
epithelial cells, presence of a nucleolus, variable chromatin
pattern, and rounded luminal spaces consisting of
trabecular rods of cells perpendicularly aligned to the
longer duct axis. Numbers of Ki-67- and ER-a positive
cells were increased in aberrant compared to normal
tissues, regardless of dose. [Results in treated compared
to control groups were not reported.] The study authors
concluded that fetal bisphenol A exposure is rats is
sufficient to induce development of preneoplastic and
neoplastic mammary lesions.
Strengths/Weaknesses: Relevance of endpoints is a
strength, as is the use of multiple dose levels. Weaknesses
include an unstated number of dams (and by inference, a
small number of these, and thus, because of dam-related
effects, a small overall n), the uncertainty of the response
rate of histopathology in the controls, and the use of 50%
DMSO as vehicle.
Utility/Adequacy for CERHR Evaluation: This study
was inadequate due to small sample size, route of
administration, and lack of clarity on statistical analysis.
Durando et al. (2007), supported by Universidad
National del Litoral, Argentine National Agency for the
Promotion of Science and technology, and NIH, examined
the effects of prenatal bisphenol A exposure on
susceptibility to mammary tumors in rats. Wistar rats
were fed Cooperación (Buenos Aires, Argentina) and
housed in stainless steel cages. [It was not clear if
bedding was used.] On GD 8–23 (GD 1 5 day of vaginal
sperm), 11–14 dams/group were s.c. dosed by osmotic
pump with the DMSO vehicle or 0.025 mg/kg bw/day
bisphenol A [purity not indicated]. Pups were delivered
on GD 23 and weaned on PND 21. It was not indicated if
day of birth was considered PND 0 or 1. During the
study, body weights and day of vaginal opening were
monitored. Offspring were killed before puberty (PND
30), after puberty (PND 50), or in adulthood (PND 110
and 180). In mammary gland stroma and epithelium,
proliferation was assessed by BrdU incorporation and
apoptotic cells were identified by TUNEL method.
Morphometric analyses were conducted in sectioned
mammary glands. Mast cells were identified by immunostaining
for proteinase. At least 6 offspring/group/
time point were evaluated. [No littermates were used in
the evaluation at any given time point (A. Soto,
personal communication, March 2, 2007).] Additional
offspring were examined for responsiveness to chemically-induced
mammary preneoplastic or neoplastic
lesions. On PND 50, N-nitroso-N-methylurea was administered
to 10–16 offspring from the vehicle control
group at 25 or 50 mg/kg bw and 21 offspring from the
bisphenol A group at 25 mg/kg bw. Based on findings
from a pilot study, 25 mg/kg bw was considered a
subcarcinogenic dose and 50 mg/kg bw was considered
a positive control. During the study, rats were palpated
for tumors. Rats that received 50 mg/kg bw N-nitroso-
Birth Defects Research (Part B) 83:157–395, 2008