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

were culled to 10 pups/litter on PND 7. One female
offspring/litter, from 6–10 litters/group, was killed on
PND 20 and 30 and at 4 months of age. The 4-month-old
mice were killed on proestrus. Another group of mice
[number not specified] was killed on the first proestrus.
Mammary glands were collected for evaluation of
mammary structures at 20 and 30 days and 4 months
of age and day of first proestrus. Mammary glands were
also collected from 30-day-old mice for analysis of DNA
synthesis by BrdU incorporation, expression of ERa and
progesterone receptor using immunohistochemistry
techniques, apoptosis by TUNEL method, and Wnt4
mRNA by RT-PCR. Plasma 17b-estradiol levels were
measured in mice killed at first proestrus. In an
experiment to monitor response to 17b-estradiol, 1
pup/litter (n 5 10/group) was ovariectomized at 25 days
of age and implanted with a s.c. pump supplying vehicle
or 0.5 mg 17b-estradiol/kg bw/day on PND 25–35. Mice
were killed following 17b-estradiol treatment for examination
of mammary structures. Statistical analyses
included ANOVA and Dunn post-hoc test. If the data
were not normally distributed, statistical analyses were
done by Kruskall–Wallis and Mann–Whitney test.
In 30-day-old mice, bisphenol A exposure increased
numbers of terminal end buds at both doses and area of
terminal end buds at the high-dose. Percentages of
apoptotic cells were decreased on PND 30 in mice from
both bisphenol A dose groups. The percentage of stromal
cells undergoing proliferation on PND 30 was reduced in
the high-dose bisphenol A group. The number of
epithelial cells expressing progesterone receptors was
increased in both dose groups on PND 30, but there were
no treatment-related changes in ERa receptor expression.
Clusters of progesterone receptors were often observed
in the ductal epithelium of bisphenol A-treated mice.
Slopes of the correlation between age of first proestrus
and mammary length were significantly reduced in the
high-dose group, suggesting slower ductal invasion of
stroma. There were no significant differences in plasma
17b-estradiol levels in mice killed at first proestrus.
Trends for increasing expression of mRNA for Wnt4, a
mediator of lateral branching downstream from progesterone
receptors, did not attain statistical significance.
The number of lateral branches in mammary gland at 4
months of age was significantly increased at the low but
not the high-dose. In mice exposed to the high-dose of
bisphenol A during perinatal development and 17bestradiol
during postnatal development compared to
mice who were exposed to 17b-estradiol but not bisphenol
A, there were increases in numbers, area, and
size of terminal end buds, terminal end bud numbers/
ductal area, and terminal end bud area/ductal area.
The study authors concluded that ‘‘yperinatal exposure
to environmentally relevant [bisphenol A] doses
results in persistent alterations in mammary gland
morphogenesis.’’
Strengths/Weaknesses: This study was a follow-up on
the study of Markey et al. (2005) and tested the same
doses using a similar schedule for effects on mammary
tissue. The administration of very low doses is a strength.
The statistics appear to be inappropriate in not accounting
for the significant number of comparisons made. A
critical weakness is the uncertainty of the DMSO
concentration as a vehicle and therefore pump
performance.
Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A
305
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for the evaluation process given
exposure uncertainties.
3.2.8.2 Male reproductive endpoints:: Nakahashi
et al. (2001), supported by the Japanese Ministry of
Education, Science, Sports, and Culture, examined the
effect of neonatal bisphenol A exposure on adult sperm
count in mice. On the first 5 days of life, 10–15 neonatal
SHN mice/group were injected [route not indicated]
with sesame oil/DMSO vehicle or with bisphenol A
[purity not reported] in sesame oil at 0.0005 or 0.050 mg/
day. [Assuming a neonatal mouse weights 2 g, the mice
received doses of 0.25 and 25 mg/kg bw/day.] A group of
12 mice received 0.050 mg/day bisphenol A in sesame oil
in combination with 100 IU retinol acetate in DMSO
vehicle. In a second exposure protocol, pregnant mice
were fed a vitamin A-deficient diet (Low Vitamin A diet;
Clea Japan) from 3 days before gestation to PND 5. After
PND 5, the dams were fed commercial diet (CE-7, Clea
Japan). On the first 5 days of life, their pups (n 5 7–9/
group) were injected with bisphenol A at 0 (sesame oil)
or 0.0005 mg/day. Male offspring from both studies were
weaned at 20 days of age and fed the CE-7 diet. Mice
were killed at 14 weeks of age and epididymal sperm
counts were obtained. [No information was provided
about caging and bedding materials. Numbers of litter
represented were not indicated. Procedures for statistical
analyses were not discussed.]
A 35% reduction in sperm counts was observed in
mice from the 0.050 mg/day group compared to the
control group. A significant reduction in sperm counts
was not observed in the group co-treated with 0.050 mg/
day bisphenol A and retinol acetate. Administration of a
vitamin A-deficient diet to dams had no effect on sperm
counts in their offspring, but sperm counts were reduced
in mice born to mothers fed a vitamin A-deficient diet
and injected with 0.0005 mg/day bisphenol A in the
neonatal period. The study authors concluded that
vitamin protects infants from the effects of environmental
xenoestrogens.
Strengths/Weaknesses: The subcutaneous route of
administration, lack of clarity on exposure issues, lack
of husbandry and statistical information are weaknesses.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for inclusion and not useful.
Aikawa et al. (2004), supported by the Japanese
Ministry of Education, Science, Sports, and Culture,
examined the effects of neonatal bisphenol A exposure
on sperm endpoints in adult mice. Unless otherwise
specified, dams were fed CE-7 and CA-1 (Clea Japan
Inc.). [No information was provided about caging or
bedding materials.] In the first experiment, SHN mice
were s.c. injected with bisphenol A, bisphenol A plus
retinol acetate, or vehicle for 5 days beginning on the day
of birth. Doses of each compound were 0.5 or 50 mg/day
bisphenol A [purity not reported] (n 5 10–14/group),
50 mg bisphenol A plus 100 IU retinol acetate/day (n 5 5),
and vehicle control (sesame oil for bisphenol A and or
DMSO for retinol acetate; n 5 11). [Assuming a neonatal
mouse weighs 2 g, these bisphenol A doses would be
0.25 and 25 mg/kg bw/day.] In another group, pregnant
mice were fed a low vitamin A diet from 3 days before
gestation to PND 5 and were fed a normal vitamin Acontaining
diet (CE-7 and CA-1) beginning on Day 6
following parturition [number/group not stated]. Pups