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

292 CHAPIN ET AL.
has been informed that there were 20–30/group (A.
Soto, personal communication, March 2, 2007).] Fetal
mammary glands were mounted whole or sectioned to
examine mammary gland development in 36–40 offspring/group.
Immunohistochemistry techniques were
used to measure expression of Ki67 and Bax in mammary
structures from 4–8 offspring/group. Mammary collagen
localization was assessed using Masson Trichrome stain
in 6–17 mice/group. Expression of mRNA for ERa, ERb,
adipocyte lipid binding protein, Col-l, and PPARg were
measured by RT-PCR in mammary glands from 4–6
offspring/group. Litter was accounted for in design and
analyses by assigning 1 individual/litter to each group or
endpoint. Statistical analyses included t-tests, ANOVA,
Mann–Whitney U non-parametric tests, and w 2 tests.
Morphometric analysis revealed significantly higher
ductal area and extension in the bisphenol A group than
in controls. In the control group, females positioned next to
two females in utero had significantly fewer branching
points than females positioned next to 1 or 2 males; this
difference was not observed in the bisphenol A group. In
fetuses that were not positioned next to a male, significantly
more branching points were observed in the
bisphenol A than in the control group. Control females
positioned next to 2 males had significantly larger
epithelial duct area than control females not positioned
next to a male; this difference was not observed in the
bisphenol A group. In bisphenol A-treated females positioned
next to 1 male, ductal extension was significantly
greater than in control females positioned next to 1 male.
In the bisphenol A group, epithelial cells were less
rounded, more evenly spaced, and more dense than
in controls. Bisphenol A did not significantly affect Ki67
(a proliferation marker) expression in mammary epithelium.
Lumen formation was observed in 6 of 16 control
mice and 0 of 10 bisphenol A-exposed mice. Significantly
decreased numbers of Bax-positive (apoptotic) cells were
observed in the inner epithelial cord (not in contact with
basement membrane) of bisphenol A-exposed than
control mice. Optical density of histological staining
was significantly lower in the fat pad of the bisphenol Aexposed
than control group. Fat pads of the bisphenol A
group compared to control group were found to be
significantly less cellular, contain more Bax-positive cells,
and have more vacuoles at a distance o1 mm from the
epithelial compartment. Study authors interpreted the
effect as increased epithelial penetration and advanced
maturation of fat pads. No significant differences were
observed for PPARg or adipocyte lipid binding protein
mRNA expression. Density of collagen deposits was
lower in the entire mammary gland but higher in the
periductal stroma (within 10 mM of the epithelium) of the
bisphenol A than the control group. Bisphenol A
exposure did not affect collagen type I, ERa, or ERb
mRNA expression. ERa protein expression in the stroma
was also unaffected by bisphenol A exposure. Study
authors concluded that advanced maturation of fat pad
and changes in extracellular matrix may be the cause of
altered growth, cell size, and lumen formation in
mammary epithelium of mouse fetuses exposed to
bisphenol A.
Strengths/Weaknesses: Strengths of this study are the
rigor with which the measurements were made, and the
fact that the authors were trying to quantify endpoints
that are difficult to measure (e.g., the relationship of the
stroma to the epithelium). The relevance of the endpoints
is a strength as is the low dose used. The single dose and
subcutaneous route of administration are weaknesses. A
critical weakness is inappropriate statistical analysis of a
complex study design that may have produced too many
positive findings and a lack of statistical accounting for
litter effects (i.e.,. 36–40 pups presented in Table 1 of
study and only 20–30 litters treated).
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for the evaluation process
because of insufficient control for litter effects.
Honma et al. (2002), supported by the Japanese
Ministry of Education, Culture, Sports, Sciences, and
Table 79
Effects in Mice Exposed to Bisphenol A During Prenatal Development a
Dose (mg/kg bw/day)
Endpoint 0.002 0.020 BMD10 BMDL10 BMD1SD BMDL1SD
Female body weight
Weaning k 10% k 7% 0.065 0.017 0.088 0.021
PND 60
Male body weight
2 k 4% 0.054 0.021 0.11 0.021
Birth 2 k 5% 0.054 0.020 0.031 0.015
PND 60
Anogenital distance
2 k 6% 0.048 0.020 0.044 0.020
Females at weaning m 6% 2
Males on PND 60 m 6% m 8% 0.035 0.020 0.035 0.020
Age at vaginal opening b
2 k 1.3 days
Body weight at vaginal opening b
k 10% k 11%
Age at 1st estrus b
2 k 1 day
Estrous cycle length m 1.3 day m 1 day 0.021 0.007 0.12 0.021
Cornified cells in vaginal smear m 3.1 days 2 0.17 0.020 0.44 0.021
Lymphocytes in vaginal smear k 2.2 days 2 0.26 0.020 0.26 0.020
a Honma et al. (2002).
b Value estimated from a graph by CERHR; data from graphs were not modeled.
m,k Statistically significant increase, decrease; 2 no significant effect.
Birth Defects Research (Part B) 83:157–395, 2008