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

288 CHAPIN ET AL.
method of oral dosing was not described.] At 8 weeks of
age (Day 77) 5 mice/group/sex were randomly selected
and immunized by i.p. injection with hen egg lysozyme.
Representation of litter was not specified. Blood was
collected and spleens were removed 3 weeks following
immunization (Day 98). Serum levels of hen egg
lysozyme-specific immunoglobulin G (IgG), IgG1, and
IgG2A were measured by ELISA. Spleen cell suspensions
were prepared, and proliferation was assessed by
incorporation of 3 H-thymidine following a 72-hr incubation
with hen egg lysozyme. Spleen cell suspensions
were also prepared for measurement of interferon-g and
interleukin-4 secretion by ELISA. An additional 6 mice/
group/sex were killed at 8 weeks of age (Day 77).
Spleens were removed and expression of CD3 1 CD8 1
and CD3 1 CD4 1 molecules on splenic lymphocytes was
examined using monoclonal antibodies and flow cytometry.
Thymus and spleen were fixed in 4% formaldehyde
and examined histologically. Data were analyzed
by Mann–Whitney U-test. It was not clear if the litter of
origin was accounted for in statistical analyses.
Bisphenol A treatment had no significant effect on
pregnancy rate, sex ratio, or body weight of offspring.
There were several significant immune responses for
male mice. [Results in female mice were said to be
similar to those observed in male mice but the data
were not show by study authors.] At bisphenol A doses
Z0.03 mg/kg bw/day, production of anti-hen egg
lysozyme IgG2a following immunization was increased.
Effects observed at Z0.3 mg/kg bw/day included
increases in production of anti-hen egg lysozyme IgG
and secretion of interferon-g and interleukin-4. Additional
findings at the high-dose (3 mg/kg bw/day) were
increases in spleen cell proliferation and production of
anti-hen egg lysozyme IgG1 following immunization.
Augmentation of interferon-g and interleukin-4 secretion
following incubation of spleen cells with hen egg
lysozyme was examined in the high-dose group only
and found to be increased. [Increases in CD3 1 CD8 1 and
CD3 1 CD4 1 expression on lymphocytes were reported
in males and females exposed to bisphenol A, but the
doses at which the effects occurred were not specified.]
No histopathological alterations were reported for the
spleen or thymus. The study authors explained that
effects on IgG2a and interferon-g were indicators of T
helper 1 immune responses and effects on IgG1 and
interleukin-4 were indicators of T helper 2 responses.
They concluded that the findings suggest that prenatal
exposure to bisphenol A may upregulate immune
responses in mice.
Strengths/Weaknesses: The oral route of administration
and the wide range of doses are strengths.
Weaknesses include small sample size (n 5 5), lack of
clarity regarding statistical handling of factors such as
litter and sex effects.
Utility (Adequacy) of CERHR Evaluation Process:
This study is inadequate for the evaluation process due
to the reasons stated above.
Berger et al. (2007), supported by The Natural Sciences
and Engineering Research Council of Canada, examined
the effect of bisphenol A exposure on ovum implantation
and pup survival in mice. CF-1 mice were housed in
polypropylene cages and were fed Harlan Teklad 22/5
rodent feed, which was stated to contain soy. [No
information was provided about bedding materials.]
On GD 1–4 or 5 [described as GD 1–5 in Methods
section and GD 1–4 in study figures and tables] (GD
0 5 day of vaginal plug), 31 mice in the control group
were s.c. injected with peanut oil vehicle and 5–15 mice/
group were s.c. injected with bisphenol A (97% purity) at
0.0005, 0.0015, 0.0046, 0.0143, 0.0416, 0.125, 0.375, 1.125,
3.375, or 10.125 mg/animal/day. In a second experimental
group, BPA was administered through a diet containing
3% or 6% BPA added to peanut butter and chow. In a
third experimental group maintained on chow, BPA was
administered at 0.11, 1.0, 3.0, or 9.0% in separate
offerings of peanut butter alone. Pregnancy disruptions
in orally exposed mice are discussed in Section 3.2.5.1.
[In the first experimental group, if it is assumed that
the mice weighed 0.02 kg at the start of gestation
(USEPA, 1988), CERHR estimated bisphenol A intakes
of 0.025, 0.075, 0.23, 0.72, 2.1, 6.3, 19, 56, 170, and 500 mg/
kg bw/day.] Mice were allowed to litter. Pups were
counted on the day of parturition and observed for
survival for 5 days. Pups were weaned at 28 days after
birth and at that time, body weight and sex ratio were
determined. Data were analyzed by ANOVA, w 2 test, and
Newman–Keuls multiple comparisons. [It was not clear
if all offspring data were analyzed on a pup or litter
basis.] A study examining implantations in s.c.-treated
females is discussed in Section 4.2.1.1 Percent of females
giving birth was significantly decreased in the
10.125 mg/day group (B28% vs. 97% in control group).
Numbers of pups born were significantly decreased in
the 3.375 and 10.125 mg/day group (B8 and 2 pups in
each of the dose groups and 13 pups in the control
group). There were no treatment-related effects on pup
weight or sex ratio at weaning. [As discussed in Section
3.2.5.1, it appears that with oral exposure, pregnancy
disruption occurred at higher bisphenol A levels
(68.8 mg/day, 3440 mg/kg bw/day) than with s.c. exposure
(10.125 mg/day, B500 mg/kg bw/day)]. The study
authors concluded that the amount of bisphenol A
required for pregnancy disruption was higher than
typical environmental levels but that it is not known if
bisphenol A could have additive or synergistic effects
with other environmental estrogens.
Strengths/Weaknesses: A strength of the subcutaneous
study is that it examined a wide range of
bisphenol A dose levels. The comparison of the differential
effects of s.c. and oral routes of bisphenol A
administration is also a strength. Weaknesses include the
limited/unequal number of mated mice in each dose
group, absence of maternal data to ascertain the potential
impact of maternal toxicity on pregnancy, methodological
deficiencies regarding fertility assessment, and the
use of a diet that contains phytoestrogens.
Utility (Adequacy) for CERHR Evaluation Process:
Due to the limited number of mated mice per dose level
(n 5 5–15), methodological concerns, absence of key
statistical information as well as maternal information,
this study is inadequate for the CERHR evaluation
process.
3.2.5.2 Studies with neurobehavioral endpoints:
Narita et al. (2006), supported by the Japanese Ministry
of Health, Labor, and Welfare, and Ministry of Education,
Culture, Sports, Science, and Technology, conducted a
series of studies to examine the effects of bisphenol A on
the dopaminergic system of mice exposed during
development. Only brief details were provided about
Birth Defects Research (Part B) 83:157–395, 2008