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

278 CHAPIN ET AL.
include small sample size (5 treated dams) and lack of
adequate experimental and statistical control for litter
effects.
Utility (Adequacy) for CERHR Evaluation Process:
Despite certain strengths, this study is inadequate for the
evaluation process for the reasons cited above.
Shikimi et al. (2004), supported by the Japan Society
for the Promotion of Science for Young Scientists,
examined the effects of bisphenol A exposure on
Purkinje cell development in rats. [No information was
provided about feed or composition of caging and
bedding materials.] At 6–9 days of age, 4 male or female
Fisher rats/group received bisphenol A [purity not
provided] at 0 (sesame oil vehicle), 0.050, or 0.500 mg/
day by injection into the cerebrospinal fluid near the
region of the cerebellum. During the same time period,
additional groups of 4 rats received 0.500 mg/day
tamoxifen, 0.500 mg/day bisphenol A10.500 mg/day
tamoxifen, or 5 mg/day estradiol benzoate through the
same exposure route. [Both male and female rats were
treated, but it was not indicated if there were equal
numbers in each group; both sexes were apparently
evaluated together.] At 10 days of age, pups were killed
and vermal cerebella were removed and sectioned.
Purkinje cells were examined morphologically following
identification by calbindin-D28 K immunostaining. Data
were analyzed by ANOVA, followed by Duncan multiple
range test.
Treatment with the high-dose of bisphenol A increased
Purkinje fiber length. There was no effect on crosssectional
soma area or Purkinje cell number as a result of
bisphenol A treatment. Co-treatment with tamoxifen
inhibited the increase in dendritic length that was
observed following treatment with bisphenol A alone.
Estradiol benzoate also induced an increase in dendritic
length of Purkinje fibers that was blocked by tamoxifen.
Treatment with tamoxifen alone also reduced dendritic
fiber length. The effects of octylphenol were also
examined and an increase in dendrite length was
observed. The study authors concluded that bisphenol
A induced Purkinje dendritic growth, possibly through
the ER.
Strengths/Weaknesses: The use of estradiol benzoate
as a positive control is a strength of this study.
Weaknesses are the injection into cerebrospinal fluid.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for the evaluation process due
to uncertainties surrounding the route of administration
(i.e., difficulty of relating a cerebrospinal injection to
human exposures).
Zsarnovszky et al. (2005), supported by NIH, NIEHS,
and the American Heart Association, evaluated the
effect of intracerebellar injection of bisphenol A on the
development of activated extracellular signal-regulated
kinase (ERK)-positive cells in cerebellar sections in
Sprague–Dawley rats. Neonatal rats on PND 4–19
underwent a single direct injection under anesthesia of
bisphenol A or 17b-estradiol under stereotactic
guidance into cerebellar folia 6 and 7. [For bisphenol
A, only PND 10 results were given. The number of
animals at each age was not specified, but a figure
legend indicated at least 6/dose group. The purity of the
chemicals was not specified. The day of birth
was not defined.] Concentrations of the chemicals were
10 -12 –10 -6 M [bisphenol A concentrations of
0.23 ng/L to 0.23 mg/L]. Uninjected, mock-injected, and
vehicle-injected controls were used. Brains were removed
and fixed 6 min after the onset of the injection.
Sections were processed for immunohistochemistry
using an antibody that recognized activated ERK.
Quantitative analysis was performed on images of
folium 9. Statistical analysis was performed using
ANOVA with post-hoc Tukey–Kramer multiple
comparison test. Response to different chemicals and
different concentrations on PND 10 were compared
using 2-factor ANOVA with post-hoc Bonferroni test.
Adult rats were also treated but were not included in the
quantitative analysis.
The qualitative appearance of the immunostained
sections was similar after bisphenol A and 17b-estradiol.
In the 10 -12 –10 -9 M dose range, the quantitative
responses to the two chemicals were similar. Activated
ERK-positive cells increased with a median effect
concentration of 7.46 pM for 17b-estradiol and 3.25 pM
[0.74 ng/L] for bisphenol A. Both chemicals were described
as having an inhibitory effect at higher doses.
[The data graph shows drop-offs to control densities at
10 -9 and 10 -10 M, with a second increase in density at
10 -7 and 10 -5 M.] Co-administration of 10 -10 M17bestradiol
with bisphenol A 10 -12 –10 -10 M [0.23–23 ng/L]
resulted in a concentration-dependent decrease in activated
ERK-positive cells compared to the administration
of 17b-estradiol alone. The authors concluded that 17bestradiol
regulates ERK signaling in the developing
cerebellum and that bisphenol A can mimic and also
inhibit this estrogenic effect, with potentially adverse
affects on brain development and function.
Strengths/Weaknesses: The use of 17b-estradiol as a
positive control is a strength of this study. Weaknesses
are the intracerebellar injection and the administration on
a per pup basis.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for the evaluation process due
uncertainties surrounding the route of administration
(i.e., difficulty of relating a cerebrospinal injection to
human exposures).
3.2.5 Mouse—oral exposure only during
pregnancy.
3.2.5.1 Studies without neurobehavioral endpoints:
Morrissey et al. (1987), supported by NTP/
NCTR, examined the effects of prenatal bisphenol A
exposure in rats and mice in studies conducted according
to GLP. The studies are also available as NTP publications
for rats (NTP, 1985c) and mice (NTP, 1985b). The
study was conducted in two sets of rats and mice and
data were pooled for each species. [The data for rats
were discussed in Section 3.2.1.] Animals were fed
Purina 5002 diet, housed in polypropylene or polycarbonate
cages with stainless steel wire lids with Ab-Sorb-Dri
cage bedding. Pregnant CD-1 mice were randomly
assigned to groups of Z10 animals in each set of the
study, for a total of Z20 animals/dose. On GD 6–15 (GD
0 5 sperm or plug), mice were gavaged with bisphenol A
at 0 (food-grade corn oil), 500, 750, 1000, or 1250 mg/kg
bw/day. Doses were based on results of preliminary
studies and were expected to result in 10% maternal
mortality at the high-dose and no toxicity at the low
dose. The purity of bisphenol A was 495%, and 2,4 0 ­
bisphenol A was reported as an impurity. Concentrations
of dosing solutions were verified. Pregnant animals were
Birth Defects Research (Part B) 83:157–395, 2008