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

256 CHAPIN ET AL.
comparisons conducted by the Duncan multiple range
test. [In the part of the study in which dams were
dosed, it appears that offspring were considered the
statistical unit.]
In the first study, rats were gavaged with bisphenol A
[purity not given] at 0, 0.0024, 0.010, 100, or 200 mg/kg
bw/day from PND 21–35. The two lowest doses were
selected to represent environmental exposures, and the
higher doses were selected to compare the effects
between low and high-doses. Rats were killed at the
end of treatment and blood was collected for measurement
of serum LH, testosterone, and 17b-estradiol levels.
Leydig cell cultures were prepared for measurement of
ex vivo testosterone production with and without the
addition of LH, testosterone precursors, or metabolizing
enzymes. Additional weanling rats were exposed to
bisphenol A at 0 or 0.0024 mg/kg bw/day on PND 21–35.
At the end of treatment, mRNA for LHb, ERb, and ERa
was measured in pituitary using an RT-PCR technique.
All endpoints were reported for 7–12 rats/group.
Compared to rats in the control group, rats exposed to
bisphenol A at 0.0024 mg/kg bw/day had significantly
lower levels of serum LH [by 62%] and testosterone [by
40%]. Serum 17b-estradiol levels were decreased in rats
exposed to 0.0024, 0.010, and 100 mg/kg bw/day bisphenol
A [by B30, 40, and 25% in each respective dose
group]. There were no effects on basal ex vivo
testosterone production by Leydig cells. In Leydig cells
obtained from rats exposed to 0.0024 mg/kg bw/day
bisphenol A, testosterone production was significantly
reduced when cells were exposed to LH or CYP450 17ahydroxylase/17–20
lyase. In Leydig cells obtained from
rats exposed to 0.0024 or 0.010 mg/kg bw/day bisphenol
A, testosterone production was significantly reduced
following exposure of the cells to pregnenolone or
progesterone. No effects on blood hormone levels or ex
vivo testosterone production were observed at higher
doses. Significant effects observed in pituitaries obtained
from rats exposed to 0.0024 mg/kg bw/day bisphenol A
were decreased LHb mRNA and increased ERb mRNA.
The study authors concluded that the decreased serum
LH level resulted from bisphenol A effects on the
pituitary and that decreased LH stimulation of Leydig
cells was the cause of reduced serum testosterone levels.
In the second experiment, 7 dams/group were
gavaged with bisphenol A at 0 or 0.0024 mg/kg bw/
day from GD 12–PND 21. Male offspring received no
further treatment following weaning. Males were randomly
selected from each dam and killed on PND 90.
Endpoints evaluated in 10–12 male offspring/group
included serum LH and testosterone levels, ex vivo
testosterone production by Leydig cells, testosterone
levels in testicular interstitial fluid, and seminal vesicle
and prostate weight. Significant (Po0.01 or 0.05) effects
observed in 90-day-old males that had been perinatally
exposed to bisphenol A compared to the control group
included increased body weight [10%], decreased relative
weight (to body weight) of paired testes [17%] and
seminal vesicles [17%], reduced testicular testosterone
level [B39%], and reduced basal and LH-induced ex
vivo testosterone production.
In the third experiment, 10–12 rats/group were
gavaged with bisphenol A at 0 or 0.0024 mg/kg bw/
day from PND 21–90. Within 24 hr following treatment,
rats were killed and examined for the same endpoints
described for the second experiment. Significant (Po0.01
or 0.05) effects compared to the control group included
increased serum LH level [117%], decreased seminal
vesicles weight [absolute: 15%, relative: 16%], reduced
testicular testosterone level [B24%], and decreased basal
and LH-induced ex vivo testosterone production. For the
second and third experiments, the study authors concluded
that bisphenol A exposure inhibits androgen
production by Leydig cells.
Strengths/Weaknesses: Significant strengths of this
report were the sequential nature of the work, in which
later studies built on the previous data, and the clear
expertise that the authors brought to this endeavor.
Experiment 1 provided a helpful examination of postnatal
effects following adolescent exposure and examined
hormonal levels under stimulated and unstimulated
conditions, thus separating pituitary from target organ
contributions to serum levels. In Experiment 2, the
sample size of 7 dams/prenatal treatment group and
the examination of 10–12 offspring/group raise questions
about the adequacy of the sample size with respect
to the number of litters represented and the number of
offspring used to represent each litter. In Experiment 3,
10–12 rats/group were treated from PND 21–90 (through
adolescence and into early adulthood) and then examined
according to endpoints common to Experiments 1
and 2. Weaknesses include an inadequate number of
animals to obtain confidence about the hormonal
changes (indeed, LH was decreased in the first experiment
and increased in the third), the lack of histopathology
evaluation, and lack of an estrogenic positive control.
Utility (Adequacy) for CERHR Evaluation Process:
Experiments 1 and 3 are adequate but of limited utility
because of the mechanistic nature of the endpoints
examined. Experiment 2 is inadequate for consideration
due to inappropriate statistics that failed to account for
litter effects.
Masutomi et al. (2004), supported by the Japanese
Ministry of Health, Labour, and Welfare, examined the
potential effects in rats of neonatal bisphenol A exposure
through maternal dietary intake on the number of
offspring pituitary cells positive for LH, FSH, and
prolactin. The authors exposed 5–8 pregnant CD(SG)IGS
dams from GD 15–PND 10 to soy-free diet containing: (1)
genistein 20, 200, or 1000 ppm; (2) diisononyl phthalate
400, 4000, or 20,000 ppm; (3) methoxyclor 24, 240, or
1200 ppm; (4) 4-nonylphenol 60, 600, or 3000 ppm; or (5)
bisphenol A [96.5% purity] 60, 600, or 3000 ppm. Ethinyl
estradiol at 0.5 ppm was also administered to a positive
control group and the regular soy-free diet to a control
group. [Only the bisphenol A-treated group will be
considered here. Feed consumption and dam body
weight were not reported, but would be expected to
have changed dramatically over the treatment period,
making it difficult to estimate the bisphenol A doses
received by the rats.] After weaning, offspring were
placed on CRF-1 rodent chow. Animals were housed in
polycarbonate cages with wood-chip bedding.
During postnatal week 3 or 11, offspring were killed
and anterior pituitary glands from 5 male and 5 female
offspring/group were harvested. Immunohistochemistry
using paraffin-embedded sections for LH, FSH, and
prolactin was conducted and the percentage of cells
positive for LH, FSH, and prolactin was determined in 2
sections/gland. Statistical analyses were performed by
Birth Defects Research (Part B) 83:157–395, 2008