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

Technology, examined the effect of prenatal bisphenol A
exposure on the reproductive system of female mice.
Mice were fed commercial diet (CE-2, CLEA, Tokyo,
Japan). [No information was provided about bedding or
caging materials.] Ten ICR/Jcl mice/group were s.c.
injected with bisphenol A [purity not reported] in
sesame oil at 0, 0.002, or 0.020 mg/kg bw/day on GD
11–17 (GD 0 5 vaginal plug). Additional mice were
injected with diethylstilbestrol at 0.02–2 mg/kg bw/day.
Pups were sexed, counted, and weighed at birth. At 22
days of age, offspring were weaned and litter sizes were
adjusted to 8 pups. Male and female offspring were
weighed during the postnatal period. Anogenital distance
was measured in males and females at 22 and 60
days of age. Females were monitored for vaginal opening.
Vaginal smears were obtained for 30 days following
vaginal opening. Female offspring were mated with
untreated males from 90 to 120 days of age. F 2 pups were
counted and sexed at birth. The litter was considered the
experimental until in statistical analyses. Data were
analyzed by ANOVA and Student or Welch t-test.
Statistically significant findings are summarized in
Table 79. There were no effects on gestation duration,
number of pups/litter, or sex ratio. Body weights were
slightly lower in high-dose males at birth, both dose
groups of females at weaning, and high-dose males and
females at 60 days of age. Anogenital distance was
increased in low-dose females at weaning and both dose
groups of males at 60 days of age. Age of vaginal opening
and first estrus was accelerated in the high-dose group,
and body weight at vaginal opening was lower in both
dose groups. Estrous cycle length was increased in both
dose groups. Total days that cornified cells were present
in vaginal smears was increased and total days that
lymphocytes were detected was decreased in the lowdose
group. In F1 offspring there were no significant
effects on mating, number of F2 pups/litter, or sex ratio
of F2 pups. Results in mice dosed with diethylstilbestrol
were similar to those observed in mice dosed with
bisphenol A. The study authors concluded that prenatal
exposure to low doses of bisphenol A results in early
vaginal opening in mice but did not affect female
reproductive function.
Strengths/Weaknesses: Strengths are that this study
represents one of the few studies that appropriately
examines the onset of puberty in the mouse as an
endpoint, it uses low dose levels of bisphenol A,
relatively large sample sizes, and effectively uses a
positive control at 3 dose levels. The lack of AGD
measurement at birth and difficulty of measurement at
PND 60 are weaknesses. he Expert Panel was unable to
confirm the statistical significance of the effects shown in
Table 2 of the manuscript.
Utility (Adequacy) for CERHR Evaluation Process:
The study is adequate for inclusion but of limited utility
due to statistical questions about body weight and AGD
and subcutaneous route of exposure.
Iwasaki and Totsukawa (2003), support not indicated,
examined the effect of prenatal bisphenol A exposure on
reproductive development of female mice. ICR mice
were fed F1 diet (Funabashi, Chiba, Japan) and housed in
polycarbonate cages containing an unspecified chip
bedding. On GD 7–18 (GD 0 5 day of copulatory plug),
6 dams/group received bisphenol A [purity not reported]
at 0 (DMSO vehicle) 0.00025, 0.025, or 2.5 mg/kg
Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A
293
bw/day by s.c. injection. A positive control group of
mice received 100 mg/kg bw/day 17b-estradiol [route
not specified]. Dams were weighed during the study.
Pups were counted and sexed on PND 0, and pup
viability was determined on PND 4. Pups were weaned
on PND 21, and male pups were killed and discarded.
Female pups (24–41/group) were observed for vaginal
opening. On PND 21, 1 pup/litter(4/group) from the
low- and mid-dose group was injected with 3 mg/kg bw/
day 17b-estradiol for 2 days and then killed. Uterine
weights were assessed and expression of the ERa gene in
uterus was determined using a colorimetric method.
Statistical analyses included ANOVA, ANOVA on ranks
(Kruskall–Wallis test), and Dunnett test. [It was not clear
if the litter or offspring was considered the statistical
unit.]
Weight gain was described as increased in all treated
dams compared to control dams, but there was no
evidence of a dose–response relationship and statistical
significance was not achieved. Pup birth weight was
significantly lower [6%] in the low-dose group compared
to the control group. There were no differences in litter
size at birth. Pup viability on PND 4 was significantly
reduced [by 26%] in the low-dose group. Age of vaginal
opening was significantly delayed by 3 days in the lowdose
group, but significantly accelerated by 2.2 days in
the high-dose group. Following 17b-estradiol exposure,
uterine weight was significantly decreased [by B85%] in
the low-dose bisphenol A group and significantly
increased [by B29%] in the mid-dose bisphenol A
group. Although expression of ERa mRNA was observed
at 132% of control levels in the mid-dose bisphenol A
group following exposure to 17b-estradiol, the effect did
not attain statistical significance. Expression of ERa gene
was not detectable in the low-dose bisphenol A group
following 17b-estradiol exposure. No significant effects
were reported in mice treated with 17b-estradiol. The
study authors concluded that ‘‘The levels tested in this
study appear to be dangerous.’’
Strengths/Weaknesses: The use of 3 dose levels,
including low doses, and the use of 17b-estradiol as a
positive control are strengths of this study. Weaknesses
include the use of DMSO as a vehicle, the subcutaneous
route of administration, the small sample size, lack of
significant effects detected in the 17b-estradiol positive
control group, and the failure to account for litter in
statistical analyses.
Utility (Adequacy) for CERHR Evaluation Process:
The study is inadequate for the evaluation process.
Nakamura et al. (2006), supported by grants from the
Japanese government, examined the effects of prenatal
exposure to bisphenol A on the morphology and
expression of certain genes related to brain development
in the mouse neocortex. In the first experiment ICR/Jc1
mouse dams were injected subcutaneous with either 0
(sesame oil vehicle) or 20 m/kg bw/day bisphenol A
[purity not indicated] daily from GD 0 (defined as the
day that a vaginal plug was detected) until GD 10.5, GD
12.5, GD 14.5 or GD 16.5. [No information was provided
on feed, caging materials, bedding.] Dams were then
given a single i.p. injection of 5-bromo-2 0 -deoxyuridine
(BrdU). Fetuses were collected either 1 hr following BrdU
treatment (to assess precursor cell proliferation) or 2 or 3
days following BrdU treatment (to assess neuronal
migration and differentiation). Brains were fixed in 4%