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

with 28 dams each were given the tocopherol-stripped
corn oil vehicle. Because results from the two vehicle
control groups were statistically equivalent, data from
the two groups were pooled. A positive control group of
28 mice was given 0.2 mg/kg bw/day diethylstilbestrol.
Dosing solutions were dripped into the animals’ mouths
using a micropipette. Concentrations of dosing solutions
were verified before dosing. Animals were fed certified
rodent chow 5002. Water was provided in glass bottles
with Teflon seals. Cages were made of polypropylene
with steel lids. Corn cob bedding was used. Music was
played at low volume to provide background noise.
Dams were monitored for clinical signs, food intake,
body weight gain, and fertility endpoints. Pups were
counted and sexed at birth (PND 0) and monitored for
survival and weight gain until weaning on PND 22.
Litters were culled to 8 pups on PND 4, leaving as many
males as possible. At weaning, no more than 4 males/
litter (65–95 males/group) were randomly selected to
continue in the study and housed individually. The
males were monitored for body weight gain and feed
intake until they were killed on PND 90. Brain, liver,
kidneys, and reproductive organs were weighed. Daily
sperm production and epididymal sperm counts were
determined and a histopathological examination of testes
was conducted. The litter was considered the experimental
unit in statistical analyses. Data were analyzed by
Levene test, ANOVA, Dunnett test, rank transformation,
Wilcoxon rank sum test with Bonferroni correction,
Fisher exact probability test, and binomial distribution
test.
There were no clinical signs or significant differences
in body weight gain or feed intake in dams. The numbers
of dams that died of unknown causes during the study
were: 2 receiving vehicle controls; 1 dosed with diethylstilbestrol;
3 dosed with 0.0002 mg/kg bw/day bisphenol
A; and 1 each in the 0.002 and 0.020 mg/kg bw/day
bisphenol A groups. The number of total pups/litter was
significantly lower than controls in the 0.2 mg/kg bw/
day bisphenol group (mean7SD 5 9.6073.85 compared
to 12.3773.02 in the control group). In communications
with the animal vendor, it was determined that litter size
in the control group exceeded typical litter sizes (9–10
pups), and the study authors therefore concluded that
the effect was not treatment-related. Bisphenol A had no
significant effects on gestation index or duration,
percentage of male pups at birth, or pup survival and
body weight during the lactation period. The same
endpoints were unaffected in the diethylstilbestrol
group.
Terminal body weights were increased [by 7%] in the
0.020 mg/kg bw/day group and [by 5%] in the 2 mg/kg
bw/day group. Bisphenol A did not affect absolute or
relative (to body or brain) weights of reproductive organs
including prostate, preputial gland, seminal vesicle, or
epididymis. Non-dose-related effects were observed for
brain and kidney weights, and the study authors
concluded that the effects were not treatmentrelated.
There were no significant effects on cauda
epididymal sperm concentration, daily sperm production,
or efficiency of sperm production. Testicular
histopathology was not affected by bisphenol A treatment.
[Data were not shown by authors.] Reproductive
development of male offspring was also unaffected by
diethylstilbestrol. The study authors noted that the
Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A
281
diethylstilbestrol dose was considered the ‘‘maximum
effect’’ oral dose by vom Saal but was lower than doses
affecting male offspring in other studies. The study
authors also noted that the effects of bisphenol A on
prostate weight and sperm production reported by vom
Saal et al. (1998) and Nagel et al. (1997) were not repeated
in this study. They concluded that bisphenol A should
not be considered a selective reproductive or developmental
toxicant.
[The NTP Statistics Subpanel (NTP, 2001) concluded
that the statistical methods used by Cagen et al. (1999a)
were appropriate. Although the Subpanel agreed with
the study author conclusions, they noted that (1) a
significant ANOVA is not a requirement for Dunnett
test; and (2) a Bonferroni correction of the Wilcoxonrank
sum test was not needed because the study
authors already required significance by ANOVA,
which was sufficient.]
Strengths/Weaknesses: The attempt to replicate the
studies of vom Saal et al. (1998) and Nagel et al. (1997),
the use of litter analysis, the large sample sizes, and the
agreement of the NTP Subpanel with the author
conclusions are strengths. With respect to this study as
a replication, weaknesses include design differences
relating to strain, dietary differences, age at evaluation,
and the use of solo housing rather than small group
housing. The lack of response of the positive control DES
group is problematic.
Utility (Adequacy) for CERHR Evaluation Process:
This study is inadequate for the evaluation process due
to absence of response of the positive control group.
Ashby et al. (1999), support not indicated [2 authors
from industry], examined the effects of prenatal bisphenol
A exposure on the mouse reproductive system.
The study attempted to duplicate the findings reported
by vom Saal et al. (1998) and Nagel et al. (1997). Both
generations of CF-1 mice were fed RM1 diet containing
6.5% soy during periods when they were not pregnant or
lactating, and dams were fed RM3 diet containing 18.5%
soy during pregnancy and lactation. On post-conception
days 11–17, 8 dams/group were dosed with bisphenol A
(99% pure) at 0, 0.002, or 0.020 mg/kg bw/day. The
negative control group was administered the tocopherol
stripped corn oil vehicle. A positive control group of 7
dams received diethylstilbestrol at 0.2 mg/kg bw/day. A
naïve group of 7 dams was not weighed or dosed. The
dosing solution was slowly expelled from a pipette
placed in the animals’ mouths. Day of vaginal plug
detection was designated post-conception day 1, however.
females that had no vaginal plugs but gained
43.5 g were arbitrarily considered to be 10 days
pregnant. Females with vaginal plugs and those that
gained 43.5 g were distributed evenly among treatment
and control groups. Females that gained 41 but o3.5 g
were considered to be pregnant, but because the day of
pregnancy could not be determined, they were assigned
to the naïve control group. Dams were allowed to litter.
All female offspring were weighed and monitored for
vaginal opening. Females were killed at B44 weeks of
age, and liver, kidney, and reproductive organs were
weighed. Male pups were housed as littermates until
PND 112 (day of birth designated as PND 1). To
determine the effects of housing, B3 males from 4–7
litters/group (11–21 males/group) were randomly selected
and housed separately from PND 112 until study