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

isphenol A exposure on the development of Xenopus
laevis embryos. Three different sets of experiments were
conducted. Data were analyzed by ANOVA followed by
Fisher protected least significant difference test. From 3–
96 hr following fertilization, embryos were exposed to
bisphenol A [purity not indicated] at 1, 2.5, 5, 10, 15, 20,
25, or 30 mM (0.3, 0.6, 1.1, 2.3, 3.4, 4.6, 5.7, or 6.8 mg/L).
Each exposure was replicated 3 times. Negative control
groups consisted of the ethanol vehicle, medium alone,
or dilution medium. Rates of normal embryo development
were equivalent in the 3 different negative control
groups. In groups exposed to Z20 mM bisphenol A, there
was a significant decrease in normal embryos and a nonsignificant
increase in mortality rate. Teratogenicity was
characterized by short body length, microcephaly, flexure,
edema, and abnormal gut coiling. Increases in
embryo abnormalities were also observed following
exposure to Z10 mM 17b-estradiol or nonylphenol.
To determine sensitive stages, embryos were exposed
to control media or 20 mM [4.6 mg/L] bisphenol A for 45–
48-hour periods ranging from 3–48 hr post-fertilization,
12–60 hr post-fertilization, 24–72 hr post-fertilization, 36–
84 hr post-fertilization, or 48–96 hr post-fertilization.
Body length, gross malformations, and distance between
eyes were measured at 96 hr following exposure. [The
Methods section indicated that 59–71 embryos were
examined in the bisphenol A group for each time
period of exposure. However, a figure in the study
reported the sample size as 3/time period.] During the
period of 3–48 hr following fertilization, statistically
significant effects in the bisphenol A group included
decreased body length and increased incidences of
microcephaly, flexure, edema, and abnormal gut coiling.
No increases in abnormal effects were observed following
exposure at later time periods. Abnormalities were
observed following exposure to 17b-estradiol or nonylphenol
at early or late stages.
In the third part of the study, embryos were exposed to
20 mM [4.6 mg/L] bisphenol A from 3–96 hr following
fertilization. RNA was isolated from whole embryos and
subjected to analysis by cDNA microarray. Results
obtained in microarray analyses were confirmed by
PCR analysis. The sample size was reported as 2. The
microarray analysis revealed 179 upregulated and 103
downregulated genes following exposure of embryos to
bisphenol A. The study authors identified 27 genes in
which expression was changed following exposure to
bisphenol A, nonylphenol, or 17b-estradiol. The identified
genes included: KNP-Ia, CmaB, XIRG, a-skeletal
tropomyosin, apelin, cyclin G1, Ube213, HGF, toponin C2,
ribosomal protein L9, and Rattus norvegicus similar to
CG10042-PA. The other genes were not identified. The
study authors concluded that these findings might
provide clues to deciphering mechanisms of teratogenic
effects associated with bisphenol A and the other
compounds examined in this study.
Strengths/Weaknesses: The inclusion of 17b-estradiol
as a comparator was a strength and the high bisphenol A
concentration is a weakness.
Utility (Adequacy) for CERHR Evaluation Process:
This study is not useful for the evaluation process.
Pickford et al. (2003), supported by the Bisphenol A
Global Industry group, the Society of the Plastics
Industry, the Bisphenol A Sector Group of the European
Chemical Industry Council, and the Japan Chemical
Birth Defects Research (Part B) 83:157–395, 2008
BISPHENOL A
311
Industry Association, examined the effects of bisphenol
A exposure on development of frog gonads. Beginning at
Stage 43/45 (B2 days post-hatching, 4 days postfertilization,
exposure day 0) and continuing through
Stage 66, Xenopus laevis larvae were exposed to bisphenol
A [purity not indicated] at nominal concentrations of 0
(water control), 1.0, 2.3, 10, 23, 100, or 500 mg/L in a flowthrough
test system [culture ware not discussed]. Actual
concentrations were verified as 0.83, 2.1, 9.5, 23.8, 100,
and 497 mg/L. A positive control group was exposed to
2.7 mg/L 17b-estradiol. There were 4 replicate test
vessels/dose, with each containing 40 larvae (i.e., 160
larvae/test condition). Larvae were observed daily for
mortality, behavior, and appearance. Growth and development
were assessed on all larvae of a replicate tank on
exposure days 32 and 62 (36 and 68 [66?] days postfertilization).
Froglets were killed and observed at
completion of metamorphosis (Stage 66). Total length
was measured, sex was determined, and testes and
ovaries were assessed for abnormalities such as asymmetry,
complete absence, presence of melanocytes,
irregular shape, segmentation or fragmentation, vacuoles,
and ambiguous sexual morphology. Data were
analyzed by Fisher exact test, ANOVA, Wilcoxon rank
sum test, G test, and w 2 test. Following exposure to
bisphenol A, there were no significant differences in
survival, distribution of developmental stages on Day 32
or 62, time to completion of metamorphosis (Stage 66), or
length of Stage 66 froglets. Bisphenol A exposure
did not affect sex ratio or abnormalities in testis or ovary
[data were not shown by authors for testis and
ovary effects]. In contrast, exposure to 17b-estradiol
resulted in an increase in ratio of females to males and
testicular and ovarian abnormalities. The study authors
identified a no-observed-effect concentration of 500 mg/L
for bisphenol A.
Strengths/Weaknesses: The use of a wide range of
exposure levels is a strength, but the incomplete data
presentation with missing organ weight data and the lack
of histological evaluations are weaknesses.
Utility (Adequacy) for CERHR Evaluation Process:
This study may have utility for environmental assessment,
but is not useful for human risk assessment.
Levy et al. (2004), supported by the Ministry of
Environment and Traffic of Baden-Württemberg, evaluated
the effect of bisphenol A on gonad development in
Xenopus laevis tadpoles. Tadpoles (n 5 40/group) were
exposed beginning at Stages 42/43 to ethanol vehicle or
to bisphenol A (499% purity) or 17b-estradiol, both at
concentrations of 10 -8 or 10 -7 M [bisphenol A concentrations
2.3 and 23 lg/L. Actual concentrations were
90–105% of target concentrations after addition of
bisphenol A to the media but decreased to low levels
by the end of the 48-hr period between media changes.
Culture ware was not discussed.] After completion of
metamorphosis, froglets were killed for examination of
gonads. Tadpoles not completing metamorphosis were
killed after 120 days of chemical exposure for examination
of gonads. In a second experiment, bisphenol A
concentrations were 10 -8 ,10
-7 ,or10
-6 M [2.3, 23, or
228 lg/L] and the 17b-estradiol positive control used a
concentration of 10 -7 M. In a third experiment, 50
tadpoles/group were treated for 2 weeks with ethanol
vehicle, bisphenol A 10 -7 M [23 lg/L], or10
-7 M17bestradiol
after which whole-body homogenates were