Bisphenol A
Hazard assessment of Bisphenol A
[Bisphenol A, CAS No. 80-05-7]
Chemical name
: Bisphenol A
Synonyms
: 2,2-bis (p- hydroxyphenyl) propane, 4,4'-(1-methylethylidine)
diphenol, 4,4'-isopropylidenediphenol, BPA
Molecular formula
: C15 H16O2
Molecular weight
: 228.29
Structural formula
:
CH3
C
HO
OH
CH3
Appearance
: White flakes 1)
Melting point
: 150 - 155°C 1)
Boiling point
: 220°C (533 Pa) 1)
Specific gravity
1)
: d 25
25 = 1.195
Vapor pressure
: 5.3 × 10-6 Pa (25°C) 1)
Partition coefficient
: Log Pow = 3.32 (observed value) 1)
Degradability
: Hydrolyzability: No Report
Biodegradability: Slightly degradable (BOD=0%, 14 days)
Solubility
2)
: Water, 120 mg/l (25°C) 1) , Soluble in aqua alkaline solution
Organic solvent: Soluble in acetone, ethanol, ether and
benzene. Slightly soluble in carbon tetrachloride.
Amout of production/
import
: 150,697t in 1998 (manufactured: 149,984t, imported: 713t)5)
Usage
: Raw material for epoxy resin and polycarbonate resin. Raw
material for phenol resin and antioxidant, etc.1).
Applied laws &
regulations
: Law Concerning Reporting, etc. of Release Special Chemical
Substances to the Environment and Promotion of the
Improvement of Their Management
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Bisphenol A
1)
HSDB, 2001;
2)
Tsusanshou Koho (Daily), 1977;
3)
Ministry of International Trade and Industry,
1999.
1.
Toxicity Data
1) Information on adverse effects on human health
Mild skin irritation is reported by contact with BPA dust (Handbook of Industrial
Poisoning).
There is a report that positive patch test reactions to BPA and resins
containing 0.014 or 0.015% of BPA were demonstrated, but not to major component of the
resins, when allergic reaction tests were conducted for a female dental technician who had
used for 4 years dental composite resin, major component of which was epoxide of BPA
containing trace amount of BPA, and had dermatitis at her hands. In this case the patient
showed positive reaction also to formaldehyde contained in the resin as an impurity.
Thus, synergy between BPA and formaldehyde was suspected. Furthermore, the
composition of the resin actually in use was not know and it was not clear which
substance caused the dermatitis as well as whether synergy between BPA and
formaldehyde existed (Jolanki et al., 1995).
There is no report on the carcinogenicity of this substance in humans.
2) Information on endocrine system and reproductive system
(1) In vitro test result related to receptor binding (Attachment-1)
In a receptor binding test, BPA binds to estrogen receptor (ER) in human and rat ( its
affinity is 1/500 - 1/15,000 of that to estradiol (E2)) (Sheeler et al., 2000; Blair et al.,
2000; Nagel et al., 1997; CERI, 2001). The genetic activation dependent on ERE
(estrogen responsive sequence) was also noted in a reporter gene assay using yeast
introduced with human ER (including two-hybrid assay) and animal cells in which human
or rat estrogen receptors were introduced, and the extent of activation was 1/600 1/130,000 of that of E2 (Sheeler et al., 2000; Nishihara et al., 2000; Coldham et al., 1997;
Gaido et al.,1997; Hiroi et al., 1999; Legler et al., 1999; CERI, 2001; Yamasaki et al.,
2001). The EC 50 of BPA was 3.1 × 10-6 M in a human ER dimer formation test using
yeast two hybrid assay, indicating that concentration of 26,000 times higher than that of
E2 (1.2 × 10-10 M) was needed for dimer formation of estrogen receptor (Sheeler et al.,
2000). In an experiment to investigate influence on endogenous estrogen responsive
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Bisphenol A
gene, induction of pS2, etc. was noted but at the concentration of 100,000 - 1,000,000
times higher than that of E2 (Jorgensen et al., 2000).
BPA (1 nM) activated the gene in a
reporter gene assay using promoter domain of prolactin gene (Steinmetz et al., 1997).
(2) in vivo test results in mammals (Attachment-2, 3)
The results of short term test to detect estrogenic action in mammals and the results
of reproductive and fertility toxicity test are shown in Attachment-2 and 3 respectively.
Uterotrophic assay (according to the OECD guideline draft) has been conducted
using rats and mice (Attachment-2).
The uterotrophic assay by subcutaneous administration of BPA at 0, 0.02, 0.2, 0.8, 2
and 8 mg/kg/day for 4 days in ovariectomized female B6C3F1 mice (35 - 60 days old)
revealed increased uterus weight at 0.8 mg/kg/day or more (Papaconstantinous et al.,
2000). On the other hand, no changes in uterus weight occurred in uterotrophic assay by
subcutaneous administration of BPA at 0, 0.01, 0.1, 1, 10 and 100 mg/kg/day for 3 days in
juvenile female CD-1 mice (21 days old) (Mehmood et al., 2000).
The uterotrophic assay of BPA at 0, 40, 160 and 800 mg/kg/day by gavage as well as
by subcutaneous administration at 0, 8, 40 and 160 mg/kg/day both for 3 days in juvenile
female SD (18 days old) or DA/Han (130g body weight) rat revealed increase in uterus
weight by oral administration at 160 mg/kg/day or more and by subcutaneous
administration at 8 mg/kg/day or more (Yamasaki et al., 2000; Diel et al., 2000).
Furthermore, when subcutaneous administration of BPA at 0, 2, 20 and 200 mg/kg/day
was conducted for 3 days in juvenile female SD rats (20 days old), increase in uterus
weight occurred at 20 mg/kg/day or more (Yamasaki et al., 2001). In another
uterotrophic assay in which a capsule corresponding to 0.3 mg/kg/day was subcutaneous ly
implanted in ovariectomized female SD rats (7 - 8 week old) and F344 rats (7 - 8 week
old), increase in uterus weight and increase in height of uterine epithelial cells occurred in
F344 rats, but nothing abnormal was noted in SD rats (Steinmetz et al., 1998). By oral
administration by gavage of BPA at 0, 100, 200 and 400 mg/kg/day for 3 days in juvenile
female Long Evans rats (21 days old), increase in uterine weight was noted at 200
mg/kg/day or more when the animals had been examined after 6 hours, but nothing
abnormal was detected after 24 hours from the final administration (Laws et al., 2000).
When oral administration by gavage of BPA at 0, 500, 750, 1,000 and 1,250
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Bisphenol A
mg/kg/day was conducted in female CD-1 mice (age unknown) from day 6 to 15 of
gestation, relative liver weight of dams increased at 500 mg/kg/day or more, and
suppression of body weight increase, decreased pregnant uterus weight and increased
embryo resorption in the dams as well as decreased fetal body weight were noted at 1,250
mg/kg. However, no deformation was observed (Morrissey et al., 1987).
When BPA at 0, 2,500, 5,000 and 10,000 ppm (corresponding to 0, 437, 875 and
1,750 mg/kg/day respectively) in diet were administered to male and female CD-1 mice
(age unknown) in a 2-generation reproductive test in which F0 males or females given BPA
were mated with untreated females or males, decrease in number of offsprings born and
their survival were noted at 875 mg/kg/day or more, as well as decreased body weight
(females), increased liver and kidney weights (males and females), decreased seminal
vesicle weight and spermatozoic motility, and increased mortality of offsprings before
weaning at 1,750 mg/kg/day in regard to F0 generation, while increased liver and kidney
weights (males and females) and decreased epididymis and seminal vesicle weights were
observed at 437 mg/kg/day or more for F1 generation (Reel et al., 1997).
By oral administration of BPA at 0, 160, 320, 640 and 1,280 mg/kg/day in female
SD rats (age unknown), decreased body weight at 160 mg/kg/day or more and death at
1,280 mg/kg/day occurred in the dams, but nothing abnormal was observed in fetuses
(Morrissey et al., 1987).
When BPA at 0, 1,000, 3,000 and 9,000 ppm (equivalent to 0, 50, 150 and
450 mg/kg/day, respectively), or BPA at 0, 100, 250, 500, 750 and 1,000 ppm (equivalent
to 0, 5, 13, 25, 38 and 50 mg/kg/day, respectively) in diet were administered to male and
female SD rats (age unknown) for 17 weeks in a single generation reproductive test,
decreased body weight at 150 mg/kg/day or more occurred in F0 generation, while
decreased body weight at 50 mg/kg/day or more was noted in F1 generation. In the
repeated study the decreased body weight was observed at 50mg/kg/day or more in F0, but
not at 50mg/kg/day in F1 (German Chemical Society, 1995).
(3) Investigation of the effect at low dose (Attachment-4)
Since 1998, the effect of a low dose of BPA has been discussed.
When oral administration of BPA at 0, 0.002 and 0.02 mg/kg/day was conducted in
female CF-1 mice (age unknown) from day 11 to 17 of gestation, increased prostate and
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Bisphenol A
preputial gland weights, and decreased epididymis weight at 0.002 mg/kg/day, and
increased prostate weight and decreased sperm production rate at 0.02 mg/kg/day were
noted in F1 (Nagel et al., 1997; vom Saal et al., 1998). However, reliability of this report
was in doubt, because 1) the changes observed in 0.002 mg/kg/day group (e.g. increased
preputial gland weight) was not observed in 0.02 mg/kg/day group, 2) the study was not
conducted in compliance with GLP and 3) the number of animals per group was too small.
To demonstrate the reproducibility, similar tests were conducted, but the results of the first
test were not reproducible (Ashby et al., 1999; Cagen et al., 1999a). When oral
administration of BPA at 0 and 0.0024 mg/kg/day was conducted in female CF-1 mice
(age unknown) from day 11 to 17 of gestation, decreased body weight on the weaning day,
and curtailed number of days before vaginal opening and before onset of estrus cycle were
noted in F1 , and the relative positions of male and female fetuses were reported to be
involved in the extent of these effects (Howdeshell et al., 1999), but the results of tests to
confirm reproducibility have not been disclosed yet.
When 1 ppm of BPA was incorporated in water and given to rats for 8 - 9 weeks
from the time before mating, during pregnancy up to weaning, testis weight decreased in
F1 males (Sharpe, 1996, though the report was presented at the 10th International Congress
of Endocrinology, San Francisco, June 1996, the complete report has not yet been
published). However, when BPA at 0, 0.01, 0.1, 1.0 and 10 ppm was incorporated in
water and administered in a reproductive test to female Wistar rats (10 weeks old) for total
10 weeks including 2 weeks before mating, 2 weeks during cohabitation, 21 - 22 days
during pregnancy and 22 days during nursing and in about 90-day old males the
reproductive organ and accessory reproductive organ weights, number of sperms and
histopathology of testis were examined, no influence of BPA was observed (Cagen et al.,
1999b). As described in the above, the results are divided as to the effect of low dose
BPA. Whereas researchers such as vom Saal insisted that the effects were noted by
administration during gestation, other reports indicated that the results were not
reproducible. The low dose effect of BPA is still in the controversial stage.
Based on these background, the reports indicating the presence of a low dose of BPA
and those indicating no influence were reviewed and discussed at NTP Endocrine
Disrupters Low Dose Peer Review Meeting (NTP, USA, 10th - 12th October, 2000).
Prior to the Meeting, the low dose panel of NTP requested the statistics subpanel to
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Bisphenol A
conduct statistic analysis of the data submitted again to review whether the facts
discovered or those not discovered as the results of experiments correctly reflected the raw
data. The subpanel of BPA discussed the analysis results presented by the statistics
subpanel and the review report was presented at the Meeting, and concluded as in the
following.
The subpanel of BPA considered that the data submitted (the researchers who denied
the effects of BPA submitted the whole data, while those who although submitting a part
of data insisted on the effects were all statistically reliable. With this as the basis, the
subpanel placed importance on the report that the results assumed to exist the low dose
effect (increased uterus weight and elevated serum prolactin level, etc.) were obtained not
only from the experiment in mice conducted by vom Saal but also from the experiment in
rats conducted by Ben-Jonathan (subcutaneous embedding of implant containing BPA), as
well as on the report that there was difference in reaction between the strains of rats (raw
data were not submitted in this report). Bearing these in mind, the subpanel indicated the
following: "There is definite evidence that BPA at a low dose influenced specific
endpoints such as prostate weight. However, considering the result that the low dose
effect was not reproducible in the experiments conducted at a plural number of
laboratories under the same conditions, this subpanel is not persuaded that a low dose
effect of BPA has been conclusively established as a general or reproducible findings.”
That is, according to the statement of the specialist panel, the low dose effect of BPA is a
phenomenon observed under considerably limited experimental conditions at present, and
therefore this phenomenon is not considered as a general phenomenon.
In addition, the toxicological significance and a long term involvement of low dose
effect reportedly observed in prostate and female reproductive organs as examples are
unclear, and were not taken up as the subjects of discussion in the peer review. As a
mechanism of low dose effect of BPA, the theory that some fetuses are susceptible to the
effect depending on implantation position in the uterus (a male sandwiched between 2
females, or a male adjacent to a female is susceptible to female endogenous estrogen) has
been proposed, but this is a task to be clarified in the future. The subpanel
simultaneously suggested a possibility that the analysis based on introduction of new
technology including genomix, proteomix, etc. as well as the dose-tissue concentration at
a low dose may provide useful data for solution of the problem (NTP, 2001).
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At NTP's Low Dose Peer Review in October 2,000, at International Congress related
to the low dose effect held in Berlin in November the same year (Bisphenol A: Low dose
effects - high dose effects; Berlin, Germany, 18th - 20th November, 2000) and at the 3rd
International Symposium on Environmental Endocrine Disrupters 2000 (Yokohama, Japan,
16th - 18th, December, 2000), Tyl et al. reported on a 3 generation reproductive toxicity
test in rats (Tyl et al., 2000). When a test was conducted using BPA at low doses of 0,
0.015, 0.3, 4.5, 75 ppm (equivalent to 0, 0.001, 0.02, 0.3, 5 mg/kg/day, respectively), as
well as at a dose of 750, 7,500 ppm (equivalent to 50, 500 mg/kg/day, respectively) known
to manifest toxicity, no general toxic effects on parent animals for each generation were
observed for low dose groups and that no abnormality was found in fertility and
development of offspring. On the other hand, at the high dose group of 7,500 ppm
abnormality was found in fertility of parent animals for each generation and in
development of offspring. And at the dose of 750 ppm, suppression in body weight
increase was observed for parent animals of each generation, however, no abnormality
was found in fertility of the parental animals and in development of offspring. Based on
these findings, NOAEL in the rat 3-generation study was estimated to be 75 ppm
(equivalent to 5 mg/kg/day) for systemic toxicity to parental animals and 750 ppm
(50mg/kg/day) for reproductive toxicity.
When oral administration by gavage of BPA at 0, 0.0002, 0.002, 0.02 and 0.2
mg/kg/day was conducted in male and female SD rats from the time before the mating of
F0 (10 weeks and 5 weeks before mating in the case of males and females respectively) up
to weaning of F2 in a 2-generation reproductive test, nothing abnormal was reported in
reproductive function of parent animal and in development & growth of offsprings in each
generation (Ema et al., 2001). The BPA subpanel assessment of these tests was
considered by the NTP low dose effect panel as the data that were statistically reliable,
showing many endpoints without however much emphasis placed as the experiment to
investigate the low dose action. In this regard, the subpanel maintained that a multigeneration reproductive test is intended to investigate reproductivity and is not appropriate
for fully assessing the effect of a substance on development & growth process of
offsprings,
unless administration is conducted at the window period using pregnant
animals (NTP, 2001).
Apart from the low dose effect discussion, HSE (Health and Safety Executive) in
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UK proposed to EU in May 2001 to attach a label with a warning that BPA corresponds to
category 2 of reproductive toxicity substance (substance that is regarded to harm the
fertility of human) and is an R60 (may harm fertility) to relevant products (HSE Bootle,
2001). The basis of the proposal is the results of a test in mice conducted by NTP (Reel
et al., 1997) and a 3-generation reproductivity test in rats conducted by Tyl et al (2000).
In the former, the middle and high doses of 5,000 and 10,000 ppm (equivalent to 875 and
1,750 mg/kg/day, respectively) developed decrease in the number of offspring born (by
5% and 9% respectively) and the number of surviving offspring per litter (by 20% and
48% respectively). As to the latter in which the doses 0.015-7,500 ppm (equivalent to
0.001 - 500 mg/kg/day) were given, decrease in the number of siblings per litter was
observed in F1 to F3 generations at 7,500 ppm (the number of surviving offspring per litter
in the control group and a group administered 500 mg/kg/day of BPA: 14.3 and 11.5
respectively in F1 , 14.6 and 10.8 respectively in F2 , 14.8 and 10.9 respectively in F3 ).
After mentioning that the above dose was known to cause toxicity in the dams including
inhibition on body weight increase and degeneration of renal tubule, HSE still seriously
considered the reproductive disorder obtained from mice (HSE Bootle, 2001). Later, the
evaluation of reproductive toxicity of BPA by EU was so amended at the conference of
January 2002 for BPA to be a substance toxic to reproduction category 3 (substances
which cause concern for human fertility) with risk phrase of R62 (possible risk of
impaired fertility). As to the developmental effect, the view was given that at least one
year would be needed before more researchers are completed (HSE Health Directorate,
2002).
Recently, the results suggesting the low dose action of BPA have been reported.
When oral administration by gavage of BPA at 0, 0.02, 0.2, 2, 20 and 200 mg/kg/day
was conducted in male SD rats (13 weeks old) for 6 days, decrease in daily
spermatogenesis was noted in all the BPA groups. When oral administration by gavage
of BPA at 0, 0.000002, 0.00002, 0.0002, 0.002, 0.02, 0.2 and 2.0 mg/kg/day was
conducted for 6 days under the same conditions, decrease in daily spermatogenesis was
also noted at 0.002 mg/kg/day or more (Sakaue et al., 2001).
Effects of BPA on development of cultured mouse two-cell embryoes were studied.
The rate of in vitro development of two-cell embryoes to eight-cell embryoes was
significantly increased by exposure to BPA at concentration of 3 nM (94%; 172/182)
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compared with the rate in the control group (88%; 334/378). The rate of development to
the blastocyst stage in 48-h cultures of two-cell embryoes were also significantly
increased (69.2%; 126/182) compared with the rate in control group (58.7%; 222/378).
On the other hand, the frequency of development to blastocyst stage at 48-h was
significantly decreased by exposure to BPA at 100 μM (31.2%; 93/298), although
development to eight-cell embryoes at 24-h was not inhibited by 100 μM BPA (Takai et
al., 2000).
The effects of exposure to BPA early in life on sexual differentiation in brain and
behavior in Wistar rats were studied. Mother rats during pregnancy and lactation were
administered at approximately 1.5 mg/kg BPA per day in drinking water.
The control
female offspring showed higher activity, lower avoidance memory, and larger locus
coeruleus than the male controls, while the BPA-exposed group did not show any sexual
dimorphism. These results may suggest that BPA affects sexual differentiation in brain
and behavior from late fatal period to first week after birth in rats (Kubo et al., 2001).
3) Information on general toxicity
(1) Acute toxicity (Table 1) (German Chemical Society, 1995)
Table 1 Acute toxicity test
Mouse
Oral LD50
Percutaneous LD 50
Intraperitoneal LD50
Subcutaneous LD50
1,600 - 5,280 mg/kg*
Rat
3,250 - 5,660 mg/kg*
−
200 mg/kg
−
400-800 mg/kg
−
2,400 mg/kg
Rabbit
2,230-4,000
mg/kg
Guinea pig
4,000 mg/kg
3,000-6,400
mg/kg
−
150 mg/kg
−
−
−
*: There are differences between the literatures cited.
(2) Repeated-dose toxicity (Attachment-5)
When BPA at 0, 2,000, 5,000, 10,000, 20,000 and 40,000 ppm (equivalent to 0, 500,
1,000, 2,200, 5,500 and 14,600 mg/kg/day, respectively in males, and 0, 600, 1,300, 2,500,
6,300 and 22,000 mg/kg/day, respectively in females) in diet and administered to male
and female B6C3F1 mice (6-week-old) for 13 weeks, no changes attributable to BPA was
observed at 2,000 ppm. However, decreased RBC and Ht at 5,000 ppm or more,
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decreased Hb concentration, cystic dilation of renal tubules, fibrosis in the periphery of
cyst, degeneration and regeneration of tubular epithelium and increased hyaline cast at
10,000 ppm or more, suppression on body weight increase, increased liver weight,
decreased ovary weight, fibrous osteodystrophy in femoral bone and sternum, and atrophy
of myofibril at 20,000 ppm or more, and leanness, death assumed to be attributable to food
rejection, increased platelet and kidney weight, and enhanced extramedullary
hematopoiesis in spleen at 40,000 ppm were observed (Furukawa et al.; 1994).
When BPA in diet was administered for 2 years to mice, large polyploidy hepatocytes
were noted in liver of male B6C3F1 mice (5 weeks old) at 1,000 and 5,000 ppm
(equivalent to 150 and 250 mg/kg/day respectively). Decreased body weight was observed
in both male and female at 5,000 ppm (NTP, 1982).
When BPA at 0, 250, 500, 1,000, 2,000 and 4,000 ppm (equivalent to 0, 13, 25, 50,
100 and 200 mg/kg/day, respectively) in diet was administered to male and female F344
rats (age unknown) for 91 days, dilation of cecum in males and females, and hyaline clots
in bladder of males were observed at 250 ppm (equivalent to 13 mg/kg/day) or more,
while body weight decreased at 1,000 ppm (equivalent to 50 mg/kg/day) (NTP, 1982).
When oral administration by gavage of BPA at 0, 40, 200 and 1,000 mg/kg/day was
conducted in male and female SD rats (5 weeks old) for 28 - 32 days in compliance with
the revised OECD test guideline 407 (enhanced TG 407), the changes mainly in kidney,
liver and intestinal tract were observed at 200 mg/kg/day or more, while abnormal estrus
cycle was noted at 1,000 mg/kg/day (CERI, 2000).
When BPA in diet was administered for 2 years to F344 rats both male and female (5
weeks old) at 1,000 and 2,000 ppm (equivalent to 74 and 148 mg/kg/day to male,
equivalent to 74 and 135 mg/kg/day to female, respectively), decrease in body weight and
in food intake were observed in both male and female at 5,000 ppm (NTP, 1982)(EPA
calculated 1000 ppm to be 50 mg/kg/day).
When BPA in diet at 0, 1,000, 3,000 and 9,000 ppm (equivalent to 25, 75, 225
mg/kg/day) was administered to male and female dogs (beagle) for 90 days, liver weight
increased at 9,000 ppm (equivalent to 225 mg/kg/day) (German Chemical Society, 1995;
General Electric, 1976b).
Concerning the inhalation exposure, when male and female F344 rats (age
unknown) was exposed to BPA at 0, 10, 50 and 150 mg/m3 for 6 hours/day for 9 days,
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slight irritation in the anterior nasal cavity was noted at 50 mg/m3 or more, and body
weight of males decreased at 150 mg/m3 (German Chemical Society, 1995; Dow
Chemicals Co., 1985a; Dow Chemicals Co., 1985b). When male and female F344 rats
(age unknown) were exposed to BPA at 0, 10, 50 and 150 mg/m3 for 6 hours/day,
5 days/week for 13 weeks, decreased body weight, dilated cecum, inflammation in nasal
cavity and respiratory mucosa, and hyperplasia of squamous epithelium were noted at
50 mg/m3 or more, while liver and kidney weights decreased at 150 mg/m3 (German
Chemical Society, 1995; Dow Chemicals Co., 1988).
4) Information related to mutagenicity/genotoxicity and carcinogenicity
(1) Mutagenicity/genotoxicity (Table 2)
Concerning the in vitro tests, the results of sister chromatid exchange test, murine
lymphoma test, chromosome aberration test and reverse mutation test using Salmonella
typhimurium, Escherichia coli and yeast were all negative regardless of metabolic
activation. There is a report that mutagenicity of BPA was tested using human Rsa cells,
which has been utilized for identification of novel mutagens. In the BPA exposed cells
base substitution mutations at K-ras codon 12 were detected. (Takahashi et al., 2001).
Concerning the in vivo tests, result of DNA adduct formation test using rat was positive,
but this was assessed to have questionable toxicological significance including
carcinogenicity, because the covalent index was too small to induce carcinoma (Atkinson
& Roy, 1995b).
Table 2 Results of mutagenicity, genetic toxicity tests
Test method
in vitro
Reverse
mutation test
Used cell/strain/Animal species
Result*
Salmonella typhimurium TA98, TA100,
TA1535, TA1537 0.33-333.3 µg/plate S9(+/-)
References
German Chemical
Society, 1995
−
Haworth et al.,
1983
German Chemical
Society, 1995
Salmonella typhimurium TA1538 0.1-1.0
mg/mL S9(+/-)
−
Shell Oil Co.,
1978
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Test method
Used cell/strain/Animal species
Result*
Salmonella typhimurium TA97, TA98, TA100,
TA102 5-1000 µg/plate S9(+/-)
References
German Chemical
Society, 1995
−
Takahata et al.,
1990
German Chemical
Society, 1995
E. coli WP2, WP2 uvrA 0.1-1.0 mg/mL S9(+/-)
−
Yeast S. cerevisiae 0.01-0.5 mg/mL S9(-)
−
Chromosomal
aberration test
Cultured liver epithelial cells of rat (RL1) 1030µg/mL S9(-)
Shell Oil Co.,
1978
HSDB, 2001
−
CHO cell, 20-50 µg/mL S9(+/-)
−
Mouse
lymphoma test
Shell Oil Co.,
1978
German Chemical
Society, 1995
Shell Oil Co.,
1978
German Chemical
Society, 1995
Ivett et al., 1989
German Chemical
Society, 1995
Mouse lymphoma cells L5178Y, 5-50 µg/mL
S9(+/-)
−
Sister chromatid
exchange test
in vivo
CHO cell, S9(+/-)
S9(-): 0.8-25 µg/mL, S9(+): 30-50 µg/mL
DNA adduct
formation test
* −: negative
CD rat (male)
200 mg/kg, single intraperitoneal administration,
oral administration by gavage at 200 mg/kg/day
× 4, 8, 12 and 16 days
＋: positive
−
＋
Myhr & Caspary,
1991
German Chemical
Society, 1995
Ivett et al., 1989
Atkinson & Roy,
1995b
(2) Carcinogenicity (Table 3)
No carcinogenicity of BPA was observed in tests in which BPA was administered at
1,000 and 2,000 ppm to F344 rats, at 1,000 and 5,000 ppm to male B6C3F1 mice, and at
5,000 and 10,000 ppm to female B6C3F1 mice for 103 weeks (NTP, 1982).
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Table 3 Carcinogenicity assessment by national and international organizations
Organization
Classification
Significance
Reference
EPA
−
No evaluation
IRIS, 2002
EU
−
No evaluation
ECB, 2000
NTP
−
No evaluation
NTP, 2000
IARC
−
No evaluation
JARC, 2001
ACGIH
−
No evaluation
ACGIH, 2001
Japan Society for
Occupational Health
−
No evaluation
Japan Society for Occupational
Health, 2001
5) Information on immune system
Currently, there is no report related to effect of this substance on immune system.
6) Fate and Metabolism
In clinical study, 8 mg (a piece) or 32 mg (8 mg/piece × 4) of dental sealant that
contained BPA was attached to the teeth of 40 healthy volunteers, and their saliva and
blood were examined after 1 and 3 hours, and after 1, 3 and 5 days. Though 5.8 - 105.6
ppb of BPA was detected in some saliva samples after 1 and 3 hours, the level was already
lower after 3 hours and nothing was detected thereafter. Since BPA was not detected in
the blood, it seemed that BPA that overflowed from dental sealant was not absorbed in the
circulating blood or was present at a level below the detection limit (Fung et al., 2000).
It is reported that BPA is absorbed from digestive tract and that the whole amount is
excreted. When single oral administration of 800 mg/kg of BPA whose C-2 position of
propyl group was labeled with 14 C was conducted in rats, 28% of the dose was excreted
into urine (mainly as glucuronic acid conjugate) and 56% into feces (20% unchanged,
20% hydroxide and 16% unknown compound) but no carbon dioxide was detected.
During 2 days after administration, the excretion into urine and feces reached 80% of the
dose administered. After 8 days, there was no radioactivity in the rat, suggesting the half
life of about 1 day (German Chemical Society, 1995; Knaak et al., 1966).
The results of oral, intraperitoneal or subcutaneous administration of 14 C-labeled
BPA (4, 4'-isopropylidene-2- 14 C diphenol or 2,2-bis(p-hydroxyphenyl)-2- 14 C-propane) at
10 and 100 mg/kg to male and female F344 rats (8 - 9 weeks old) indicated that the
340
Bisphenol A
pharmacokinetics became different by administration route and between male and female.
The maximum blood concentration was observed within 1 hour after oral and
intraperitoneal administration, while the peak was noted at 4 hours after subcutaneous
administration.
The compound was quickly excreted to reach the level below detection
limit within 72 hours after subcutaneous and intraperitoneal administration and within 18
hours after oral administration.
The bioavailability and plasma radioactivity
concentration were the highest by subcutaneous administration, followed by
intraperitoneal administration. The levels were markedly low after oral administration
probably because of
low absorption of BPA from digestive tract and conjugation in the
first pass effect in liver. The plasma radioactivity was noted mainly as glucuronic acid
conjugate in the case of oral administration, but mainly unchanged BPA was detected after
intraperitoneal and subcutaneous administration. Other than the unchanged BPA, 4
metabolites were noted by these 2 administration routes. The hydroxide reported in the
past experiment was very small in quantity. Depending on the dose, it was assumed that
hydroxylation occurs after other metabolic pathways have become saturated. Most of the
radioactive compound was excreted into feces unchanged by each route. BPA was
mainly excreted into urine as monoglucuronide. Urinary excretion was about twice
higher in females by each administration route. The residue of BPA and its metabolites
was very small and only 1.3%, 0.8% and 0.4% of the radioactivity administered remained
after 7 days from subcutaneous, intraperitoneal and oral administration respectively
(Pottenger, 2000).
An in vitro experiment demonstrated that sulfate conjugation took place in BPA by
recombinant human sulfate transferase. Furthermore, sulfate conjugation of BPA was
observed in an experiment in which BPA and sulfuric acid were added to HepG2 cell
derived from human liver cancer, suggesting that sulfate conjugation of BPA proceeds in
living body (Suiko et al., 2000).
It was demonstrated that when BPA was allowed to react with an oxidant in vitro,
bisphenol- o-quinone was generated, and that when this was incubated with DNA, binding
to DNA occurred (Figure 1)(Atkinson and Roy, 1995b). When single intraperitoneal
administration of BPA at 200 mg/kg was conducted in rats in an experiment or when oral
administration by gavage at 200 mg/kg/day for 4, 8, 12 and 16 days was conducted in
another experiment, covalent binding to DNA occurred in the liver (Atkinson and Roy,
341
Bisphenol A
1995a). Based on these results, it was assumed that, after BPA was metabolized to 5hydroxybisphenol in liver, the reactive metabolites bisphenol semiquinone and 4,5bisphenol- o-quinone were produced (Figure 1) and they were bound to DNA. However,
based on the calculation of index of covalent binding to DNA, this reaction was not
intense so that no carcinogenicity was induced (German Chemical Society, 1995).
H3C
H3C
CH 3
HO
OH
(1)
H3C
CH 3
HO
OH
HO
O
OH
HO
(2)
(3)
H3C
glucuronic
acid conjugation
グルクロン酸抱合体
sulfate
conjugation
硫酸抱合体
CH 3
CH 3
O
OH
O
(4)
(1) Bisphenol-A
(2) 5-hydroxybisphenol
(3) Bisphenol semiquinone
(4) 4,5-bisphenol-o-quinone
Fig. 1: Metabolic pathway of bisphenol-A
2.
Hazard Assessment at present
There is no report on the effect of BPA on endocrine system and reproductive system
of humans.
To investigate the effect on endocrine system, many in vitro tests, as well as in vivo
tests (uterotrophic assay) have been conducted, and the results indicated that BPA has
estrogenic action. However, in in vitro tests its activity mediated by estrogen receptors is
weak (its receptor binding affinity is 1/500 - 1/15,000 of that of E2 and its transcription
activation capability 1/600 and 1/130,000 of that of E2).
Concerning the effect on reproductive system, a test was conducted in compliance
342
Bisphenol A
with the revised OECD test guideline 407 (enhanced TG 407). The results indicated
various toxicological changes at 200 mg/kg/day or more, and at 1,000 mg/kg/day
abnormal estrus cycle suggesting endocrine disruption. Regarding reproductive and
developmental toxicity or fertility toxicity, there are reports ranging from single generation
reproductive tests to 3-generation reproductive tests. In single generation reproductive
test in rats body weight decrease in F1 was observed at 50 mg/kg/day, but no reproductive
toxicity was observed. In 2-generation reproductive test in mice declined seminal vesicle
weight and spermatozoic motility, etc. in F1 was observed at 437 mg/kg/day or more.
In a
3-generation reproductive test in rats at lower doses of 0.015, 0.3, 4.5 and 75 ppm
(equivalent to 0.001, 0.02, 0.3 and 5 mg/kg/day, respectively) and higher doses of 750,
7500 ppm (equivalent to 50, 500 mg/kg/day, respectively), suppression of body weight
increase was observed for parental animals of each generation at 50 mg/kg/day. But no
abnormality was found in fertility of parental animals and in development of offspring.
Reproductive and developmental toxicity was observed at the dose equivalent to 500
mg/kg/day. At present NOAEL for reproductive and developmental toxicity and fertility
toxicity is estimated to be 50 mg/kg/day.
On the other hand, the problem related to toxicity of BPA is the effect on fetus and
offspring by administration during gestation as was reported in some animal experiments.
According to positive reports, changes were observed at considerably low doses of
0.002 mg/kg/day by oral administration and at 1 ppm mixed in drinking water. However,
reproducibility tests conducted for various administration periods and at many doses in a
larger number of animals and test parameters all indicated negative results.
In the final report of NTP Peer Review results about low dose effects given in
October 2,000 (published on May 14, 2001), all the data submitted regarding the low dose
effects of BPA on prostate, etc. were considered reliable whether the result was positive or
negative.
The subpanel concluded that at present the effect is observed only under
considerably limited conditions and therefore this is hardly considered to be the general
phenomenon. In this regard, the subpanel indicated the following: "There is definite
evidence that BPA at a low dose influenced specific endpoints such as prostate weight.
However, this subpanel is not persuaded that a low dose effect of BPA has been
conclusively established as general or reproducible findings." Even though BPA does
have the low dose effect, current findings are unable to prove that its action is harmful.
343
Bisphenol A
As the information related to the harmful nature of BPA in humans, mild skin
irritation and allergic dermatitis were reported. The patient of dermatitis showed positive
allergic reaction to both BPA and formaldehyde, accordingly substances causing the effect
as well as synergy of both substances was not identified,.
As repeated oral dose toxicity, the effects mainly on large intestine, cecum, liver and
kidney were noted in animal experiments. Anemia was also observed. Additionally,
body weight decrease was observed at dose of 50 mg/kg/day and above in a 2-years rat
oral chronic toxicity test, and EPA estimated LOAEL as 50 mg/kg/day.
Mutagenicity test results were mostly negative and carcinogenicity was also negative.
There is no report on carcinogenicity of this substance in humans.
3.
Risk assessment and other necessary future measures
BPA has weak estrogenic activity (receptor binding affinity is 1/500 - 1/15,000 of E2
and its transcription activation capability 1/600 and 1/130,000 of E2) and reproductive &
developmental toxicity was observed at high doses of approximately not lower than 500
mg/kg/day in oral tests. Since sufficient scientific findings in in vitro as well as in vivo
animal test data were already obtained to assess the effect of BPA on endocrine system and
reproductive system for humans, there seems to be no urgent need to plan additional tests
in the future.
Though it is necessary to collect further information on so-called "low dose effects"
represented by BPA from academic point of view, it seems unnecessary to take any
specific measure other than the above, considering the view expressed by NTP Low Dose
Effect Panel that the low dose effect of BPA at present is a phenomenon observed under
considerably limited experimental conditions and it is hardly considered to be the general
phenomenon.
On the other hand, since effects of BPA on reproductive & developmental toxicity
was observed, independent of the existence of endocrine disruption, risk assessment based
on hazard and exposure assessment is to be conducted and appropriate risk management is
to be examined.
344
Bisphenol A
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349
Bisphenol A
Attachment-1 Results of in vitro test on receptor binding
Items
ER binding
test
Test methods and conditions
Method, Experiment to investigate
the effect of serum in a binding test
(Relative binding affinity-serum
modified access assay, RBA-SMA)
Result
IC50:
No serum
BPA, 8.57×10-6 M
(E2: 5.64×10-10 M)
Containing serum
BPA, 3.94×10-5 M
(E2: 3.96×10-9 M)
Receptor, Human ER
IC50
BPA: 7.1×10-5 M
(E2: 5.0×10-9 M)
Method, Competitive binding test IC50
using [3H]-E2 as a ligand, Receptor, BPA, 1.17×10-5 M
ER derived from rat's uterine
(E2: 8.99×10-10 M)
cytoplasm
Human ER binding test
IC50: 8.3×10-7 M
(recombinant ER α ligand domain) (E2: 1.6×10-9 M)
RBA: 0.20%
Yeast twoMethod: Human ER dimer
EC50
hybrid assay formation test using yeast twoBPA: 3.1×10-6 M
hybrid assay
(E2:: 1.2×10-10 M)
Cells: yeast transfected with Gal4
DNA binding domain/human ER
ligand binding domain gene, Gal4
activation domain coactivator TIF2
gene and ß-galactosidase reporter
gene
Cells: Estrogen responsive
recombinant yeast
Reporter
gene assay
using
recombinant
yeast
Cells: Estrogen responsive
recombinant yeast
Cells: Estrogen responsive
recombinant yeast
Reporter
gene assay
using
recombinant
cultured cells
Cells: GH3 cells transfected with
reporter gene in which 5’ nontranscriptive domain (2.5 kb) of
prolactin gene positioned in the
upstream of luciferase gene
Cells: HeLa cells transfected with
ER α or ER ß expression element
and ERE/CAT reporter gene
REC10
BPA: 3×10-6 M
(E2: 3×10-10 M)
EC50
BPA: 3.40×10-6 M
(E2: 2.25×10-10 M)
Relative estrogenic
activity of BPA when
that of E2 was assumed
as 100 is 0.005.
EC50
BPA: 2.2×10-6 M
(E2: 1.0×10-9 M)
As in the case with E2
(1 pM), luciferase
activity increased with
BPA (1 nM).
Conclusion
Weak affinity
(Serum-free:
1/15,000)
(Serum: 1/9,900 of
E2)
References
Nagel et al.,
1997
Weak affinity
(1/14,000 of E2)
Sheeler et al.,
2000
Weak affinity
(1/13,000 of E2)
Blair et al.,
2000
Weak affinity
(1/500 of E2)
CERI, 2001
Sheeler et al.,
Positive of gene
2000
transcription
activity(1/26,000 of
E2)
Nishihara et
Positive of gene
al., 2000
transcription
activity
(1/10,000 of E2)
Positive of gene
transcription
activity
(1/15,000 of E2)
Positive of gene
transcription
activity
(1/20,000 of E2)
Positive of gene
transcription
activity
(1/2,200 of E2)
Positive of gene
transcription
activity
(1/1,000 of E2)
BPA showed agonist
Positive of gene
activity at > 10-9 M to
transcription
activity
both ER α and ERß.
As to ERß, antagonist
activity was observed at
10-6 M .
350
Gaido, 1997
Coldham et al.,
1997
Sheeler et al.,
2000
Steinmetz et
al., 1997
Hiroi et al.,
1999
Bisphenol A
Items
Test methods and conditions
Method: Reporter gene assay
mediated by estrogen receptor
Cells: T47D cells transfected with
estrogen responsive element and
luciferase gene
Cells: HeLa cells transfected with
human ER expression gene and ER
responding sequence.
Concentration: 10-11 10-5 M
Cells: HeLa cells transfected with
rat ER expression gene and ER
responding sequence.
Concentration: 10-11 - 10-5 M
Method: Experiment to determine
the secretion of prolactin in GH3
cells cultured in the presence of
BPA and E2
Result
EC50
BPA: 7.70×10-7 M
(E2: 6×10-12 M)
Conclusion
Positive of gene
transcription
activity
(1/130,000 of E2)
References
Legler et al.,
1999
PC50:
BPA: 2.9×10-7 M
(E2: <10−11 M)
Positive
(1/29,000 of E2)
CERI, 2001
PC50:
BPA: 6.0×10-7 M
(E2=<10−11 M)
Positive
(1/600 of E2)
Yamasaki et
al., 2002
BPA and E2 dosePromoting prolactin Steinmetz et
al., 1997
dependently promoted
secretion
prolactin secretion
within the range of 10-8 10-6 M and 10-12 - 10-9
respectively.
Steinmetz et
Method: Experiment by single
The expression of c-fos Promoting gene
al., 1998
intraperitoneal administration of
increased by 14 times in expression
BPA to F344 and SD rats at 18.75, uterus and vagina of
37.5, 75, 150 and 200 mg/kg
F344 at 2 hours after
administration of BPA
(50 mg/kg).
Jorgensen,
Method: Experiment to investigate The concentration of
Promoting gene
2000
effect on endogenous estrogen
BPA required to induce expression
5
6
responding gene expression level pS2 gene was 10 - 10
(the expression levels of pS2, TGF times that of E2.
ß3, monoamine oxidase A (MAOA), α1-antichymotrypsin (α1-ACT)
were quantitated by PCR method)
Diel et al.,
Method: After administration of
Inhibition on expression Promoting gene
2000
BPA at 5, 50 and 200 mg/kg to
of AR, ER and PR gene expression
ovariectomized DA/Han rats for
and an increase in the
3 days, uterus was collected and
expression of C3 gene
gene expression in the tissue was were noted in 200 mg/kg
determined by Northern blot
group.
method and semi-quantitative PCR
method
ER: Estrogen receptor; E2: 17ß-estradiol; REC10: Concentration corresponding to 10% of E2 activity
value at 10-7 M PC50: Concentration corresponding to 50% of the maximum activity of E2; IC50: 50%
inhibitory concentration; RBA: Relative binding affinity (%)
Changes in
gene and
protein
expression
351
Bisphenol A
Attachment-2 Results of short-term detection test of estrogenic activity in mammals
Animal
species
Mouse
(B6C3F1
, female)
Mouse
(CD-1,
female)
Rat
(F344,
female)
Rat
(F344 or
SD,
female)
Rat
(Alpk:A
P/SD,
female)
Rat
(SD,
female)
Administration
method
s.c.
(uterotro
trofic
assay,
ovariecto
mized
mice)
s.c.
(uterotro
trofic
assay,
juvenile
mice
i.p.
(uterine
growth
assay,
ovariecto
mized
rat)
s.c.
capsule
implant
(uterotro
trofic
assay,
ovariecto
mized
rat)
s.c.
(uterotro
trofic
assay,
ovariecto
mized
rat)
p.o.
gavage
(uterotro
trofic
assay,
juvenile
rat)
s.c.
(uterotro
trofic
assay,
juvenile
rat)
Administration
period
35 - 60 days
old, for 4 days
Dose
Result
References
Papaconstantinous et
al., 2000
0, 0.02, 0.2, 0.8,
2, 8 mg/kg/day
Increased uterus weight at
0.8 mg/kg/day or more
21 days old
3 days
0, 0.01, 0.1, 1, 10,
100 mg/kg/day
No increase in uterus weight.
No changes in lactoferrin,
peroxidase activity or BrdU
labeling index in uterine mucus
epithelium
Mehmood et
al., 2000
7-8 weeks old
Single dose
0, 18.75, 37.5, 75,
150, 200 mg/kg
Significant increase in BrdU
labeling index in uterine and
vaginal epithelia at 37.5 mg/kg or
more
Steinmetz et
al., 1998
7-8 weeks old
3-day
implantation
Corresponding
to 0.3 mg/kg/day
8-10 weeks
old
3 days
33 mg/rat/day
In F344 rat increased uterus
weight, hypertrophy and
hyperplasia of uterus, mucous
fluid secretion from uterus,
epithelial hyperplasia and
keratinization of vagina.
The height of uterine epithelial
cells increased by 2.5 times.
No influence on SD rat.
Increased uterus weight
18 days old
3 days
0, 40, 160, 800
mg/kg/day
Increased uterus weight at
160 mg/kg/day or more
18 days old
3days
0, 8, 40, 160
mg/kg/day
Increased uterus weight at
8 mg/kg/day or more
352
Ashby et al.,
2000
Yamasaki et
al., 2000
Bisphenol A
Animal
species
Rat
(Long
Evans,
female)
Rat
(SD,
female)
Administration
method
p.o.
gavage
(uterotro
trofic
assay,
juvenile
rat)
p.o.
gavage
(uterotro
trofic
assay,
ovariecto
mized
rat)
s.c.
(uterotro
trofic
assay,
juvenile
rat)
Administration
period
Dose
Result
References
21 days old
3 days
Comparative
test by autopsy
at 6 hours and
24 hours after
final
administration
60 days old
3 days
0, 100, 200 , 400
mg/kg/day
Increased uterus weight at
200 mg/kg/day or more
The above result was obtained
after 6 hours but no difference
from the control was noted after
24 hours
Laws et al.,
2000
0, 100 mg/kg/day
No influence on uterus weight
20 days old
3 days
0, 2, 20, 200
mg/kg/day
Increased uterus weight at
20 mg/kg/day or more
353
Yamasaki et
al, 2002
Bisphenol A
Attachment-3 Results of reproductivity and reproductive toxicity test in mammals
Animal
species
Mouse
(CD-1,
female)
Mouse
(CD-1,
male
and
female)
Administration
method
p.o.
gavage
By
feeding
Administration
period
Dose
Result
References
Age unknown
Day 6 - 15 of
gestation
(sacrificed on
day 17 of
gestation)
0, 500, 750, 1,000,
1,250 mg/kg/day
Morrissey
et al., 1987
2-generation
reproductive
test from
1week before
F0 mating to F2
weaning
0, 2,500, 5,000,
10,000 ppm
(Corresponding to
0, 437, 875, 1,750
mg/kg/day)
Dams: Increased relative weight of
liver at 500 mg/kg/day or more,
suppression on body weight
increase and decreased pregnant
uterus weight at 1,250
mg/kg/day
Fetus: Increased embryo
resorption, decreased body
weight at 1,250 mg/kg group
No deformation was noted
F0: Decreased number of offspring
born and surviving at 875
mg/kg/day or more; at 1,750
mg/kg/day or more decreased
body weight, increased liver and
kidney weights, decreased
seminal vesicle weight and
spermatozoic motility, and
increased mortality of offspring
before weaning
F1: Increased liver and kidney
weights (both males and
females) and decreased
epididymis and seminal vesicle
weights at 437 mg/kg/day or
more
Mating of F0
generation
(males or
females) with
untreated
animals.
Rat (SD,
female)
p.o.
gavage
Rat (SD,
male
and
female)
By
feeding
Day 6 - 15 of
gestation
(sacrificed on
day 20 of
gestation)
Single
generation
reproductive
test for 17
days in F0 (age
unknown) and
for 90 days in
F1
0, 160, 320, 640,
1,280 mg/kg/day
0, 1,000, 3,000,
9,000 ppm
(Corresponding to
0, 50, 150, 450
mg/kg/day)
Both mating of males in the
high dose group with untreated
females and that of females in
the high dose group with
untreated males resulted in a
decrease in the number of
offspring born
Parent animal: Decreased body
weight at 160 mg/kg/day or more,
death at 1,280 mg/kg/day
Fetus: No abnormality
F0: Decreased body weight at
150 mg/kg/day or more
F1: Decreased body weight at
50 mg/kg/day or more
Reel et al.,
1997
Morrissey
et al., 1987
German
Chemical
Society,
1995
General
Electric,
1976a
354
Bisphenol A
Administration
method
Rat (SD,
By
male
feeding
and
female)
Animal
species
Administration
period
Single
generation
reproductive
test for 17
days in F0 (age
unknown) and
for 90 days in
F1
Dose
Result
0, 100, 250, 500,
750, 1,000 ppm
(Corresponding to
0, 5, 13, 25, 38, 50
mg/kg/day)
F0: Decreased body weight at
50 mg/kg/day or more
F1: No effects
References
German
Chemical
Society,
1995
General
Electric,
1978
355
Bisphenol A
Attachment-4 Results of toxicity tests at low doses
Animal
species
Administration
method
Timing of
administration
Administration
period
Age unknown
Day 11 - 17 of
gestation
Dose
Test method and result
References
0, 0.002, 0.02
mg/kg/da
y
F1: Examined when 6 months old
Increased prostate weight at
0.002 and 0.02 mg/kg/day
Nagel et al.,
1997
F1: Examined when 6 months old
Increased preputial gland
weight, decreased epididymis
weight at 0.002 mg/kg/day
Decreased sperm production
rate at 0.02 mg/kg/day
As to F1 females on the whole,
increased body weight on the
weaning day, and curtailed
number of days before vaginal
opening and before onset of estrus
cycle were noted in all F1 females.
However, these findings were
most clear-cut when no male fetus
was positioned next to female
fetus. Even though these
findings were obtained when a
male was positioned on one side
only, none of the above results
was noted when a female was
sandwiched between the males
vom Saal et
al., 1998
No changes in reproductive organ
and accessory reproductive organ
weights, number of sperms, sperm
production rate and vaginal
opening day in F1
F1 mice were examined when they
were 90 days old.
Nothing abnormal in number of
sperms, sperm production rate,
reproductive organ and accessory
reproductive organ weights, and
histopathological examination of
testis
Ashby et
al., 1999
Mouse
(CF-1,
Female)
p.o.
gavage
Mouse
(CF-1,
Female)
p.o.
gavage
Age unknown
Day 11 - 17 of
gestation
0, 0.002, 0.02
mg/kg/da
y
Mouse
(CF-1,
Female)
p.o.
gavage
0, 0.0024
mg/kg/da
y
Mouse
(CF-1)
p.o.
gavage
Age unknown
Between day
11 - 17 of
gestation
For the purpose
of investigating
the effects of the
position of
female fetuses
in uterus
(influence of
hormone of
adjacent fetus),
cesarean section
was performed
on day 19 of
gestation and
offsprings were
nursed by
untreated
parents
Age unknown
Day 11 - 17 of
gestation
Mouse
(CF-1)
p.o.
gavage
Age unknown
Day 11 - 17 of
gestation
0, 0.0002, 0.002,
0.02, 0.2
mg/kg/da
y
0, 0.002, 0.02
mg/kg/da
y
356
Howdeshell
et al., 1999
Cagen et
al., 1999a
Bisphenol A
Animal
species
Rat
(SD,
female)
Administration
method
p.o.
gavage
Timing of
administration
Administration
period
13 weeks old
6 days
Dose
Test method and result
References
0, 0.02, 0.2, 2, 20,
200 mg/kg/day
The testis was weighed and
spermatogenic function was tested
when the rats were 14 weeks old
and 18 weeks old.
Daily spermatogenic function as
well as daily spermatogenic
function per testis weight
decreased at 0.02 mg/kg/day or
more of BPA
F1 rats were examined when they
were 90 days old
Testis weight decreased and
number of sperms decreased
Sakaue et
al., 2001
0, 0.01, 0.1, 1.0,
10 ppm
(BPA intake
Corresponding to
0, 0.001-0.004,
0.008-0.038,
0.100-0.391,
0.775-4.022
mg/kg/day)
F1 rats were examined when they
were 90 days old
No changes in the reproductive
organ and accessory reproductive
organ weights, number of sperms,
sperm production rate and
histopathological examination of
testis
Cagen et al,
1999b
0, 3.2, 32, 320
mg/kg/da
y
F0: No abnormality in fertility and
reproductive organ weights
F1: No abnormality in the time of
sexual maturity of females, and
reproductive organ weights of
females and males
No abnormality in reproductive
function of parent animal and
development & growth of
offspring in each generation
Kwon et al,
2000
0, 0.000002,
0.00002, 0.0002,
0.002, 0.02, 0.2, 2
mg/kg/da
y
Rat
Drinking
water
Rat
(Wistar,
female)
Drinking
Rat
(SD,
female)
p.o.
gavage
Rat
(SD)
25
rats/sex/
group
p.o.
gavage
water
Administration
for 8 to 9 weeks
(before mating,
during
pregnancy and
lactation)
10 weeks old.
10 weeks of
administration
(2 weeks before
mating, 2 weeks
during
cohabitation,
21 - 22 days
during
pregnancy and
22 days during
nursing)
From day 11 of
gestation to the
weaning (up to
20 days after
birth)
1 ppm
2-generation
reproductivity
test
Male: 5 weeks
old
Females: 10
weeks old.
From the time
before mating of
F0 (from 10
weeks before
and 5 weeks
before in the
case of males
and females
respectively) up
to the weaning
of F2
0, 0.0002, 0.002,
0.02, 0.2
mg/kg/da
y
357
Sharpe,
1996
Ema et al.,
2001
Bisphenol A
Animal
species
Administration
method
Rat
(SD)
30
rats/sex/
group
By
feeding
Mouse
B6C3F1
Early
preimpla
ntation
embryos
In
culture
medium
0.1%
ethanol
solution
Rat
Wistar
Female
N=
5/group
Drinkin
g Water
Timing of
administration
Administration
period
3-generation
reproductivity
test
Start
administrating
F0 at 7 week old
10 weeks before
the mating of
F0 - 12 weeks
after the
weaning of F3
Method using
embryos culture
system: The
developmental
rates of two-cell
embryos to
eight-cell or to
blastocysts in
the presence of
BPA with or
without 100 nM
Tamoxifen.
During
pregnancy and
21 days after
delivery, only to
the dams
Dose
Test method and result
0, 0.015, 0.3, 4.5,
75, 750, 7,500
ppm
(male: equivalent
to 0, 0.001, 0.02,
0.3, 5, 50, 500
mg/kg/day,
female:
equivalent to 0,
0.0009, 0.018,
0.27, 4.5, 45, 450
mg/kg/day)
Suppression of the body weight
increase in the F1 - F3 generation
parent animals and F3 at 750 ppm
or more.
At 7,500 ppm, decreased
implantations, total number of
offspring and of surviving
offspring in regard to F1 - F3,
decreased ovary weight in F1 - F3
females, degeneration of renal
tubule in the kidney and chronic
hepatitis in the liver of F0 - F2
females.
0, 100 pM, 300
For 24-h incubation :
pM, 1 nM, 3 nM, The rate of development of twocell embryos to eight-cell
10 nM, 10 μM,
embryos was significantly
100μM
increased by 3 nM BPA.
References
Tyl et al.,
2001
Takai et al.,
2001
For 48-h intervals:
The rates of development of twocell embryoes to the blastocyst
stage were significantly increased
by 1 nM and 3 nM BPA, and was
significantly decreased by 100 μ
M BPA.
0, 5 mg/l
(equivalent to 0,
1.5 mg/kg/day)
Rates of development of BPAexposed two-cell embryoes to
eight-cell embryos were not
altered by 100 nM Tamoxifen.
Decreased blastocyst formation by
100 μM BPA was cancelled by
100 nM Tamoxifen.
F1 (6-week age) both male and
female, brain morphological
analysis and behavior test.
0 mg/ l: Female offspring showed
higher motor activity, lower
avoidance memory, and larger
locus ceruleus (LC) than the male.
5 mg/ l: Disappearance of
sexually dimorphic pattern both in
behavior and LC
358
Kubo et al.,
2001
Bisphenol A
Attachment-5 Repeated dose toxicity test results
Animal
species
Mouse
(B6C3F1,
male and
female)
Mouse
(B6C3F1,
male and
female)
Administr
ation
method
By
feeding
By
feeding
Administrati
on period
Dose
Result
References
6 weeks old
13 weeks
0, 2,000, 5,000, 10,000,
20,000, 40,000 ppm
(Male : Corresponding to
0, 500, 1,000, 2,200,
5,500, 14,600 mg/kg/day
Female : Corresponding to
0, 600, 1,300, 2,500,
6,300, 22,000 mg/kg/day)
Furukawa
et al., 1994
5 weeks old
2 years
Male: 0, 1,000, 5,000 ppm
(Corresponding to 0, 150,
750 mg/kg/day)
Female: 0, 5,000, 10,000
ppm
(Corresponding to 0, 750,
1,500 mg/kg/day)
5,000 pm or more:
Decreased RBC and Ht
10,000 ppm or more:
Decreased Hb, cyst-like
dilation of urinary tubule,
fibrous hyperplasia in the
periphery of cysts,
degeneration and
regeneration of tubular
epithelium and increased
vitreous urinary casts
20,000 ppm or more:
Suppression of body
weight increase, increased
liver weight, decreased
ovary weight, fibrous
osteodystrophy of femoral
bone and sternum and
atrophy of myofibrils
40,000 ppm: Leanness,
death assumed to be
attributable to food
rejection, increased
platelet count and kidney
weight, and enhanced
extramedullary
hematopoiesis in spleen
Increased polyploidy
hepatocytes in liver of males
at 1,000 ppm or more,
decreased body weight of
males at 5,000 ppm and that
of females at 5,000 ppm or
more
359
NTP, 1982
Bisphenol A
Animal
species
Rat
(SD, male
and
female)
Administr
ation
method
p.o. by
gavage
(OECD
enhanced
TG 407)
Administrati
on period
Dose
Result
References
5 weeks old
28 - 32 days
0, 40, 200, 1,000
mg/kg/day
CERI,
2000
0, 250, 500, 1,000, 2,000,
4,000 ppm
(Corresponding to 0, 13,
25, 50, 100, 200
mg/kg/day)
0, 1,000, 2,000 ppm
(Male: Corresponding to
74, 148 mg/kg/day,
Female: Corresponding to
74, 135 mg/kg/day)
0, 10, 50, 150 mg/m3
200 mg/kg/day or more;
Inhibition on body weight
increase, decreased ALT
(males only),
cholinesterase and T3
(these 2 parameters in
females only), swollen
cecum, decreased heart
weight, hyperplasia of
intestinal mucosa, dilation
of intestinal lymph
vessels
1,000 mg/kg/day: Death,
persistent telogen in
estrus cycle test,
prolonged partial
activated thromboplastin
time, decreased Hb
concentration and Ht,
increased r-GTP and
alkaliphosphatase,
decreased triglyceride,
increased chlorine, T4,
liver and kidney weights,
decreased prostate and
thyroid gland weights,
degeneration and necrosis
of urinary tubule in
kidney
Decreased body weight at
1,000 ppm or more.
Hyaline clots (males only) in
bladder and dilation of
cecum at 250 ppm or more
Decreased body weight and
food consumption at
1,000 ppm or more
Rat
(F344,
male and
female)
By
feeding
Age
unknown
91 days
Rat
(F344,
male and
female)
By
feeding
5 weeks old
2 years
Rat
(F344,
male and
female)
10
rats/group
Inhalation
Age
unknown
Exposure for
6
hours/days,
for 9 days
Slight irritation in anterior
nasal cavity at 50 mg/m2 or
more.
Decreased body weight of
males at 150 mg/m3
NTP, 1982
NTP, 1982
German
Chemical
Society,
1995
Dow
Chemicals
Co.,
1985a, b
360
Bisphenol A
Animal
species
Rat
(F344,
male and
female)
10
rats/group
Dog
(beagle)
Administration
method
Inhalation
By
feeding
Administration period
Age
unknown
Exposure for
6 hours/
days, 5 days/
weeks,
for 13 weeks
Age
unknown
90 days
Dose
0, 10, 50, 150 mg/m3
0, 1,000, 3,000, 9,000
ppm
(Corresponding to 0, 25,
75, 225 mg/kg/day)
Result
Decreased body weight,
dilation of cecum,
inflammation in nasal cavity
and respiratory mucosa and
hyperplasia of squamous
epithelium at 50 mg/m3 or
more.
Decreased liver and kidney
weights at 150 mg/m3
Increased liver weight at
9,000 ppm
References
German
Chemical
Society,
1995
Dow
Chemicals
Co., 1988
German
Chemical
Society,
1995
General
Electric,
1976b
361