Endocrine activity of alternatives to BPA found in

Transcription

YRTPH 3213
No. of Pages 10, Model 5G
8 January 2015
Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx
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Contents lists available at ScienceDirect
Regulatory Toxicology and Pharmacology
journal homepage: www.elsevier.com/locate/yrtph
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Endocrine activity of alternatives to BPA found in thermal paper
in Switzerland
Daniela M. Goldinger a,1, Anne-Laure Demierre a,⇑,1, Otmar Zoller b, Heinz Rupp b, Hans Reinhard b,
Roxane Magnin b, Thomas W. Becker c, Martine Bourqui-Pittet a
a
b
c
Federal Office of Public Health, Division Chemical Products, 3003 Bern, Switzerland
Federal Food Safety and Veterinary Office, 3003 Bern, Switzerland
PhaToCon (Pharm/Tox Concept) GmbH, 82152 Martinsried, Germany
a r t i c l e
i n f o
Article history:
Received 21 October 2014
Available online xxxx
Keywords:
BPA alternatives
Thermal paper
Endocrine activity
H295R steroidogenesis assay
VirtualToxLab™
a b s t r a c t
Alternatives to bisphenol A (BPA) are more and more used in thermal paper receipts. To get an overview
of the situation in Switzerland, 124 thermal paper receipts were collected and analyzed. Whereas BPA
was detected in most samples (n = 100), some alternatives, namely bisphenol S (BPS), PergafastÒ 201
and D-8 have been found in respectively 4, 11 and 9 samples. As no or few data on their endocrine activity
are available, these chemicals and bisphenol F (BPF) were tested in vitro using the H295R steroidogenesis
assay. 17b-Estradiol production was induced by BPA and BPF, whereas free testosterone production was
inhibited by BPA and BPS. Both non-bisphenol substances did not show significant effects. The binding
affinity to 16 proteins and the toxicological potential (TP) were further calculated in silico using VirtualToxLab™. TP values lay between 0.269 and 0.476 and the main target was the estrogen receptor b
(84.4 nM to 1.33 lM). A substitution of BPA by BPF and BPS should be thus considered with caution, since
they exhibit almost a similar endocrine activity as BPA. D-8 and PergafastÒ 201 could be alternatives to
replace BPA, however further analyses are needed to better characterize their effects on the hormonal
system.
Ó 2015 Published by Elsevier Inc.
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1. Introduction
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Bisphenol A (BPA) is a high production volume chemical. It is
widely used as monomer in the manufacturing of polymer products such as polycarbonate, epoxy resins and also as an additive
in plastics. Additionally, BPA is found in the paper industry as color
developer in thermal paper (Geens et al., 2012a).
Human exposure to BPA is widespread and many data are available suggesting adverse effects at low-dose. Its association with
several diseases is frequently discussed (Geens et al., 2012a;
Vandenberg et al., 2010, 2013), and its endocrine activity has been
widely investigated, including e.g. effects on steroidogenesis
(Zhang et al., 2011). However, many uncertainties remain and
controversial discussions are still ongoing.
Due to the ubiquity of BPA, its hormonal activity and the related
uncertainties, EFSA recently focused a Scientific Opinion on this
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⇑ Corresponding author at: Federal Office of Public Health, Division Chemical
Products, Schwarzenburgstrasse 165, CH-3003 Bern, Switzerland. Fax: +41 58 464
90 34.
E-mail address: [email protected] (A.-L. Demierre).
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These authors contributed equally to this work.
substance (EFSA, 2013, 2014). There, EFSA evaluated BPA exposure
and the risks for human health. In most cases, diet was found to be
the main source of exposure, whereas thermal paper was the second source (EFSA, 2013). Several reports from the USA (US EPA,
2014), Denmark (Lassen et al., 2011) and Sweden (KEMI, 2012) also
identified thermal paper as a source of exposure to BPA. Moreover,
some countries or states such as Japan (2001), Taiwan (2011) and
Connecticut, USA (2015) prohibited the use of BPA in thermal
paper following the precautionary principle. In June 2014, France
submitted a restriction proposal to the European Chemicals Agency
(ECHA) to ban the use of BPA in thermal paper in concentrations
equal or higher than 0.02% (ECHA, 2014). Accordingly, safer alternatives to replace BPA are required.
At the beginning of 2014 the US EPA published a final report on
‘‘Bisphenol A alternatives in thermal paper’’ (US EPA, 2014), identifying nineteen substances as potential BPA substitutes. These nineteen substances were selected according to their physical and
chemical properties and/or because they are already commercially
used. No clearly safer alternative to BPA could be identified in the
report, as only limited toxicological information on these replacement substances is available. Analyses or structural similarities
of most of these alternatives led to some doubts concerning their
http://dx.doi.org/10.1016/j.yrtph.2015.01.002
0273-2300/Ó 2015 Published by Elsevier Inc.
Q1 Please cite this article in press as: Goldinger, D.M., et al. Endocrine activity of alternatives to BPA found in thermal paper in Switzerland. Regul. Toxicol.
Pharmacol. (2015), http://dx.doi.org/10.1016/j.yrtph.2015.01.002
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D.M. Goldinger et al. / Regulatory Toxicology and Pharmacology xxx (2015) xxx–xxx
innocuity. Indeed, it was reported that most of them have moderate to high probability to impact human health or aquatic toxicology endpoints.
To obtain an overview of the Swiss situation, we performed a
market analysis of thermal paper receipts in the region of Bern,
Switzerland. Between September 2013 and January 2014, 124 thermal paper receipts were randomly sampled and analyzed. We
focused the follow-up studies on the alternative substances found
during this Swiss market analysis. Another potential alternative
bisphenol F (BPF) has been included to these analyses. The studies
included BPA as control compound, bisphenol S (BPS), BPF, D-8
(also known as WinCon-8) and PergafastÒ 201 (Table 1).
Data about these alternatives are scarce, particularly for the
non-bisphenols, and are mostly limited to in vitro studies. BPS
has been shown to bind to the estrogen receptor (ER) in vitro
(Laws et al., 2006; Yamasaki et al., 2004), elicit estrogen induced
gene transcription (Chen et al., 2002; Nishihara et al., 2000) and
induce cell proliferation in MCF7 cancer cells (Kuruto-Niwa et al.,
2005). There is only one in vivo uterotrophic study available suggesting a potential for estrogenic activity (Yamasaki et al., 2004).
The available in vitro and in silico assays indicate that BPF can
bind to estrogen receptors (ERs) (Blair et al., 2000; Coleman
et al., 2003; Yamasaki et al., 2004), trigger estrogen induced gene
transcription (Chen et al., 2002; Hashimoto and Nakamura, 2000;
Miller et al., 2001), induce progesterone receptors (PgR)
(Kitamura et al., 2005; Perez et al., 1998), and induce cell proliferation in MCF7 cancer cells (Coleman et al., 2003; Stroheker et al.,
2004). Additionally, BPF has been shown to exhibit in vitro androgenic and anti-androgenic effects (Cabaton et al., 2009; Kitamura
et al., 2005; Stroheker et al., 2004). BPF was shown to have estrogenic and anti-estrogenic activity in some in vivo studies with
female rats (Akahori et al., 2008).
For D-8 there is only limited evidence of endocrine activity. D-8
was negative for estrogenic activity in two ER binding assays and
one competitive ER binding assay (Terasaki et al., 2007), and positive for anti-estrogenicity in a competitive binding assay in the
presence of 17b-estradiol (Kuruto-Niwa et al., 2005).
There is only one in vitro study available suggesting that PergafastÒ 201 is non-estrogenic with a relative potency substantially
low compared to 17b-estradiol (US EPA, 2014).
The H295R steroidogenesis assay is part of the Conceptual
Framework of the OECD for the testing and assessment of endocrine disrupting chemicals (OECD, 2011). This assay allows the
detection of change in the level of both estradiol and testosterone.
The alteration in the concentration of hormones can result from
different interactions of the chemicals with steroidogenic function,
such as binding to an enzyme involved in the steroidogenesis pathway, modulating the steroid metabolism, or affecting the transcription of the enzymes, for example by binding the chemical to
hormone receptor.
In this study, we evaluated in a first step which alternatives to
BPA are present in thermal paper receipts on the Swiss market.
Secondly the found chemicals and BPF were tested in vitro for their
influence on the 17b-estradiol and free testosterone level using the
H295R steroidogenesis assay under GLP conditions. In parallel,
binding affinity to 16 proteins involved in the hormonal system
and the toxicological potential of the substances were determined
using the in silico tool VirtualToxLab™.
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2. Materials and methods
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2.1. Chemicals and materials
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D-8 was obtained from Connect Chemicals GmbH (Ratingen,
Germany) and PergafastÒ 201 from BASF (Bradford, Great Britain).
For the chemical analysis bisphenol A-propane-D6 (BPA-D6) was
purchased from Cambridge Isotope Laboratories (Tewksbury,
USA) and bisphenol S (BPS) was from TCI Europe (Zwijndrecht,
Belgium). Methanol, LC–MS ChromasolvÒ, and the following substances for the H295R steroidogenesis assay such as bisphenol A
(BPA), bisphenol S (BPS), bisphenol F (BPF), forskolin and prochloraz were purchased from Sigma Aldrich (St. Louis, USA). Ultrapure
water was obtained from an ElgaPurelab ultra water purification
system (Labtec Services, Villmergen, Switzerland).
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2.2. Market analysis in Switzerland
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Thermal paper receipts (cashier receipts, ATM receipts, parking
tickets, bus tickets etc.) were randomly sampled in Switzerland
between September 2013 and January 2014, mostly in the Bern
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Table 1
List of substances tested.
CAS #
Chemical name
Substance
name
Molecular
formula
80-05-7
2,2-Bis(4-hydroxyphenyl)propane
Bisphenol A (BPA)
C15H16O2
620-92-8
Bis(4-hydroxyphenyl)methane
Bisphenol F (BPF)
C13H12O2
80-09-1
Bis(4-hydroxyphenyl)sulfone
Bisphenol S (BPS)
C12H10O4S
232938-43-1
N-(p-Toluenesulfonyl)-N0 (3-p-toluenesulfonyloxyphenyl)urea
PergafastÒ 201
C21H20N2O6S2
95235-30-6
4-Hydroxyphenyl-40 -isopropoxyphenyl-sulfone
D-8 (WinCon-8)
C15H16O4S
Structure
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area. A subsample of each receipt was tested on a hotplate at
140 °C to confirm it was thermal paper. Thermal paper samples
(n = 124) were wrapped in aluminium foil and kept in the dark
until further processing. Samples were first analyzed for their
BPA content. All receipts without BPA (n = 23) and 14 randomly
selected receipts containing BPA were screened for their content
of possible alternative substances. The main substances detected
in the screening method were quantified during a further analysis
step.
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2.3. Extraction of thermal paper
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The extraction was carried out according to Geens et al.
(2012b). About 25 mg of thermal paper were cut into small strips,
accurately weighted and suspended in 2 mL of methanol. Extraction was performed by two cycles of vortex (30 s) followed by sonication (10 min). The solution was diluted 50 times with methanol
(dilution 1). This solution was diluted with water by a factor of 2
(1 + 1) and used for the screening assay. Dilution 1 was further
diluted by a factor of 1000 with methanol for the quantitation of
PergafastÒ 201, D-8 and BPS. For quantitation of BPA 0.05 mL of
dilution 1 was mixed with 0.05 mL of BPA-D6 internal standard
(IS) solution. If the concentration was above the calibration range,
dilution 1 was further diluted by a factor of two and reanalyzed.
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2.4. Screening of chemicals by liquid chromatography high resolution
mass spectrometry (LC–HRMS)
The chromatographic system consisted of a Shimadzu Prominence binary gradient system (Shimadzu, Reinach, Switzerland),
degasser, auto sampler and column heater. Chromatographic
separations were performed on a Kromasil C18 (125 2 mm,
3.5 lm particle size) analytical column (Macherey–Nagel, Düren,
Germany). The flow rate was 0.2 mL/min and the column temperature maintained at 30 °C. A gradient program was used starting
with 10% methanol in water and ramped linearly over the course
of 12 min to 90% methanol, held for 5 min at this condition, then
re-equilibrated for 2.5 min at 90% water. Injection volume was
5 lL resulting in 500 pg of standards on column.
Mass spectrometric detection was achieved with a Bruker
maXis 4G Qq-TOF mass spectrometer (Bruker, Bremen, Germany),
equipped with an electrospray ionization interface operated in
negative ion mode. Source parameters were: plate offset 500 V,
capillary voltage 4.5 kV, dry temperature 200 °C, nebulizer gas
pressure 150 kPa and nitrogen dry gas flow rate 8 L/min. Internal
calibration was achieved by incorporating sodium formate solution
as calibrant at the beginning of every run with a loop injection. For
instrument control, data acquisition and processing, Compass 1.5
and TargetAnalysis 1.3 were used.
Identification of compounds was accomplished by high resolution mass determination (deviation 61 mDa allowed); positives
were additionally verified by comparing retention time to the
standard compound.
The screening method was designed to detect 17 of the 19 alternative substances mentioned in the report of US EPA (2014) (as no
adequate information was available for 2 patented substances) and
13 additional bisphenols. Table S1 of Supplementary data lists the
targeted analytes under investigation.
2.5. Quantitation of chemicals by liquid chromatography tandem mass
spectrometry (LC–MS/MS)
The chromatographic system consisted of a Shimadzu UFLC binary gradient system (Shimadzu, Reinach, Switzerland) with pumps
LC-30AD, vacuum degasser DGU-20A, thermostated column
compartment CTO-20 and autosampler SIL-30A. Separations were
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performed on a Kinetex XB-C18, 100A (100 2.1 mm, 1.7 lm particle size) with a precolumn (Phenomenex, Torrance, USA). The
injection volume was 1.0 lL and the column temperature maintained at 50 °C. A gradient program was used starting with 50%
methanol in water and ramped linearly over the course of 3 min
to 95% methanol, held for 1 min at this condition, then re-equilibrated for 3 min at 50% methanol. The flow rate was 0.3 mL/min
for the BPA determination and 0.4 mL/min for the determination
of PergafastÒ 201, BPS and D-8.
MS/MS analysis was carried out on an API 5000 system (AB
Sciex, Framingham, USA) equipped with a turboIon spray source
(ESI). The following instrumental settings were used: source tem- Q3
perature 600 °C, curtain gas 31, collision gas 7, gas 1 50, gas 2 70,
ionspray 4500 V. Measurements were carried out using multiple
reaction monitoring (MRM) in negative mode. The used MRM transitions and dwell times are listed in Table S2 of Supplementary
data.
BPA was quantified using the internal standard method and
linear regression. A five point calibration curve was used with calibration points between 145 and 2900 ng/mL, corresponding to
about 1.16–23.2 mg of BPA in the paper using our usual procedure.
For quantification of PergafastÒ 201, D-8 and BPS external calibration was used.
Individual stock solutions of 1 mg/mL for each compound were
used. Mixed working solutions of 0.5, 1.0, 2.5, 5 and 10 ng/mL in
methanol were used for the analysis, corresponding to a range of
about 2–40 mg/g substance in the paper. As PergafastÒ 201 was
often unstable in methanolic solution at room temperature, standards and extracts for determination of PergafastÒ 201 were
always freshly produced and processed in less than 20 h.
In order to confirm the concentration of PergafastÒ 201 in the
thermal papers, this substance was additionally quantified with
an LC/UV method. This method and the performance of the quantitation are detailed in Supplementary data.
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2.6. H295R steroidogenesis assay
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Steroidogenesis assay was performed under GLP conditions, following the OECD TG 456 (H295R Steroidogenesis Assay). It has
been done at MDS (Molecular Diagnostic Services) Inc., San Diego,
USA, under coordination and supervision of PhaToCon GmbH,
Martinsried, Germany.
The effects of the substances on the level of estradiol and testosterone were tested in the H295R human adrenocortical carcinoma
cell line (ATCC No. CRL-2128, Manassas, USA) as previously
described (Hecker et al., 2011; OECD, 2011).
In brief, cells were cultured in T75 tissue culture flasks (Thermofisher Cat #156499, Waltham, USA) at 37 °C and 5% CO2. Cells
were seeded in 48-well plates (Corning Cat #353078, Tewksbury,
USA) at a density of 106 cells per well and incubated at 37 °C and
5% CO2. After 24 h, cells were exposed in triplicate for 48 h to various concentrations of the test substances dissolved in 0.1% DMSO.
Control wells contained the same amount of DMSO (0.1%) as
exposed cells. Concentrations of BPA, BPF, BPS, D-8 and PergafastÒ
201 were 0.1, 0.3, 1, 3, 10, 30 and 100 lM. Forskolin (an inducer of
steroid hormone production) and prochloraz (an inhibitor) were
used as positive controls at concentration of 1 and 10 lM for
forskolin, and 0.1 and 1 lM for prochloraz. After 48 h of exposure,
medium was collected from each well and stored at 80 °C for hormone analysis. Complete medium was added back to each well and
cell viability was analyzed using CellTiter 96Ò AQueous One Solution Cell Proliferation Assay (Promega Cat #G3581, Madison, USA).
17b-Estradiol was measured using the Estradiol Ultrasensitive
ELISA (ALPCO Cat #20ESTHUU-E01, Salem, USA). The analytical sensitivity of the assay was 1.4 pg/mL, calibration range was
0–200 pg/mL. All data generated were within the validated range
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of the assay and met the validity criteria. Samples were diluted in
the range of 1:9–1:45 to reach the validated range of the assay. Free
testosterone was measured using the Coat-a-Count Free
Testosterone solid-phase 125I RIA (Siemens Healthcare Diagnostics,
Cat #TKTF2, Tarrytown, USA). The analytical sensitivity of the assay
was 0.15 pg/mL, calibration range was 0.55–50 pg/mL. All data generated were within the validated range of the assay and met the
validity criteria. Data processing and statistical analysis of estradiol
and testosterone values was performed as described below.
The positive controls forskolin and prochloraz behaved as
expected. Data can be found in Table S3 of Supplementary
materials.
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2.7. VirtualToxLab™
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VirtualToxLab™ is an in silico tool which was used to predict the
endocrine and metabolic disruption potential of BPA, BPF, BPS, D-8
and PergafastÒ 201. It calculates the toxic potential (TP) and the
binding affinity (binding constant K) of any molecule to 16 proteins: 10 receptors (androgen, estrogen a, estrogen b, glucocorticoid, liver X, mineralocorticoid, progesterone, peroxisome
proliferator-activated receptor c (PPARc), thyroid a and thyroid
b), 4 members of the cytochrome P450 enzyme family (1A2, 2C9,
2D6 and 3A4), 1 transcription factor (aryl hydrocarbon receptor)
and 1 potassium ion channel (hERG). The VirtualToxLab™ concept
is described in Vedani et al. (2014).
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2.8. Statistical analysis
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Hormone data from the H295R steroidogenesis assay was illustrated graphically with GraphPadÒ Prism 5 (GraphPad Software,
San Diego, USA). Due to the small number of replicates, normality
and variance were evaluated on the combined dataset for the three
assays. Data distribution for normality was assessed with the
Kolmogorov–Smirnov test and the variance homogeneity with
the Bartlett test. Differences between treatments were assessed
by analysis of variance (ANOVA one-way) followed by Dunnett’s
test to compare treatment means with respective controls. If the
data was not normally distributed, differences between treatments
were assessed by the Kruskal–Wallis test followed by Dunn’s
multiple comparison test. Results are given as mean ± standard
deviation. Differences were considered significant at p < 0.05.
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3. Results
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In total 124 thermal paper receipts were analyzed. All receipts
contained only one single developer substance in relevant
amounts. The results are summarized in Table 2. BPA was found
most often (range: 5.6–30.4 mg/g), and only three alternative substances namely BPS, PergafastÒ 201 and D-8 were detected in the
range of 3.3–13.2 mg/g. The papers containing D-8 contained also
traces of BPS in the range of 0.01–0.13 mg/g.
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Table 2
Occurrence and concentrations of the different developer substances found in thermal
paper receipts in Switzerland.
Chemical
BPA
BPS
PergafastÒ 201
D-8
Occurrence
Range conc.
Median conc.
Mean conc.
n
(%)
(mg/g)
(mg/g)
(mg/g)
100
4
11
9
(81)
(3)
(9)
(7)
5.6–30.4
8.3–12.6
3.3–8.2
3.4–13.2
14.5
10.0
4.6
12.0
13.5
10.2
5.4
11.2
3.2. Cytotoxicity of BPA and its alternatives
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Cytotoxicity was not observed in most of the tested concentrations. Only the highest concentration (100 lM) showed a significant decrease in viability for BPA (23% ± 8.3%), PergafastÒ 201
(43% ± 6.1%) and D-8 (26% ± 5.0%) (Table 3).
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3.3. Effects on steroidogenesis in vitro
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A significant increase of 17b-estradiol concentration was seen
for BPA and BPF (Fig. 1, Table 4). Despite a 23% ± 8.3% drop in viability in the 100 lM treatment with BPA (Table 3), a statistically
significant increase of 17b-estradiol level was observed in a dosedependent manner. Thus, the effect at 100 lM was taken into
account when assessing the overall response of BPA. Although
the lowest observed effect concentration (LOEC) for BPA and BPF
were both at 30 lM, BPF seemed to be more potent than BPA, since
the increase in 17b-estradiol concentration was 15% higher at
30 lM.
Overall, BPS, PergafastÒ 201 and D-8 did not show any significant effects on 17b-estradiol level. Significant effects were only
seen at concentrations that did not meet the viability requirements
(Fig. 1, Table 3, Table 4).
Concerning effects on free testosterone level, a decrease was
seen for BPA and BPS, whereas no significant effects were seen
for BPF and D-8. The LOEC for BPA was observed at 1 lM and for
BPS at 30 lM, indicating that BPA is more potent than BPS
(Fig. 2, Table 4). A significant effect for D-8 was only seen at the
highest concentration that did not meet the viability requirements
(Fig. 2, Table 4). A significant decrease of free testosterone level
was observed with PergafastÒ 201. This substance was not considered as an inhibitor though, since these observations were not
dose-dependent and were near background level (Fig. 2, Table 4).
In conclusion, BPA and BPF were found to increase the level of
17b-estradiol, and BPA and BPS were reported to decrease the free
testosterone concentration. BPS, PergafastÒ 201 and D-8 were
shown to have no effect on the 17b-estradiol level. The free testosterone concentration was not significantly affected by BPF, PergafastÒ 201 and D-8.
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Using VirtualToxLab™ we calculated the toxic potential (TP)
and the binding affinities of BPA, BPF, BPS, PergafastÒ 201 and
D-8 to 16 proteins (Table 5). The TP was derived from the normalized binding affinities towards the 16 target proteins. The values
range from 0 (none) to 1 (extreme) and could be interpreted as
toxic alert. The TP values calculated for BPS, D-8, BPF and BPA lay
between 0.380 and 0.476, showing a moderate risk of binding
the receptors. Only PergafastÒ 201 has a low risk of binding, with
a TP value of 0.269 (Vedani et al., 2014).
The main target for BPA, BPF, BPS and D-8 was the estrogen
receptor b with binding affinity values ranging from 84.4 nM to
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Table 3
Cytotoxicity of BPA, BPF, BPS, PergafastÒ 201 and D-8. Results are expressed as
percentage of control cells (DMSO 0.1%). Data not meeting the viability requirements
are shown in bold.
lM
BPA
BPF
BPS
PergafastÒ 201
D-8
0.1
0.3
1
3
10
30
100
101 ± 2.5
105 ± 0.2
104 ± 2.2
104 ± 2.4
104 ± 1.2
102 ± 2.7
77 ± 8.3
106 ± 3.2
111 ± 3.9
110 ± 1.0
111 ± 3.4
110 ± 5.0
108 ± 7.9
105 ± 6.8
106 ± 2.2
109 ± 2.7
106 ± 0.9
110 ± 2.2
107 ± 6.6
104 ± 3.3
94 ± 6.7
104 ± 3.4
103 ± 3.5
103 ± 5.7
103 ± 1.7
101 ± 5.0
99 ± 3.0
57 ± 6.1
105 ± 4.6
105 ± 2.8
106 ± 6.5
105 ± 6.8
103 ± 7.3
102 ± 2.7
74 ± 5.0
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Fig. 1. Concentration of 17b-estradiol in H295R cell culture medium after 48 h of exposure to BPA, BPF, BPS, PergafastÒ 201 and D-8. Values are expressed as average relative
change ± SD. Asterisks (⁄) indicate statistically significant difference to control (p < 0.05). Shaded bars indicate concentrations that did not meet the viability requirements.
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1.33 lM. PPARc was the main target for PergafastÒ 201 with a
binding affinity of 22 lM.
377
4. Discussion
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379
Currently BPA is the most commonly used color developer in
thermal paper. Many studies investigated the concentration of
380
BPA in such paper (Biedermann et al., 2010; Environmental
Working Group (EWG), 2010; Geens et al., 2012a; KEMI, 2012;
Lassen et al., 2011; Liao and Kannan, 2011; Lu et al., 2013;
Mendum et al., 2011; US EPA, 2014). In these studies BPA was
found with a detection frequency of 44–100% with a concentration
of up to 28 mg/g. Our BPA results (frequency of 81% with a range of
6–30 mg/g) are in line with the detection frequencies and concentrations found worldwide.
In recent years, BPA has increasingly fallen into disrepute and as
consequence, alternative substances have been developed for
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390
YRTPH 3213
8 January 2015
6
Table 4
Summary of steroidogenesis testing. The overall response is presented for each substance’s effect on 17b-estradiol and free testosterone production. LOEC = Lowest observed
effect concentration. Max change = Average maximal change observed for any concentration of the substance.
17b-Estradiol
Bisphenol A
Bisphenol F
Bisphenol S
PergafastÒ 201
D-8
*
Free testosterone
Overall response
LOEC (lM)
Max. change
Overall response
LOEC (lM)
Max. change
Inducer*
Inducer
None
None
None
30
30
–
–
–
1.85
2.80
–
–
–
Inhibitor
None
Inhibitor
None
None
1
–
30
–
–
0.29
–
0.33
–
–
The 100 lM BPA treatment is taken into account as a statistically significant increase in 17b-estradiol was observed despite the low viability of the treated cells.
424
thermal paper. BPA was not found in 19% of the thermal papers
collected in our study. Recently some Swiss retailers announced
stopping the use of thermal papers containing BPA. Therefore, it
was not astonishing to find thermal papers with alternatives. BPS
has been detected in 3% of the collected papers, in a range of
8–13 mg/g, which is comparable to the concentrations found in
the study of Liao et al. (2012).
Our study is one of the first to find substitutes of BPA other than
bisphenols in thermal paper, i.e. D-8 and PergafastÒ 201. Concentrations of D-8 and BPS were in a similar range, reflecting the structural similarity of these 2 substances. The average concentration of
PergafastÒ 201 is a factor of 2.5 lower than the average BPA concentration. The concentration of this substance has been determined by 2 different methods and the stability of the substance
in thermal paper has been confirmed by repetition of the analysis
after 3 months, obtaining similar results (data not shown). This
corresponds to the information in the NICNAS report (NICNAS,
2004), indicating that the concentration should be less than
10 mg/g in the end product.
Traces of BPS have been detected in all thermal papers containing D-8. As this substance is the isopropylether of BPS, the traces
found could be an impurity or decomposition product of technical
D-8.
Thermal papers are recycled (Terasaki et al., 2007). Therefore, as
it has been shown for BPA, these substances can also find their way
back to our daily life in the form of other papers such as journals or
toilet papers (Liao and Kannan, 2011). Accordingly, not only
thermal papers but also other kind of papers should be in the scope
of further investigations. Furthermore the probability that these
substances end up in the aquatic environment is high. Due to the
suspected risks of most BPA alternatives concerning environmental
endpoints, including for D-8 and PergafastÒ 201 (US EPA, 2014),
the consequences of thermal paper recycling for aquatic organisms
should be evaluated.
425
4.2. Effects on steroidogenesis in vitro
426
BPA and BPF led to an increase in 17b-estradiol concentration.
Furthermore all tested bisphenols showed a decrease in free
testosterone level. This decrease was significant for BPA and BPS,
but not for BPF, probably due to the high standard deviation found
for this last substance. Mostly one out of three replicates was not in
line with the other two therefore leading to a higher standard deviation as expected. This observation however was more related to
the assay performance and the study design than to the material
and is not regarded as biologically relevant. The effects on steroidogenesis of BPA have previously also been investigated in the
H295R assay (Rosenmai et al., 2014; Zhang et al., 2011) showing
the same tendencies as we found in our study. In addition,
Rosenmai et al. (2014) also investigated the effects of BPF and
BPS on steroidogenesis. Our results are in line with theirs. They
have also looked at the effect of BPA and its analogs on the other
hormones intermediates of the steroidogenesis pathway.
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441
Interestingly, they observed a significant increase of 17a-OH progesterone level with BPS, whereas BPA did not affect this hormone
(Rosenmai et al., 2014). In the present study, we only focused on
testosterone and 17b-estradiol levels, as the assay is only validated
for these two hormones (OECD, 2011).
The metabolic capability of H295R cell line is unknown, but it is
probably quite limited. Accordingly, substances that need to be
metabolically activated to show endocrine activity could be missed
in this assay (OECD, 2011). BPS was shown to be negative for estrogenic activity in the E-screen assay without metabolic activation
(Hashimoto et al., 2001). However, after metabolic activation
estrogenic activity could be seen. Therefore, it is possible that
BPS first needs metabolic activation to elicit estrogenic activity in
the H295R steroidogenesis assay. This issue has to be further investigated as metabolic activation can take place in the human body.
Up to now there is only limited data available on the endocrine
activity of D-8 and PergafastÒ 201. It was for the first time that the
H295R steroidogenesis assay was conducted with these two
substances.
So far, there is only one in vitro study available showing that
PergafastÒ 201 is non-estrogenic (US EPA, 2014), and it is supported by our analysis. However, we observed a significant
decrease of free testosterone level for the concentrations of
1–10 lM, but not at 30 lM. Although these values were significant,
they were weakened by uncertainties, since they are in the range of
variations of the conducted assay. This interpretation is also supported by the fact that no toxic potential is found with VirtualToxLab™. Therefore, there is no indication that PergafastÒ 201 does
exhibit hormonal activity. However, steroidogenesis could be
affected by several mechanisms such as binding to pathway
enzymes or modulation of metabolism. Thus further tests would
be required in order to make a final decision on this issue.
Although D-8 is structurally related to BPS, neither effect on the
concentration of 17b-estradiol nor free testosterone was found,
suggesting that this substance does not influence steroidogenesis.
D-8 was found to be negative for estrogenic activity in a study
conducted by Terasaki et al. (2007). They also showed that D-8 is
anti-estrogenic. However, this cannot be supported by our results.
As mentioned above, the metabolic activity is very limited in
this test system. Therefore, it could not be excluded that a metabolic activation of PergafastÒ 201 and D-8 would lead to estrogenic
activity.
442
484
This part of the study was conducted to support our findings in
the in vitro H295R steroidogenesis assay. Our prediction showed
that the main target for all compounds except PergafastÒ 201
was the estrogen receptor b (ERb). These findings are supported
by studies found in the literature, where the affinity of BPA to
the ERb is stronger than for ERa (Kolsek et al., 2014). In addition,
it was also shown that both BPF and BPS are positive for ER binding
485
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446
447
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449
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YRTPH 3213
8 January 2015
7
Fig. 2. Concentration of free testosterone in H295R cell culture medium after 48 h of exposure to BPA, BPF, BPS, PergafastÒ 201 and D-8. Values are expressed as average
relative change ± SD. Asterisks (⁄) indicate statistically significant difference to control (p < 0.05). Shaded bars indicate concentrations that did not meet the viability
requirements.
492
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498
using two other QSAR models, i.e. MultiCASE and Leadscope
(Rosenmai et al., 2014).
The binding affinity to the ERb for BPA is about two times stronger than for BPF and nearly 100 times stronger than for BPS. However, compared to 17b-estradiol, the binding affinity of BPA is still
one order of magnitude weaker (Table 5). Due to the binding affinities of BPF to the other receptors, this substance has a TP almost
similar to BPA. D-8 shows weak binding affinities for several receptors. This indicates that BPA and BPF have a higher potential than
BPS and D-8 to show hormonal activity, but at significantly higher
concentrations than the endogenous hormone 17b-estradiol.
PergafastÒ 201 shows less concern.
These in silico results have been confirmed in in vitro assays for
the bisphenols (Rosenmai et al., 2014). However further in vitro
499
500
501
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505
8
⁄
Bisphenol F
Bisphenol S
PergafastÒ 201
D-8
17b-Estradiol
0.577 lM
9.90 lM
29.0 lM
39.9 lM
31.0 lM
Not binding
5.76 lM
0.084 lM
0.203 lM
14.1 lM
64.7 lM
0.430 lM
9.80 lM
2.29 lM
37.5 lM
7.02 lM
0.476
1.96 lM
Not binding
32.7 lM
Not binding
Not binding
Not binding
3.10 lM
0.161 lM
1.57 lM
28.9 lM
56.1 lM
3.78 lM
1.22 lM
0.925 lM
2.55 lM
22.4 lM
0.447
21.1 lM
Not binding
Not binding
Not binding
Not binding
Not binding
25.7 lM
0.742 lM
10.2 lM
49.8 lM
Not binding
6.64 lM
35.1 lM
13.5 lM
78.2 lM
70.2 lM
0.380
Not binding
Not binding
Not binding
Not binding
92.7 lM
97.1 lM
Not binding
Not binding
44.3 lM
Not binding
40.7 lM
31.0 lM
22.0 lM
23.0 lM
99.2 lM
37.5 lM
0.269
7.93 lM
77.3 lM
Not binding
Not binding
Not binding
not binding
2.01 lM
1.33 lM
9.93 lM
3.18 lM
Not binding
3.58 lM
12.9 lM
20.3 lM
8.46 lM
3.83 lM
0.386
0.047 lM
16.1 lM
4.38 lM
21.5 lM
6.16 lM
39.0 lM
0.038 lM
0.004 lM
0.172 lM
3.45 lM
11.6 lM
0.034 lM
13.1 lM
0.206 lM
4.77 lM
2.71 lM
0.574
TP 6 0.3 (low), 0.3 < TP 6 0.6 (moderate), and TP > 0.6 (high).
YRTPH 3213
8 January 2015
Androgen receptor
Aryl hydrocarbon receptor
CYP450 1A2
CYP450 2C9
CYP450 2D6
CYP450 3A4
Estrogen receptor a
Estrogen receptor b
Glucocorticoid receptor
hERG
Liver X receptor
Mineralocorticoid receptor
PPAR c
Progesterone
Thyroid receptor a
Thyroid receptor b
Toxic potential
Bisphenol A
Q4
Table 5
VirtualToxLab™. Binding affinity profile (binding constant K) and estimated toxic potential (TP) of BPA, BPF, BPS, PergafastÒ 201 and D-8. The lower the concentration, the stronger the binding affinity to the target protein. Binding
affinity >100 lM are considered not binding. Strongest binding affinity and toxic potential for each compound are highlighted in bold.
YRTPH 3213
8 January 2015
508
and/or in vivo analyses are required in order to make a concluding
statement on the binding activity of D-8 and PergafastÒ 201 to hormone receptors.
509
5. Conclusion
510
529
Substitution of BPA by its structural analogs BPF and BPS should
be considered with caution, since those bisphenols exhibit almost a
similar endocrine activity as BPA in the tests applied in this study.
Although our study showed that D-8 and PergafastÒ 201 could be
good alternatives for the replacement of BPA with regards to their
in vitro effects on steroidogenesis, further studies are required to
show that there are no adverse effects on the hormonal system.
Indeed substances which influence the steroidogenesis through
the HPG axis (hypothalamic–pituitary–gonadal axis) are not recognized by the H295R steroidogenesis assay (OECD, 2011). Effects on
non-sexual hormones, such as thyroid hormones are also not covered. Endocrine disruptors can also act through other pathways
than receptor binding (Yoon et al., 2014). Further investigation
on the effect of these substances, particularly of PergafastÒ 201
on enzymes involved in steroidogenesis could give clues for the
understanding of the mechanism of toxicity. Finally, there are actually no data available on metabolic activation of D-8 and PergafastÒ
201. Therefore, further tests which cover also these and other
aspects of the hormonal system have to be conducted to perform
a final assessment on the safety of the substitutes.
530
Conflicts of interest
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531
We declare that the authors have no conflicts of interest.
532
Acknowledgments
533
534
We would like to acknowledge Connect Chemicals GmbH and
BASF for kindly providing us D-8 and PergafastÒ 201, respectively.
535
Appendix A. Supplementary data
536
538
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.yrtph.2015.01.
002.
539
References
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548
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554
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559
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Akahori, Y., Nakai, M., Yamasaki, K., Takatsuki, M., Shimohigashi, Y., Ohtaki, M.,
2008. Relationship between the results of in vitro receptor binding assay to
human estrogen receptor A and in vivo uterotrophic assay: comparative study
with 65 selected chemicals. Toxicol. In Vitro 22 (1), 225–231.
Biedermann, S., Tschudin, P., Grob, K., 2010. Transfer of bisphenol A from thermal
printer paper to the skin. Anal. Bioanal. Chem. 398 (1), 571–576.
Blair, R.M., Fang, H., Branham, W.S., Hass, B.S., Dial, S.L., Moland, C.L., Tong, W., Shi,
L., Perkins, R., Sheehan, D.M., 2000. The estrogen receptor relative binding
affinities of 188 natural and xenochemicals: structural diversity of ligands.
Toxicol. Sci. 54 (1), 138–153.
Cabaton, N., Dumont, C., Severin, I., Perdu, E., Zalko, D., Cherkaoui-Malki, M.,
Chagnon, M.C., 2009. Genotoxic and endocrine activities of bis (hydroxyphenyl)
methane (bisphenol F) and its derivatives in the HepG2 cell line. Toxicology 255
(1), 15–24.
Chen, M.-Y., Ike, M., Fujita, M., 2002. Acute toxicity, mutagenicity, and estrogenicity
of bisphenol-A and other bisphenols. Environ. Toxicol. 17 (1), 80–86.
Coleman, K.P., Toscano, W.A., Wiese, T.E., 2003. QSAR models of the in vitro estrogen
activity of bisphenol A analogs. QSAR Comb. Sci. 22 (1), 78–88.
ECHA, 2014. Annex XV Restriction Report, Proposal for Restriction, Substance
Name: 4.40 -isopropylidenediphenol (bisphenol A; BPA). Available at <http://
echa.europa.eu/documents/10162/c6a8003c-81f3-4df6-b7e8-15a3a36baf76>
(accessed October 2014).
EFSA, 2013. Draft scientific opinion on the risks to public health related to the
presence of bisphenol A (BPA) in foodstuff. Part: Exposure assessment. EFSA J.,
1–314, EFSA-Q-2012-00423.
537
9
EFSA, 2014. Draft scientific opinion on the risks to public health related to the
presence of bisphenol A (BPA) in foodstuff. Part: Assessment of human health
risks. EFSA J., 1–532, EFSA-Q-2012-00423.
Environmental Working Group (EWG), 2010. BPA in store receipts. Available at
<http://www.ewg.org/bpa-in-store-receipts> (accessed October 2014).
Geens, T., Aerts, D., Berthot, C., Bourguignon, J.P., Goeyens, L., Lecomte, P., MaghuinRogister, G., Pironnet, A.M., Pussemier, L., Scippo, M.L., Van Loco, J., Covaci, A.,
2012a. A review of dietary and non-dietary exposure to bisphenol-A. Food
Chem. Toxicol. 50 (10), 3725–3740.
Geens, T., Goeyens, L., Kannan, K., Neels, H., Covaci, A., 2012b. Levels of bisphenol-A
in thermal paper receipts from Belgium and estimation of human exposure. Sci.
Total Environ. 435–436, 30–33.
Hashimoto, Y., Moriguchi, Y., Oshima, H., Kawaguchi, M., Miyazaki, K., Nakamura,
M., 2001. Measurement of estrogenic activity of chemicals for the development
of new dental polymers. Toxicol. In Vitro 15 (4), 421–425.
Hashimoto, Y., Nakamura, M., 2000. Estrogenic activity of dental materials and
bisphenol-A related chemicals in vitro. Dent. Mater. J. 19 (3), 245–262.
Hecker, M., Hollert, H., Cooper, R., Vinggaard, A.M., Akahori, Y., Murphy, M.,
Nellemann, C., Higley, E., Newsted, J., Laskey, J., 2011. The OECD validation
program of the H295R steroidogenesis assay: phase 3. Final inter-laboratory
validation study. Environ. Sci. Pollut. Res. 18 (3), 503–515.
KEMI, 2012. Bisfenol A i kassakvitton– rapport från ett regeringsuppdrag. KEMI4/12,
1–58.
Kitamura, S., Suzuki, T., Sanoh, S., Kohta, R., Jinno, N., Sugihara, K., Yoshihara, S.,
Fujimoto, N., Watanabe, H., Ohta, S., 2005. Comparative study of the endocrinedisrupting activity of bisphenol A and 19 related compounds. Toxicol. Sci. 84
(2), 249–259.
Kolsek, K., Mavri, J., Sollner Dolenc, M., Gobec, S., Turk, S., 2014. Endocrine
disruptome – an open source prediction tool for assessing endocrine disruption
potential through nuclear receptor binding. J. Chem. Inf. Model. 54 (4), 1254–
1267.
Kuruto-Niwa, R., Nozawa, R., Miyakoshi, T., Shiozawa, T., Terao, Y., 2005. Estrogenic
activity of alkylphenols, bisphenol S, and their chlorinated derivatives using a
GFP expression system. Environ. Toxicol. Pharmacol. 19 (1), 121–130.
Lassen, C., Mikkelsen, S.H., Brandt, U.K., 2011. Migration of Bisphenol A from Cash
Register Receipts and Baby Dummies. Environmental Protection Agency, 1–67.
Laws, S.C., Yavanhxay, S., Cooper, R.L., Eldridge, J.C., 2006. Nature of the binding
interaction for 50 structurally diverse chemicals with rat estrogen receptors.
Toxicol. Sci. 94 (1), 46–56.
Liao, C., Kannan, K., 2011. Widespread occurrence of bisphenol A in paper and paper
products: implications for human exposure. Environ. Sci. Technol. 45 (21),
9372–9379.
Liao, C., Liu, F., Kannan, K., 2012. Bisphenol S, a new bisphenol analogue, in paper
products and currency bills and its association with bisphenol a residues.
Environ. Sci. Technol. 46 (12), 6515–6522.
Lu, S.Y., Chang, W.J., Sojinu, S.O., Ni, H.G., 2013. Bisphenol A in supermarket receipts
and its exposure to human in Shenzhen, China. Chemosphere 92 (9),
1190–1194.
Mendum, T., Stoler, E., VanBenschoten, H., Warner, J.C., 2011. Concentration of
bisphenol A in thermal paper. Green Chem. Lett. Rev. 4 (1), 81–86.
Miller, D., Wheals, B.B., Beresford, N., Sumpter, J.P., 2001. Estrogenic activity of
phenolic additives determined by an in vitro yeast bioassay. Environ. Health
Perspect. 109 (2), 133.
NICNAS, 2004. Pergafast 201, full public report, file No.: STD/1058. Available at
<http://www.nicnas.gov.au/__data/assets/pdf_file/0017/10394/
STD1058FR.pdf> (accessed October 2014).
Nishihara, T., Nishikawa, J.I., Kanayama, T., Dakeyama, F., Saito, K., Imagawa, M.,
Takatori, S., Kitagawa, Y., Hori, S., Utsumi, H., 2000. Estrogenic activities of 517
chemicals by yeast two-hybrid assay. J. Health Sci. 46 (4), 282–298.
OECD, 2011. OECD Guidelines for the Testing of Chemicals, Section 4: Test No. 456:
H295R Steroidogenesis Assay. Available at <http://www.oecd-ilibrary.org/
docserver/download/9745601e.pdf?expires=1410345422&id=id&accname=
guest &checksum=AF893851A46C6FB1A9F795BD800E41F7> (accessed October
2014).
Perez, P., Pulgar, R., Olea-Serrano, F., Villalobos, M., Rivas, A., Metzler, M., Pedraza,
V., Olea, N., 1998. The estrogenicity of bisphenol A-related diphenylalkanes
with various substituents at the central carbon and the hydroxy groups.
Environ. Health Perspect. 106 (3), 167.
Rosenmai, A.K., Dybdahl, M., Pedersen, M., van Vugt-Lussenburg, B.M.A., Wedebye,
E.B., Taxvig, C., Vinggaard, A.M., 2014. Are structural analogues to bisphenol A
safe alternatives? Toxicol. Sci. 139 (1), 35–47.
Stroheker, T., Picard, K., Lhuguenot, J.C., Canivenc-Lavier, M.C., Chagnon, M.C., 2004.
Steroid activities comparison of natural and food wrap compounds in human
breast cancer cell lines. Food Chem. Toxicol. 42 (6), 887–897.
Terasaki, M., Shiraishi, F., Fukazawa, H., Makino, M., 2007. Occurrence and
estrogenicity of phenolics in paper GÇÉ recycling process water: pollutants
originating from thermal paper in waste paper. Environ. Toxicol. Chem. 26 (11),
2356–2366.
US EPA, 2014. Bisphenol A alternatives in thermal paper, Final Version. Available at
<http://www.epa.gov/dfe/pubs/projects/bpa/bpa-report-complete.pdf>
(accessed October 2014).
Vandenberg, L.N., Chahoud, I., Heindel, J.J., Padmanabhan, V., Paumgartten, F.J.,
Schoenfelder, G., 2010. Urinary, circulating, and tissue biomonitoring studies
indicate widespread exposure to bisphenol A. Environ. Health Perspect.,
1055–1070.
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
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589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
621
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625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
646
647
648
649
YRTPH 3213
8 January 2015
10
650
651
652
653
654
655
656
657
Vandenberg, L.N., Hunt, P.A., Myers, J.P., vom Saal, F.S., 2013. Human exposures to
bisphenol A: mismatches between data and assumptions. Rev. Environ. Health
28 (1), 37–58.
Vedani, A., Dobler, M., Hu, Z., Smiesko, M., 2014. OpenVirtualToxLab – A platform
for generating and exchanging in silico toxicity data. Toxicol. Lett..
Yamasaki, K., Noda, S., Imatanaka, N., Yakabe, Y., 2004. Comparative study of the
uterotrophic potency of 14 chemicals in a uterotrophic assay and their receptorbinding affinity. Toxicol. Lett. 146 (2), 111–120.
Yoon, K., Kwack, S.J., Kim, H.S., Lee, B.M., 2014. Estrogenic endocrine-disrupting
chemicals: molecular mechanisms of actions on putative human diseases. J.
Toxicol. Environ. Health Part B 17 (3), 127–174.
Zhang, X., Chang, H., Wiseman, S., He, Y., Higley, E., Jones, P., Wong, C.K., AlKhedhairy, A., Giesy, J., Hecker, M., 2011. Bisphenol A disrupts steroidogenesis
in human H295R cells. Toxicol. Sci. 121, 320–327.
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