Content uploaded by Matthew A Stoner
Author content
All content in this area was uploaded by Matthew A Stoner on Oct 17, 2014
Content may be subject to copyright.
Estrogenic chemicals often leach from BPA-free
plastic products that are replacements for
BPA-containing polycarbonate products
Bittner et al.
Bittner et al. Environmental Health 2014, 13:41
http://www.ehjournal.net/content/13/1/41
R E S E A R C H Open Access
Estrogenic chemicals often leach from BPA-free
plastic products that are replacements for
BPA-containing polycarbonate products
George D Bittner
1,2*
, Chun Z Yang
1
and Matthew A Stoner
1
Abstract
Background: Xenobiotic chemicals with estrogenic activity (EA), such as bisphenol A (BPA), have been reported to
have potential adverse health effects in mammals, including humans, especially in fetal and infant stages. Concerns
about safety have caused many manufacturers to use alternatives to polycarbonate (PC) resins to make hard and
clear, reusable, plastic products that do not leach BPA. However, no study has focused on whether such BPA-free
PC-replacement products, chosen for their perceived higher safety, especially for babies, also release other chemicals
that have EA.
Methods: We used two, well-established, mammalian cell-based, assays (MCF-7 and BG1Luc) to assess the EA of
chemicals that leached into over 1000 saline or ethanol extracts of 50 unstressed or stressed (autoclaving,
microwaving, and UV radiation) BPA-free PC-replacement products. An EA antagonist, ICI 182,780, was used to confirm
that agonist activity in leachates was due to chemicals that activated the mammalian estrogen receptor.
Results: Many unstressed and stressed, PC-replacement-products made from acrylic, polystyrene, polyethersulfone, and
Tritan™resins leached chemicals with EA, including products made for use by babies. Exposure to various forms of UV
radiation often increased the leaching of chemicals with EA. In contrast, some BPA-free PC-replacement products made
from glycol-modified polyethylene terephthalate or cyclic olefin polymer or co-polymer resins did not release chemicals
with detectable EA under any conditions tested.
Conclusions: This hazard assessment survey showed that many BPA-free PC- replacement products still leached
chemicals having significant levels of EA, as did BPA-containing PC counterparts they were meant to replace. That is,
BPA-free did not mean EA-free. However, this study also showed that some PC-replacement products did not leach
chemicals having significant levels of EA. That is, EA-free PC-replacement products could be made in commercial
quantities at prices that compete with PC-replacement products that were not BPA-free. Since plastic products often
have advantages (price, weight, shatter-resistance, etc.) compared to other materials such as steel or glass, it is not
necessary to forgo those advantages to avoid release into foodstuffs or the environment of chemicals having EA that
may have potential adverse effects on our health or the health of future generations.
Keywords: BG1Luc, Bisphenol A, BPA, Estrogenic activity, MCF-7, BG1Luc, Polycarbonate plastic, Human health
Background
Plastic resins are made by polymerizing one or more
monomers in the presence of additives such as antioxi-
dants, initiators, catalysts, thermal stabilizers, etc. More
chemicals such as other antioxidants, thermal stabilizers,
plasticizers, impact modifiers, clarifiers, colorants, mold
release agents and rheology modifiers are then often
added to the resin that is then heated and molded in
various stages to make a product, or a single piece of
a multi-piece product, each often composed of differ-
ent chemicals. The final plastic product may receive
yet-more chemical additives such as inks. All such ad-
ditives are typically not part of the more chemically-
tightly-bound, polymerized-monomer backbone of the
plastic product. Since polymerization is not complete,
* Correspondence: gbittner@certichem.com
1
CertiChem, Inc., 11212 Metric Blvd, Suite 500, Austin, TX, USA
2
Department of Neuroscience, The University of Texas, Austin, TX, USA
© 2014 Bittner et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Bittner et al. Environmental Health 2014, 13:41
http://www.ehjournal.net/content/13/1/41
unpolymerized monomers such as bisphenol A (BPA)
can leach from the finished product, as can any additive.
Furthermore, the high heat and other stresses of the
manufacturing processes often generate additional leach-
able “degradation chemicals”that might have EA [1-3].
Until recently, many reusable, hard and clear, plastic
products were commonly synthesized using BPA, a
chemical known to have estrogenic activity (EA) that
leaches from polycarbonate (PC) plastic products [4-6].
PC plastics were recently not recommended for use as
baby products by the US FDA and such use has also
been limited or banned in some other jurisdictions such
as Canada, Minnesota and some EU countries; PC plas-
tics are now not as widely used as they were several
years ago in many consumer products [6,7]. However,
PC-replacement products made from other BPA-free
polymers might also release monomers having EA. Such
BPA-free polymers would include acrylic, cyclic olefin
copolymer (COC), cyclic olefin polymer (COP), glycol-
modified polyethylene terephthalate (PETG), polyether
sulfone (PES), polystyrene (PS), Tritan™copolyester [3]
and BPS [7,8] resins. PC-replacement products made
from such resins might also release other additives
that exhibit EA used in their manufacture such as
triphenyl phosphate (TPP) or degradation chemicals such
as phenols [3,9].
Chemicals are said to have EA if they mimic in vitro
and/or in vivo actions of naturally occurring estrogens
such as 17β-estradiol (E2). EA is the most-studied form
of such endocrine disruptor activity [6,10-12]. Chemicals
with EA bind to one or more estrogen receptor (ER)
subtypes [8,13,14], and can produce various adverse
health effects in mammals, such as early menarche, re-
duced sperm counts and other altered functions of re-
productive organs, obesity, and increased rates of some
cancers [6,12,15]. Fetal, infant and juvenile mammals
have been reported to be especially sensitive to low
doses of chemicals that exhibit EA [4-6,12,15].
Given these considerations, we performed a hazard-
analysis survey that examined the leaching of chemicals
with EA in over 1000 assays from 50 reusable, hard and
clear, PC-replacement products. We used two well
established in vitro mammalian cell-based assays [16,17]
validated by ICCVAM/NICEATM/OECD (BG1Luc assay)
or that has been undergoing validation (MCF-7 assay:
[18]). Leaching of more- or less-polar chemicals was
assessed by extracting PC-replacement products with
more-polar (water or saline) or less-polar (ethanol (EtOH))
solvents, or mixtures of water and EtOH. The effects of
some aspects of common-use stresses was tested by expos-
ing PC-replacement products to autoclaving, microwaving,
or UV radiation prior to solvent extraction.
We report that chemicals with EA were released
(leached) from many unstressed BPA-free PC-replacement
products (including products for babies). Stressing these
PC-replacement products, especially with various forms of
UV radiation often increased the probability and/or level
of release of chemicals with EA. However, most import-
antly, some reusable, BPA-free, hard and clear PC-
replacement products could be found that did not release
chemicals with EA, even after exposure to UV or other
stresses. Consequently, it is possible to manufacture EA-
free PC-replacement products in commercial quantities
and thereby avoid potential adverse consequences to the
environment or human health due to release of xenobiotic
chemicals with EA [6,11,12,15,19,20].
Methods
Materials
Survey of plastic products
Fifty reusable PC-replacement products made from
seven types of resins (acrylic, COC, COP, PES, PETG,
PS, Tritan™) were obtained from 2010–2013 by purchase
at retail outlets. These products included many items for
which BPA-free resins are commonly used such as baby
bottles, reusable water bottles, food storage, packaging,
cups, medical supplies, and labware. These items were
branded by well-known firms such as Camelbak, Nalgene,
Dr. Weil, Born Free, AVENT, Costar, Crate and Barrel,
Green-to-Grow, and LocknLock. There was no consist-
ent price differential for products made with different
resins. We tested each product with various (not neces-
sarily the same) combinations of extraction solvents
and stresses using two different assays, MCF-7 and
BG1Luc (see below). We often stopped testing a prod-
uct if a particular type of extraction solvent or stress
showed that the product leached chemicals having sig-
nificant levels of EA. That is, the aim of this study was
not to perform an exhaustively-complete study of re-
sponses to all possible stresses and extraction condi-
tions for a few products, but rather to survey a larger
sample of PC-replacement products and assess whether
some released chemicals with EA whereas others were
potentially EA-free.
MCF-7 and BG1Luc cells
MCF-7:WS8 (MCF-7) cells were a gift from Dr. V. Craig
Jordan, then at Northwestern Medical School, now at
Georgetown Medical School. Every 2–3monthsthe
MCF-7 cells were replaced with stocks from the same
MCF-7 primary source to maintain more uniform MCF-7
cell characteristics throughout the study. BG1Luc cells
were licensed from the University of California, Davis.
Media and medium supplements (RPMI (Roswell Park
Memorial Institute)-1640 Medium, DMEM, FBS, nones-
sential amino acids, l-glutamine, penicillin, streptomycin)
used to initially grow and maintain the MCF-7 and
BG1Luc cells were purchased from Invitrogen (Grand
Bittner et al. Environmental Health 2014, 13:41 Page 2 of 14
http://www.ehjournal.net/content/13/1/41
Island,NY,USA).InsulinwaspurchasedfromSigma
(St. Louis, MO, USA).
MCF-7 and BG1Luc cells were maintained in polystyr-
ene T-75 flasks (BD Falcon, BD Biosciences, San Jose,
CA, cat#353136) and polystyrene T-25 flasks (CytoOne,
USA Scientific, Ocala, FL, cat#CC7682-4825). MCF-7
cells were seeded into 96-well flat bottom PS poly-
styrene plates (BD Falcon, cat#353075) and BG1Luc
cells were seeded into 96-well white wall/clear bottom
plates (Greiner Bio-One, Monroe, NC, cat#655098). Etha-
nol was obtained as 100% from various sources (OmniPur,
EMD-Millipore, Billerica, MA; Acros Organics/Fisher
Scientific, Pittsburgh, PA or Sigma-Aldrich, St. Louis,
MO). Water was distilled on-site in an all-glass system and
collected directly into glass before use in extractions. Ex-
tractions were performed in borosilicate glass tubes.
Equipment
We used Labconco Class II Biosafety Hoods (Kansas City,
MO, USA) equipped with a 254 nm fluorescent bulb to en-
close EpMotion 5070 robotic workstations (Eppendorf,
Hamburg, Germany) for serial dilutions of test chemicals,
cell seeding, and media changes in 96-well plates [3]. A
Tristar Luminometer (Brethold Technology, Germany)
was used to measure luminescence in BG1Luc assays. A
Bio-Tek PowerWavex and a Bio-Rad 96-well plate reader
spectrophotometer were used to measure DNA content in
MCF-7 assays, as previously described [3,18].
Protocols to stress plastic products
There are no regulatory protocols for stressing plastic
products to test for leaching of chemicals with hormonal
activity. Hence, we devised microwave, autoclave, and
UV stresses described below to simulate various aspects
of the short- or long-term effects of such common-use
stresses of microwaving, boiling (moist high heat) or ger-
micidal or UV exposure experienced by various types of
PC plastics such as food containers, water bottles, or
baby bottles. Some of these stresses have been previously
described [3].
For most microwave stresses, 4x4 mm square pieces of
plastic were placed into glass beakers in a 1200 W
microwave oven set on “high”for two minutes, and then
allowed to rest for 30minutes. The cycle was repeated 10
times. Some samples were placed in EA-free polypropyl-
ene (PP) tubes, and then microwaved on “high”setting
for three minutes with a resting time 30minutes between
stresses. The cycle was repeated 5 times. We did not
detect consistent differences in leaching after the two
protocols.
For autoclave stresses, plastic products were enclosed
in individually crimped packets of EA-free aluminum
foil and placed in a Tuttnauer autoclave at 134°C for
8minutes.
For UV stresses, the UV radiation in sunlight is often
classified [21] as UVC (100–280 nm), UVB (280–315 nm),
and UVA (315–400 nm); visible wavelengths are from
400–750 nm. Most UVC wavelengths are filtered by the
ozone layer before reaching the earth’s surface. However,
UVC wavelengths are used in some germicidal UV devices,
e.g., to sterilize baby bottles. For UV stresses, the protocols
were as follows:
(1) Long wavelength (315–400 nm) UVA stresses that
simulate many aspects of UV in sunlight: Samples were
placed in a Q-Lab QUV unit containing UVA-340 nm
bulbs to simulate exposure to moisture-free sunlight be-
tween 295 nm and 365 nm for 80 hours at 45–50°C.
(2) Short wavelength (100–280 nm) UVC stresses that
simulate many aspects of UV in germicidal sterilizers:
Samples were placed on aluminum foil in a Labconco
Biosafety hood about 24”from a germicidal fluorescent
light (maximum intensity wavelength of 254 nm) for
24 hours.
Extracts of plastic products
Two to five grams of unstressed or stressed samples of
PC-replacement products were added to sterile glass test
tubes. The tubes were placed under a germicidal UV
light for 30 minutes to sterilize the samples before add-
ing an extraction solvent to produce a final concentra-
tion of 1.0 g/mL. Such brief UVC exposures do not alter
leaching [3]. The extraction solvents consisted of saline-
based solution (saline: RPMI-1640 Medium without phe-
nol red), 100% EtOH, 10-50% aqueous EtOH, or distilled
water. Most samples were extracted at 40°C for 240 hours
in an incubator shaker. Saline extracts were diluted 2× with
2× estrogen-free medium (EFM) in a 1:1 ratio and then
further diluted 1-4× with EFM so that the highest starting
product concentration applied to wells was 0.125-0.500 g
product/mL. [EFM was modified from cell maintenance
media (see below) by replacing 10% FBS with 1% charcoal-
stripped FBS and 4% charcoal-stripped calf serum and
phenol red-free RPMI-1640.] EtOH extracts were con-
centrated 10× by evaporation and then diluted 100×
with EFM to produce a highest starting concentration
of 0.1 g product/mL to be applied to wells.
MCF-7 assays
An MCF-7 cell line was used in a robotized version of
the MCF-7 cell proliferation assay [3,18] that has been
employed for decades in manual format to reliably assess
EA [16,22]. The assay is currently undergoing validation
for international use by ICCVAM/NICEATM [18]. Chemi-
cals with EA bind to ERs and activate the transcription of
estrogen-responsive genes, which leads to proliferation of
MCF-7 cells.
As previously described in detail [3,18], cell mainten-
ance media was used to grow and maintain the MCF-7
Bittner et al. Environmental Health 2014, 13:41 Page 3 of 14
http://www.ehjournal.net/content/13/1/41
cells. This media consisted of RPMI-1640 media with
non-essential amino acids, 10 μg/mL phenol red, 4 mM
l-glutamine, 6 ng/mL insulin, 100 units/mL penicillin,
100 μg/mL streptomycin, and 10% fetal bovine serum
(FBS). EA assays were performed in EFM. Each test ex-
tract at each concentration was added in triplicate or
quadruplicate to 96-well plates containing MCF-7 cells
in EFM. After six days exposure to test chemicals or ex-
tracts, the cell culture medium was aspirated and the
amount of DNA/well, an indication of cell numbers, was
assayed using a microplate modification of the diphenyl-
amine assay [3,18].
BG1Luc assays
The BG1Luc4E2 cell line (aka BG1Luc) responds to es-
trogenic chemicals with the induction of firefly luciferase
[17]. The BG1Luc assay has been approved as a screen-
ing method for estrogenic chemicals by OECD, EPA,
and ICCVAM/NICEATM [23]. BG1Luc cells were main-
tained in cell culture medium that consisted of phenol
red-free DMEM with 8% FBS, 100 units/mL penicillin,
and 100 μg/mL streptomycin, L-glutamine and sodium
pyruvate. Prior to assaying for EA, BG1Luc cells
were placed for 3 days in EFM that was modified
from cell culture medium by replacing 8% FBS with 4.5%
charcoal-stripped FBS and substituting phenol red-free
DMEM for phenol red-free containing DMEM. Accli-
mated cells were then seeded at 10,000 cells per well in
100 μL EFM in 96-well plates for 24 hours, followed by a
24 ± 6 hours incubation with test extracts in triplicate.
Cytotoxicity was assessed as described by ICCVAM/
NICEATM [23] and Yang et al. [18]:
Cells were visually observed under an inverted light
microscope immediately before terminating incubation.
Cellular cytotoxicity was assessed using scoring param-
eters 1 = normal cell morphology, 2 = low cytotoxicity
(10 - 50% of cells with altered morphology), 3 = moderate
cytotoxicity (50- 90% of cells had altered morphology), and
4 = high cytotoxicity (few or no cells visible). Test sub-
stance concentrations with a cytotoxicity score of 2 or
higher were excluded from further analyses.
Cell culture medium was aspirated, cells were lysed,
and luciferase was measured in an automated microplate
luminometer (Tristar Berthold Technology, Germany) with
the Promega Luciferase Assay System (Promega, Madison,
WI, USA) following the manufacturer’sprotocol.
Calculation of EA
The estrogenic effect of a test chemical or extract on cell
proliferation or luciferase activity was calculated as %
RME2, a percentage of the maximum DNA/well (or the
Relative Luminescence Unit, RLU) produced by the
maximum response relative to 17β-estradiol (E2, positive
control). This value was corrected for the background
(DNA or RLU in MCF-7 or BG1Luc, respectively) re-
sponse to the vehicle (negative) control. We incorpo-
rated both a vehicle control (VC) and “sham”control
(SC) in each experiment. The VC was the vehicle used
for that particular assay. The SC was the vehicle taken
through all steps that were used to assay the test sam-
ple/test extract. For both SCs and test extracts, %RME2
was calculated by subtraction of the VC value from the
SC or test sample value, followed by normalization of
each adjusted value to the maximum E2 value measured
in the experiment (set at 100%) and the VC (set at 0%).
Typical value of an SC was 0% ± 10% RME2. However, if
the EA of an SC were greater than 15% RME2, then the
entire experiment was rejected.
The EA of a test chemical or extract was classified as
detectable if the EA effect was greater than 15%RME2,
which was greater than three standard deviations (SD) of
the SC response for that experiment. Therefore, 15%
RME2 is a conservative measure of EA detectability.
Stimulation of MCF-7 proliferation or BG1Luc Luciferase
expression by test chemicals or extracts was confirmed as
estrogenic (rather than non-specific effects) by suppression
of the EA by co-incubation with the anti-estrogen (ICI
182,780 (ICI) at 10
−7
-10
−8
M.
We saw no examples of an unsuppressed agonist re-
sponse using either assay. That is, these in vitro assays
rarely produce false positive responses [3,18,24]. Note
also that we have limited our analyses to whether the
plastic or chemical exhibited EA that was statistically
significantly (p < 0.01) greater than any EA detected in
VC or SC samples. We have not attempted to statisti-
cally compare absolute %RME2 values for different ex-
tracts or plastic products because extraction procedures,
while similar, were not exactly the same for the two cell
lines and the chemicals, much less their concentrations,
in extracts were not known.
Results
MCF-7 and BG1Luc assays of PC-replacement products
with EA
Our assays quantified the total EA of chemicals that lea-
ched from a plastic product, but could not determine
the identity of those chemicals. The total EA of chemi-
cals extracted from 50 BPA-free PC-replacement prod-
ucts was calculated as %RME2 from concentration
(dilution)-response curves for MCF-7 and BG1Luc assays.
Concentration-dependent increases in EA for E2 (in M)
were the positive controls for MCF-7 assays (Figure 1A)
and BG1Luc assays (Figure 1B). Concentration units for
extracts of unstressed or stressed plastic products were
given in grams of product/mL (Figure 1A,B). The sta-
tistically significant reduction of %RME2 by ICI (dashed
lines, open symbols in Figure 1A,B) confirmed that any
agonist activity was due to ER activation of EA, rather
Bittner et al. Environmental Health 2014, 13:41 Page 4 of 14
http://www.ehjournal.net/content/13/1/41
than a non-specific response. Extracts of PC products
(Figure 1A,B) always exhibited the presence of che-
micals having EA, almost-certainly mostly due to leach-
ing of BPA [4-6]. Such EA was greatly reduced by ICI
(Figure 1A,B).
EA-positive concentration-response curves were ob-
served for many PC-replacement products assayed in
this survey. As representative examples from 50 prod-
ucts tested in over 1000 assays, Figure 1D and E show
that both MCF-7 and BG1Luc assays detected chemi-
cals with EA leaching from an unstressed toddler blue
cup extracted using 50% EtOH. For this particular
PC-replacement product, the MCF-7 assay was more
sensitive in detecting EA than the BG1Luc assay. For
some other products, the BG1Luc assay was more
sensitive (also see Figures 2 and 3). However, the
conclusion as to whether a PC-replacement product
released chemicals with EA was almost always the same
for the two cell lines (Figure 1C). Dose–response
curves showing leaching of chemicals with EA were
observed for both more-polar and less-polar extrac-
tion solutions, including 50% EtOH (Figure 1E) for many
products, as did saline extracts for many products (also see
Figures 2 and 3).
Figure 1 Concentration-response curves for E2 and plastic extracts. Mean and SD data for concentration (g/mL or M) –response (%RME2)
curves of luciferase activity in BG1Luc cells and MCF-7 cell proliferation by extracts of PC (A,B) or PC-replacement (C-I) products. Black lines
and associated data points show agonist activity for all data points not associated with toxicity (see Methods). Red lines and associated data
points show results of exposure of extracts to 10
−8
M ICI. Horizontal dotted lines show 15%RME2 values that are significantly (p < 0.01) greater than
the vehicle (VC) and sham (SC) controls. PC-replacement products. Panel C. Costar 3300 tissue culture plate. D. Cup.1: Camelbak kids reusable water
bottle, blue; Cup 2: Nalgene kids reusable water bottle, blue. E. Cup.1: Camelbak kids reusable water bottle, blue; Cup 2: Nalgene kids reusable water
bottle, blue. F. Zeonor COP bottle. Topas COC bottle. G. AVENT baby bottle, green; Crate and Barrel wine glass, green. H. Born Free baby bottle A;
Green to Grow baby bottle B. I. Dr. Weil baby bottle; Contigo reusable water bottle, turquoise.
Bittner et al. Environmental Health 2014, 13:41 Page 5 of 14
http://www.ehjournal.net/content/13/1/41
Some extracts of PC-replacement products did not
exhibit leaching of chemicals with EA. Figure 1F shows
adose–response curve for a 100% EtOH extract of
unstressed COC and COP bottles that exhibited no
detectableEA.Figure1Gshowsadose–response
curve for a saline extract of a UVA-stressed green
Tritan™bottle with no detectable EA and a green
acrylic wine goblet whose extract had EA. Colorless
Tritan™bottles or Tritan™bottles of colors other than
green often exhibited leaching of chemicals with EA
(Figures 1I and 3).
Survey of EA in leachates of BPA-free PC-replacement
products
As shown in Tables 1 and 2 and Figures 2 and 3, we
examined whether chemicals with EA leached into
more-polar (saline) or less-polar (EtOH) extraction sol-
vents from 50 unstressed and/or stressed reusable PC-
replacement products. Stresses included autoclaving,
microwaving, and/or UV irradiation. As previously re-
ported [3], for some products EA positive responses were
found in polar solvents, but not in non-polar or, alter-
natively, in non-polar but not in polar solvents. An
BG1Luc:Acrylic Red Goblet
UN
AU
UVA
-25
0
25
50
75
100
15
1
1
1
1
1
1
1
D
1
%RME2
MCF-7:COC D Bottle
UN
AU
MI
UVC
-25
0
25
50
75
100
15
4455
7
76
6
A
Saline
100% EtOH
50% EtOH
10% EtOH
Water
- - -
15%RME2
%RME2
MCF-7:COP Z Bottle
UN
AU
MI
UVC
UVA
-25
0
25
50
75
100
15
4344424
411
B
MCF-7:PES Baby Bottle 2
UVC
UVA
-25
0
25
50
75
100
125
15
1
2
3
F
MCF-7:PES Baby Bottle 1
UN
AU
MI
UVC
-25
0
25
50
75
100
15
1123
1
2
2
3
C
MCF-7:PES Baby Bottle 3
UVA
-25
0
25
50
75
100
125
15
2
3
G
%RME2
BG1Luc:PS Cell Culture Plate
AU
MI
UVC
-25
0
25
50
75
100
15
H
11
4
11
2
MCF-7:Polystyrene Products
Unstressed, 100% Ethanol
-25
0
25
50
75
100
125
150
15
I
Cup
Cube 1.9L
Clamshell Container
11
6
55
2
5
3
3
PS Cell Culture Plates
4
BG1Luc:PS Products
Unstressed, 100% Ethanol
-25
0
25
50
75
100
15
Cell Culture Plate
Clamshell 5" container
4
6
E
Figure 2 EA of PC-replacement products assessed by MCF-7 or BG1Luc assays. Means of highest value and SD of %RME2 from dose–response
curves of various extracts of acrylic, COC, COP, PES, PS PC-replacement products as stated in key to each panel. Number of independent assays for each
type of product extract (e.g., EtOH concentration, saline) given above the bar graph. Each assay consisted of three replicates for that extract whose SD
was typically very small (as indicated in Figure 1). Unstressed = UN, AU = autoclave stress, MI = microwave stress, UVA and UVC = long and short
wavelength UV stresses, respectively. Each assay for each product is considered by itself whether its EA is significantly greater than its VC or SC
EA, i.e. > 15%RME2 (see Methods). We do not statistically compare EA values obtained for different plastic products because extraction procedures
and concentrations, while similar, are not exactly the same. PC-replacement products. Panel A. COC Topas bottle. B. Zeonor bottle. C. Green to
Grow baby bottle. D. Crate and Barrel wine glass, red. E. Costar 3585 cell culture plate; Dart clamshell container. F. Born Free baby bottle. G. Avent
baby bottle. H. Costar tissue culture plate. I. Chinet cut crystal cup; Click Clack Cube; Dart clamshell container.
Bittner et al. Environmental Health 2014, 13:41 Page 6 of 14
http://www.ehjournal.net/content/13/1/41
unstressed or stressed product was classified as leaching
chemicals with EA if statistically significant (p < 0.01)
amounts of EA > 15%RME2 (see Methods) were de-
tected in any extraction solvent for either MCF-7 or
BG1Luc assays. Figures 2 and 3 show bar-graphed ex-
amples of the maximal EA (in %RME2) obtained from
over 1000 concentration-response curves for various
product types. Not all extracts or stresses were applied
to each of 50 products tested in this survey in which
we often stopped testing a product once a particular
protocol showed that chemicals with EA leached from
the product.
Unstressed extracts of some COC (Figure 2A), COP
(Figure 2B), PES (Figure 2F), and Tritan™(Figure 3A,C-G)
products had no detectable EA in MCF-7 (Figure 3A, C-E)
or BG1Luc (Figure 3F,G) assays. A few PC-replacement
products, such as a COC bottle (Figure 2A), a COP bottle
(Figure 2B), and a green Tritan™bottle (Figure 3B) exhib-
ited no detectable EA after one or more stresses, including
one or more UV stresses, in more-polar or less-polar sol-
vents. In contrast, one or more extracts of most PS, PES,
and Tritan™products (Figures 2C-I; 3C-L) exhibited EA in
more-polar or less-polar solvents, especially when the
product was exposed to UVA or UVC radiation. In general,
Figure 3 EA of PC-replacement products assessed by MCF-7 or BG1Luc assays. Means of highest value and SDs of %RME2 for sets
of dose–response curves for Tritan™products. Abbreviations as described for Figure 2. PC-replacement products. Panel: A. Nalgene reusable water
bottle, green. B. Nalgene reusable water bottle, green C. Dr. Weil kids reusable water bottle. D. Dr. Weil baby bottle E. Evenflo baby bottle, clear.
F. Nalgene reusable water bottle, blue. G. Camelbak kids reusable water bottle, blue. H. Camelbak kids reusable water bottle, blue. I. Camelbak reusable
water bottle, black. J. Camelbak reusable water bottle, blue. K. Lock and Lock food storage container. L. Nalgene reusable water bottle, blue.
Bittner et al. Environmental Health 2014, 13:41 Page 7 of 14
http://www.ehjournal.net/content/13/1/41
Tritan™products stressed with UVA or UVC radiation
more frequently released chemicals with statistically
significant levels (p < 0.01) of EA in both MCF-7 and
BG1Luc assays compared to unstressed Tritan™prod-
ucts.However,stressingper se did not necessarily in-
crease the release of chemicals with EA. For example,
compared to unstressed or UV-stressed products, ex-
tracts of autoclaved (AU) - or microwaved (MI) PC-
replacement products often had lower EA in BG1Luc
and MCF-7 assays (Figure 3C,D,G-K).
Although the analyses of the same extracts using the
BG1Luc and MCF-7 assays produced slightly different
%RME2 values (Figure 3A vs. B; G vs. H), the results
of both assays almost-always led to the same conclu-
sion that a given product did (Figure 3G,H) or did not
(Figure 3A,B) release chemicals with detectable EA.
Detection of EA in some but not all extracts of differ-
ent products made from the same type of resin was not
surprising, and likely resulted from different additives
and impurities (catalyst residues, thermal degradation
products, etc.) in the different resins or products and/or
differences in their processing [3], as well as differences
in the highest concentration tested (see Methods). Simi-
larly, obtaining different %RME2 EA values of the same
product for different extract solutions using the same
assay (Figures 2, 3) was not surprising because chemicals
with EA can be more-polar or less-polar and are more ef-
ficiently extracted with a solvent that is more-compatible
with their polarity. The relative ability of more-or less-
polar solvents to extract and detect chemicals with EA
from UV-stressed products is summarized in Table 1.
Water or saline extracts or water/EtOH mixtures were
more effective than 100% EtOH in detecting EA leaching
from Tritan™type products when either MCF-7 or BG-
1Luc assays were used. Conversely, for acrylic-based resins,
100% EtOH as a solvent was more effective than water-
based solvents in extracting chemicals with EA.
Table 2 summarizes the findings for total EA in the
leachates of the 50 products sorted according to the 7
types of resins used to manufacture the product. The
data summarized in Tables 1 and 2 and Figures 1, 2, and
3 demonstrate that more than one type of extract and
type of stress are needed to detect whether a given prod-
uct will leach chemicals having detectable EA. Consider-
ing all assays of unstressed products for all resin types,
Table 1 Frequency of EA Leaching from UV-stressed PC-replacement products
Ratio of EA
+
products/total products tested for UV stresses for each type of resin
MCF-7 assays BG1Luc assays Either
Product type n Saline & water 100% EtOH 10% &50% EtOH EA
+
/NT Saline & water 100% EtOH 10& &50% EtOH EA
+
/NT Overall
Acrylic 3 0/3 2/3 1/1 2/3 0/3 1/1 1/2 2/3 2/3
COC 4 0/2 0/2 0/2 0/2
COP 1 0/2 0/2 0/1 0/1
PES 3 4/4 2/2 3/3 3/3
PETG 3 1/3 0/2 1/3 1/3
PS 9 0/3 2/2 2/3
Tritan™25 16/23 7/21 7/7 20/23 4/11 6/8 7/7 12/14 23/25
Relative number of UV-stressed (GUV or QUV) products exhibiting significant (p < 0.01) EA reported by resin type versus extract solvents for each assay.
“n”is the number of products of a resin that received at least one assay for UV-stress.
“blank cell”indicates that no UV tests done for that combination of conditions’.
“EA
+
/n Total”gives the number of products for an assay type showing significant leaching of chemicals having EA (EA
+
) with respect to the total number of
products of that resin type (n).
“EA
+
/n Overall”gives the number of products showing significant leaching of chemicals having EA (EA
+
) with respect to the total number of products of that resin
type (n) using either assay type.
All combinations of product, assay, and solvent types were not used, sometimes because unstressed products of a resin type exhibited significant EA (i.e., 9/11
unstressed products made from acrylic resins exhibited EA). For some cells that combine data for two extraction solvents (water and saline, 10% & 50% EtOH), the
number of possible assays was double the number of products.
Table 2 Frequency of EA leaching from PC-replacement
products
Ratio of EA + products/total tested for each stress
Product type n UN MI AU UV Overall
Acrylic 3 2/3 2/3 2/3 2/3
COC 4 0/3 0/1 0/2 0/2 0/2
COP 1 0/1 0/1 0/1 0/1 0/1
PES 3 0/1 1/1 3/3 3/3
PETG 3 0/3 0/3 1/3 1/3
PS 11 9/11 0/1 2/3 9/9
Tritan™25 2/6 3/10 3/14 23/25 23/25
Total 50 13/25 4/16 5/24 31/40 38/46
Relative number of products made from a resin type exhibiting significant
(p <0.01) EA for a given stress or for any stress.
Row labeled “overall”gives the number of products exhibiting EA for those
products having at least three assays for at least 3 stresses (row labeled “Overall”).
Row labeled “Total”sums the data for that column.
n: number of products tested for any stress condition, blank column: no tests
made for that particular combination of product and stress, UN: unstressed,
MI: microwaved, AU: autoclaved, UV: UVA or UVC stress.
Bittner et al. Environmental Health 2014, 13:41 Page 8 of 14
http://www.ehjournal.net/content/13/1/41
13/25 released chemicals with statistically significant
(p < 0.01) levels of EA. Following microwave stress, only
4/16 of all products exhibited release of chemicals with
EA and only 5/24 exhibited EA after autoclave stress.
That is, some stresses might have reduced the release of
chemicals having EA. Autoclaving, for example, might
extract chemicals having EA so that after autoclaving, the
stressed product subsequently releases chemicals having
less total EA. In contrast, following UV stresses (UVA
and/or UVC), 32/41 products demonstrated leaching of
chemicals with EA, including most acrylic, PES, PS, and
Tritan™products. That is, UV stresses increased the
probability (and perhaps the levels) of the total EA of
chemicals leaching from PC-replacement products.
For PC-replacement products that received at least 3
assays of different types of extraction solvents or stresses
(Table 2), 38/46 exhibited significant release of chemicals
with EA in at least one extract type, including most
products made from (2/3) acrylic, (3/3) PES, (9/9) PS,
and (23/25) Tritan™resins. That is, UV stresses signifi-
cantly (p < 0.03, Chi Square Test) increased the prob-
ability that a product would release chemicals with EA
(38/46) compared to unstressed products (13/25). How-
ever, our data also showed that hard and clear, reusable,
PC-replacement products that did not release any detect-
able EA for any type of stress or extract could be manu-
factured in commercial quantities from some COC, COP,
and PETG resins.
Effects of UV radiation on EA release from BPA-free
PC-replacement products
The results described above indicated that exposure to
UV radiation could increase the EA levels of extracts
from most PC-replacement products assayed in this
study. However, the depth of UV wavelength penetration
into the product depends upon the structure of the poly-
mer and its additives. Thus, even if chemicals with EA
were produced by UV radiation in the presence of oxy-
gen and such chemicals were readily extractable at the
outer surface of a plastic container, the chemicals might
not be produced and/or released from the inner surface
of such containers.
To examine this possibility, we determined the UV/
Visible spectra in the range of 200–600 nm of blow-
molded bottles of similar thickness made from various
PC-replacement products (Table 3 and Figure 4). UV ra-
diation is often classified by wavelength as UVA, UVB,
and UVC ([21] See Methods). All products transmitted
UV radiation over a range of wavelengths typical for
each respective class of PC-replacement resins. Table 3
shows for colorless products that the PES bottle had the
lowest percent penetration of UVA + B and UVA radiation.
These data matched well with data shown in Figure 4A, in
which PES had the lowest percent transmittance of the
products tested in this study. The UV/Visible spectrum of
acrylic indicated that it had the highest transparency of the
products in this study, but had slightly lower percent trans-
mittance of the UVA + B and UVA wavelengths than PS.
The ability of UV light to penetrate these PC-
replacement products was confirmed by inserting
white UV-detecting beads that changed color when ex-
posed to light in the range of 300–360 nm into bottles
made from various resins. Different beads changed to
different colors for the same UV exposure, i.e., the
exact change color was not significant. Figure 5 shows
white beads placed in the bottle prior to exposure to
sunlight outdoors for a COP (Figure 5A) and for a Tritan™
(Figure 5C) bottle. Figure 5B and D shows that those beads
inside either bottle changed color after a few seconds of
outdoor exposure to UV in sunlight.
Product color can significantly alter UV transmission
Table 3 and Figure 4B show that red, blue, and green
colorants in Tritan™bottles could reduce the percent
transmittance relative to a colorless bottle. Figure 4B
shows transmittance data forfourdifferentcolored
Tritan™bottles of the same model, with walls 1.35 to
1.90 mm thick. The red bottle blocked most UV radiation
200–325 nm passing through the sample, with an increas-
ing amount of penetration as the wavelength increases.
The blue bottle blocked UV radiation below 325 nm, but
allowed much UV radiation to pass at 325 - 400 nm. The
green colored bottle showed the least penetration com-
pared to the colorless or other colored bottles and blocked
almost all (~99%) of the UVA and UVB radiation.
Additives in BPA-free replacement products can
exhibit EA
In addition to polymerized monomers, PC-replacement
products contain many other chemicals, including those
added during resin or product manufacturing processes.
Some of these additives might have EA and could
potentially leach from the outer surface (e.g., during
handling) or inner surface (e.g., ingestion of container
contents). MCF-7 assays showed that two unstressed
lower-molecular-weight antioxidant additives, butylated
hydroxyanisole (BHA) and butylated hydroxytoluene
(BHT), exhibited EA (Figure 6A,B). We tested other ad-
ditives by adding them to PP resins that were formed into
plaques or bottles that exhibited no detectable EA in the
absence of any additional chemicals added during manu-
facture. When one commonly used higher-molecular
weight antioxidant (processing stabilizer) was added prior
the forming the PP resin into plaques (heat stress), the
resulting plaques leached chemicals with EA. (Figure 6C).
However, another higher-molecular-weight antioxidants
did not exhibit EA even after many hours of heat stress
(Figure 6C). Similarly, many plastic colorants contained in
Bittner et al. Environmental Health 2014, 13:41 Page 9 of 14
http://www.ehjournal.net/content/13/1/41
EA-free plaques added during manufacture exhibited EA,
but others were EA-free after heat stress (Figure 6D). Our
assays also showed that other additives such as EA-
containing and EA-free UV stabilizers (Figure 6E) and inks
tested on EA-free bottles or plaques (Figure 6F) were com-
mercially available.
Discussion
Assessments of EA release from BPA-free PC-replacement
products
While the release of various chemicals from plastic
products had been well-studied [1,2], the specific leach-
ing of chemicals with hormonal activity such as EA from
many different types of unstressed or stressed plastics
has only recently been examined [3]. BPA is by far the
best studied as an estrogenic chemical released from re-
usable hard and clear PC-type products (for reviews see
[4-6]). For example, Howdeshell et al. [25] reported that
BPA with significantly detectable EA leaches from PC
animal cages. Le et al. [26] reported that BPA leaches
from PC drinking bottles. Yang et al. [3] reported that
chemicals with EA leached from a small sample of com-
mercially available PC products. However, prior to the
current survey, there were few data on whether chemicals
exhibiting EA leached from BPA-free PC-replacement
products.
Our data from more than 1000 in vitro assays showed
that many BPA-free PC-replacement products made
from various types of resins, including PETG, PS, PES,
and Tritan™resins, could leach chemicals with EA, irre-
spective of whether these products were unstressed
(13/25 in Table 2) or stressed (38/41 in Table 2) or which
in vitro assay was used. Furthermore, agonist responses
obtained using BG1Luc or MCF-7 assays were always
inhibited by ICI, confirming that the observed agonist ac-
tivity required ER activation. These data were consistent
with a much smaller set of previous data using only
MCF-7 assays showing that 14/28 unstressed products
made from PS resins leached chemicals with EA (Table 1
of reference [3]) and that that four water bottles and a baby
bottle made from Tritan™resins (identified as PETG resins
in Figure 3 of reference [3]) released chemicals with EA,
Table 3 Integrating radiometer data for HC bottles and containers
Base resin Product Color Thickness (mm) UVA+B [J/cm
2
] % UVA+B UVA [J/cm
2
] % UVA
PES Bottle Colorless 1.30 1.00 [2.90] 34.4 0.98 [2.73] 36.0
ABS Food storage Colorless 1.88 1.04 [2.38] 43.9 1.08 [2.14] 50.5
PS Food storage Colorless 0.25 2.31 [2.82] 81.9 2.23 [2.72] 82.0
Acrylic Goblet Colorless 2.31 2.19 [3.02] 72.5 2.27 [2.85] 79.8
Tritan™Bottle colorless 1.70 1.56 [2.72] 57.3 1.78 [2.57] 69.0
Toddler bottle colorless 1.35 2.07 [2.78] 74.4 2.05 [2.88] 71.3
Bottle Green 1.62 0.03 [3.03] 1.1 0.020 [2.71] 0.70
Bottle Blue 1.62 0.90 [2.80] 32.1 0.88 [2.94] 29.9
Bottle Red 1.49 0.74 [2.68] 27.7 0.78 [2.47] 31.5
An integrating radiometer was used with two sensors to measure penetration of UVA + B and UVA radiation on August 7
th
and 8
th
, 2013 in Austin, TX. The
percentage of radiation that penetrated into the interior of the containers was calculated by integrating the energy over a ten minute interval in the sunlight
without anything blocking the sensors (value inside of brackets), and then placed into the sealed containers for ten minutes. J = joules.
0
10
20
30
40
50
60
70
80
90
100
200 300 400 500 600
% Transmittance
Wavelength (nm)
colorless
red
blue
green
B
0
10
20
30
40
50
60
70
80
90
100
200 300 400 500 600
% Transmittance
Wavelength (nm)
ABS
acrylic
COC
COP
PES
PS
Tritan™
A
Figure 4 UV transmission curves for some PC replacement products. UV/Visible spectroscopy of PC-replacement products in Table 2 showing
percent light transmittance at different wavelengths. (A) UV penetration for colorless bottles of resin type given in key. (B) UV penetration for colorless
or colored bottles made from Tritan™resin.
Bittner et al. Environmental Health 2014, 13:41 Page 10 of 14
http://www.ehjournal.net/content/13/1/41
especially when stressed with UVC (germicidal) radiation.
Our much more extensive data in the survey reported
herein also demonstrated that some unstressed and
stressed colorless COC, COP, and PETG PC-replacement
products (Tables 1, 2) did not release chemicals with de-
tectable EA, indicating that it is possible to synthesize EA-
free PC-replacement products for commercial use.
UV stresses often increase leaching of chemicals with EA
While leachates obtained from unstressed products could
contain chemicals with EA, exposure to UV radiation
frequently increased the probability of leaching of chemi-
cals with EA from those same products. For example, UV
exposure produced release of chemicals with EA from all
Tritan™products, except for one green-colored bottle
(Figures 1, 2, and 3). This increased probability of EA
release could result from UV-dependent formation of
new chemical (s) with EA and/or enhanced release of
chemical (s) with EA that were already in the unstressed
product.
It is not unexpected that UV exposure might cause chem-
ical changes in a plastic. UVC wavelengths (100–280 nm)
Figure 5 UV light transmits to the interior of Tritan™bottles. UV-sensitive beads placed in colorless PC-replacement COC (A,B) or Tritan™
(C,D) bottles before (A,C) or after (B,D) a few seconds of outdoor exposure. Different beads change to a given color when exposed to UV, i.e., a
color change, but not the specific color change, shows that UV light has been captured by the beads.
Figure 6
BG1Luc:Masterbatch Colorant Plaques
Microwaved, 100% EtOH, 240 h, 40 °C
-5 -4 -3 -2 -1 0 1
-25
0
25
50
75
100
15
Blue Masterbatch Plaque
D
Red Masterbatch Plaque
log g/mL
%RME2
MCF-7:Antioxidants
200C, 100 h, 100% EtOH
-5 -4 -3 -2 -1 0
-25
0
25
50
75
100
125
15
Additive 14
C
Additive 15
log g/mL
MCF-7:UV Stabilizers
Unstressed, Saline or 100% EtOH
-6 -5 -4 -3 -2 -1 0
-25
0
25
50
75
100
15
UV Stabilizer C
E
UV Stabilizer S
+ICI
+ICI
log g/mL
BG1Luc:Inks on Polypropylene (PP)
Autoclaved, 100% EtOH
-3.0 -2.5 -2.0 -1.5 -1.0
-25
0
25
50
75
100
15
Black Ink on PP Plaque
+ICI
F
Blue Ink on PP Bottle
+ICI
Log g/mL
MCF-7:BHT
Unstressed, 100% EtOH
-8 -7 -6 -5 -4
-25
0
25
50
75
100
15
BHT
A
Log M
%RME2
- - - 15%RME2
MCF-7:BHA
Unstressed, 100% EtOH
-8 -7 -6 -5 -4
-25
0
25
50
75
100
15
BHA
B
Log M
Figure 6 Concentration-response curves for some common additives. Dose–response curves for 6 additives used in the manufacture of
various PC-replacement plastics. Key as for Figure 1. Panels A-F: antioxidants (A-C) tested as individual chemicals; colorants (D), UV stabilizers
(E) or inks (F) added to PP bottles or PP plaques. Without the additive, the PP plaques or bottles exhibited no detectable EA (data not shown).
Bittner et al. Environmental Health 2014, 13:41 Page 11 of 14
http://www.ehjournal.net/content/13/1/41
found in germicidal sterilizers are damaging to plastics;
UVB (280–315 nm), and UVA (315–400 nm) [21]
wavelengths in sunlight are also damaging to plastics,
especially in the presence of oxygen at the inner and
outer surfaces of a plastic product [27-33]. Although
UVB wavelengths have greater energy and cause more
UV degradation in plastics than UVA wavelengths, UVA
wavelengths are more commonly found at the earth’s sur-
face and can cause much degradation of PC-replacement
polymers, especially with longer exposures [27,28,30,34].
In brief, if UV wavelengths associated with photo-
oxidation are transmitted through the walls of a plastic
product, significant degradation with subsequent leach-
ing can occur on the non-exposed, inner, surface of the
plastic [29].
Our UV transmission data (Figure 4, and 5, Table 3)
demonstrated that UV wavelengths could completely
penetrate to the inner surface of various PC-replacement
bottles where chain scission or other chemical reactions
could occur in the presence of oxygen, thereby potentially
producing chemicals with EA that could leach into solu-
tions contained within the product. Product manufacturers
could attempt to minimize such UV penetration to the
inner wall of their products. For example, our data
(Figure 4B) suggest that the potential UV degradation at
the inner surface of some green bottles would be lower
compared to the colorless and blue Tritan™bottles. How-
ever, green colorants would not guarantee that a green
product would not leach chemicals with EA. As one ex-
ample, a UV-stressed green acrylic wine goblet (Figure 1G)
leached chemicals with EA, as did other green bottles
made of various resins (data not shown). Hence, rather
than trying to prevent release by using certain green color-
ants, we believe that the best solution to potential leaching
of UV-produced chemicals having EA would be to use
PC-replacement resins and additives that remain EA-free
upon exposure to UV radiation.
Additives and monomers can exhibit EA or be EA-free
PC-replacement products are manufactured in ways that
almost-always use additives in addition to the monomers
polymerized to make the resin. Such additives can leach
from the plastic product. Our data (Figure 6) showed
that some commercially-available additives such as anti-
oxidants, colorants, processing stabilizers and inks had
EA, whereas others were free of detectable EA (EA-free).
Our previously published study [13] reported that some
monomers (COC, COP, some PETG] now used to
manufacture PC-replacement products should be EA-
free and some COC, COP, and PETG products were
indeed EA-free in the present study. Use of chemicals
(monomers or additives) that exhibit EA to produce a
PC-replacement product would almost-certainly increase
the probability that the product would leach chemicals
having EA. As one possible example, TPP is an additive
used in the synthesis of Tritan™resins [35] and TPP ex-
hibits EA [9]. Hence, some of the EA we detected in ex-
tracts of Tritan™products could arise from leaching of
TPP and/or its degradation chemicals. However, our data
also suggest that EA-free additives can be found that
could be used to manufacture BPA-free PC replacement
products from resins that were also EA-free, Such a for-
tuitous circumstance might account for our data that
some COC, COP, or PETG products were EA-free.
Conclusions
The results of our MCF-7 and BG1Luc assays demon-
strate that extracts of many unstressed and/or stressed
BPA-free PC-replacement products, including most
acrylic, PES, PS and Tritan™-based products, release
chemicals that can activate ER-dependent cell signaling,
i.e., exhibit EA. In this survey of PC-replacement prod-
ucts, we quantitatively measure maximum effects of total
EA in leachates (%RME2) relative to the maximum effect
of E2) using two sensitive assays and various extraction
protocols. We recognize that such in vitro data can only
describe the existence of a possible hazard for consump-
tion of chemicals with EA leaching from plastic products,
not what risk that consumption might have to human
health. We cannot calculate that risk in part because we
and other scientists do not know how much total EA
from plastics and other sources that anyone is exposed
to, how many chemicals have EA, their relative EA, their
release when exposed to different solvents or stresses,
their metabolic degradation products or half-lives in vivo,
whether chemicals with EA from different sources are
additive or synergistic, and the appropriate levels of EA
in males versus females at different life stages.
However, whatever the potential risk, our data for
some types (COC, COP, and PETG) of BPA-free PC-
replacement products show it is possible to use resins
and additives to manufacture such PC-replacement
products, including water bottles and baby products,
in commercial quantities that are also free of detectable
EA. Such data are important because other studies have
reported that chemicals with EA in mammals can pro-
duce various adverse health effects such as early menar-
che, reduced sperm counts and other altered functions of
reproductive organs, obesity, and increased rates of some
cancers; some of these effects are produced at very low
doses in fetal, infant, and juveniles [4,6,15]. Various stud-
ies from other laboratories have also suggested that che-
micals with EA can produce measurable changes in the
health of various human populations, e.g., on the off-
spring of mothers given diethylstilbestrol [4-6,12,15].
Since plastic products have advantages (weight, cost, im-
pact strength, energy footprint, etc.) in various combina-
tions compared to other materials such as steel or glass,
Bittner et al. Environmental Health 2014, 13:41 Page 12 of 14
http://www.ehjournal.net/content/13/1/41
it is not necessary to forgo these advantages of plastic
in order to avoid release of chemicals having EA into
foodstuffs or the environment that may have potential
adverse effects on our health or the health of future
generations. That is, our data show that producing EA-free
PC-replacement products is not an impossible or extraor-
dinarily costly task.
Abbreviations
BPA: Bisphenol A; BPS: Bisphenol S; CCi: CertiChem Inc.; COC: Cyclic olefin
copolymer; COP: Cyclic olefin polymer; E2: 17β-estradiol; EA: Estrogenic
activity; EA-free: No detectable estrogenic activity; EFM: EA-free medium;
ER: Estrogen receptor; EtOH: Ethanol; ICCVAM: Interagency Coordinating
Committee on the Validation of Alternative Methods; NICEATM: National
Toxicology Program’s Interagency Center for the Evaluation of Alternative
Toxicological Methods; NIEHS: National Institute of Environmental Health
Sciences; NIH: National Institutes of Health; NSF: National Science Foundation;
OECD: Organization for Economic Cooperation and Development; PETG: Glycol-
modified polyethylene terephthalate; PES: Polyethersulfone; PP: Polypropylene;
PPi: PlastiPure Inc.; PS: Polystyrene; RPMI: Roswell Park Memorial Institute;
SC: Sham control; TPP: Triphenyl phosphate; UV: Ultraviolet; UVA: Long
wavelength UV; UVC: Short wavelength UV; VC: Vehicle control.
Competing interests
CZY is employed by CCi. CZY and GDB own stock in CertiChem (CCi) and PPi.
GDB is consulting CEO of CCi. MAS was employed by CCi. This is a research
study. No products in this paper are manufactured or sold by CCi or CCi and
most data were obtained on NIH grants to assay such products. CCi has put
its MCF-7 and BG1-Luc robotic assays in the public domain.
Authors’contributions
All authors participated in the design of the experiments. CZY was primarily
responsible for MCF-7 assays and MAS for BG1Luc assays at CCi. GDB primarily
and CZY and MAS secondarily were responsible for writing the manuscript.
CZY and MAS were primarily responsible for analyzing and plotting data. All
authors read and approved the final manuscript.
Acknowledgments
This work was supported by the following NIH/NIEHS grants: R44 ES011469,
01–03 (CZY); 1R43/44 ES014806, 01–03 (CZY); subcontract (CZY, PI) on
an NIH Grant 01–03 43/44ES018083-01 to PlastiPure (DK, SY PIs). We thank
Drs. Daniel Klein and Stuart Yaniger of PlastiPure (PPi) for preparing samples of
plastic products, UV radiation exposures, and/or data analyses. CZY is employed
by CCi. CZY and GDB own stock in CertiChem (CCi) and PPi. MAS is employed
by CCi. GDB is consulting CEO of CCi. This is a research study. No products in
this paper are manufactured or sold by CCi or PPi and most data were obtained
on NIH or NSF grants to assay such products. CCi has put its MCF-7 and
BG1-Luc robotic assays in the public domain.
Received: 16 March 2014 Accepted: 5 May 2014
Published: 28 May 2014
References
1. Begley T, Castle L, Feigenbaum A, Franz R, Hinrichs K, Lickly T, Mercea P,
Milana M, O’Brien A, Rebre S, Rijk R, Piringer O: Evaluation of migration
models that might be used in support of regulations for food-contact
plastics. Food Addit Contam 2005, 22:73–90.
2. De Meulenaer B, Huyghebaert A: Packaging and other food contact
material residues. In Handbook of Food Analysis, Vol. 2. 2nd edition. Edited
by Nollet LML, Marcel D. NY: Inc; 2004:1297–1330.
3. Yang CZ, Yaniger SI, Jordan VC, Klein DJ, Bittner GD: Most plastic products
release estrogenic chemicals: a potential health problem that can be
solved. Environ Health Perspect 2011, 119(7):989–996.
4. Vom Saal FS, Nagel SC, Timms BG, Welshons WV: Implications for human
health of the extensive bisphenol A literature showing adverse effects at
low doses: a response to attempts to mislead the public. Toxicology 2005,
212(2–3):244–252. Author reply 253–254.
5. Talsness CE, Andrade AJ, Kuriyama SN, Taylor JA, Vom Saal FS:
Components of plastic: experimental studies in animals and
relevance for human health. Philos Trans R Soc Lond B Biol Sci 2009,
364(1526):2079–2096.
6. Vandenberg LN, Colborn T, Hayes TB, Heindel JJ, Jacobs DR, Lee D-H,
Shioda T, Soto AM, vom Saal FS, Welshons WV, Zoeller RT, Myers JP:
Hormones and endocrine-disrupting chemicals: low-dose effects
and nonmonotonic dose responses. Endocr Rev 2012, 33:378–455.
doi:10.1210/er.2011-1050.
7. Barrett JR: Assessing the safety of a replacement chemical: nongenomic
activity of bisphenol S. Environ Health Perspect 2013, 121(3):a97. PMID:
23454847.
8. Vinas R, Watson CS: Bisphenol S disrupts estradiol-induced nongenomic
signaling in a rat pituitary cell line: effects on cell functions. Environ
Health Perspect 2013, 121:352–358.
9. Kojima H, Takeuchi S, Toshihiro I, Mitsuru I, Kobayashi S, Yoshida T: In vitro
endocrine disruption potential of organophosphate flame retardants via
human nuclear receptors. Toxicology 2013, 314:76–83.
10. ICCVAM: 2003 ICCVAM Evaluation of In Vitro Test Methods for Detecting Potential
Endocrine Disruptors: Estrogen Receptor and Androgen Receptor Binding and
Transcriptional Activation Assays. 2003. NIH Publication No. 03–4503. Available:
http://ntp.niehs.nih.gov/iccvam/docs/endo_docs/edfinalrpt0503/edfinrpt.pdf.
11. ICCVAM: Addendum to Evaluation of In Vitro Test Methods for Detecting
Potential Endocrine Disruptors: Estrogen Receptor and Androgen Receptor
Binding and Transcriptional Activation Assays. 2006. NIH Publication
No. 03–4503. Available: http://ntp.niehs.nih.gov/iccvam/docs/endo_docs/
edaddendfinal.pdf.
12. National Research Council: Hormonally Active Agents in the Environment.
Washington: DC, National Academies Press; 1999.
13. Hewitt SC, Deroo BJ, Korach KS: Signal transduction: a new mediator for
an old hormone? Science 2005, 307(5715):1572–1573.
14. Matsushima A, Teramoto T, Okada H, Liu X, Tokunaga T, Kakuta Y,
Shimohigashi Y: ERR gamma tethers strongly bisphenol A and 4-alpha-
cumylphenol in an induced-fit manner. Biochem Biophys Res Commun
2008, 373(3):408–413.
15. Gray J, Evans N, Taylor N, Rizzo J, Walker M: State of the evidence: the
connection between cancer and the environment. Int J Occup Environ
Health 2009, 15(1):43–78.
16. Soto AM, Sonnenschein C, Chung KL, Fernandez ML, Olea N, Serrano FO:
The E-SCREEN assay as a tool to identify estrogens: an update on
estrogenic environmental pollutants. Environ Health Perspect 1995,
103(Suppl 7):113–122.
17. Rogers JM, Denison MS: Recombinant cell bioassays for endocrine
disruptors: development of a stably transfected human ovarian cell line
for the detection of estrogenic and anti-estrogenic chemicals. In Vitro
Mol Toxicol 2000, 13(1):67–82.
18. Yang CZ, Casey W, Stoner MA, Kollessery GJ, Wong AW, Bittner GD: A
robotic MCF-7 cell proliferation assay to detect agonist and antagonist
estrogenic activity. Toxicol Sci 2013, in press. Epub 2013 Nov 9.
19. Della Seta D, Minder I, Belloni V, Aloisi AM, Dessì-Fulgheri F, Farabollini F:
Pubertal exposure to estrogenic chemicals affects behavior in juvenile
and adult male rats. Horm Behav 2006, 50(2):301–307.
20. Patisaul HB, Adewale HB: Long-term effects of environmental endocrine
disruptors on reproductive physiology and behavior. Front Behav Neurosci
2009, 3:10.
21. Sliney DH: Radiometric quantities and units used in photobiology and
photochemistry: recommendations of the Commission Internationale de
l’Eclairage (International Commission on Illumination). Photochem
Photobiol 2007, 83:425–432.
22. Leusch FD, de Jager C, Levi Y, Lim R, Puijker L, Sacher F, Tremblay LA,
Wilson VS, Chapman HF: Comparison of five in vitro bioassays to measure
estrogenic activity in environmental waters. Environ Sci Technol 2010,
44(10):3853–3860.
23. NIEHS: OECD Guideline for the Testing of Chemicals. 2012. Available:
http://ntp.niehs.nih.gov/NTP/About_NTP/SACATM/2012/September/
B_DRAFT_Updated_OECD_TG455_508.pdf.
24. ICCVAM: Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM) Background Review Document: Validation Study of the
BG1Luc4E2 Estrogen Receptor (ER) Transcriptional Activation (TA) Test
Method. 2011. Available: http://ntp.niehs.nih.gov/iccvam/docs/endo_docs/
ERTA-TMER/AppxC/BRD.pdf”Retrieved January 17, 2014.
25. Howdeshell KL, Peterman PH, Judy BM, Taylor JA, Orazio CE, Ruhlen RL,
Vom Saal FS, Welshons WV: Bisphenol A is released from used
Bittner et al. Environmental Health 2014, 13:41 Page 13 of 14
http://www.ehjournal.net/content/13/1/41
polycarbonate animal cages into water at room temperature. Environ
Health Perspect 2003, 111(9):1180–1187.
26. Le HH, Carlson EM, Chua JP, Belcher SM: Bisphenol A released from
polycarbonate drinking bottles mimics the neurotoxic actions of
estrogen in developing cerebellar neurons. Toxicol Lett 2008, 176:149–156.
27. Day M, Wiles DM: Photochemical degradation of poly (ethylene
terephthalate): II Effect of wavelength and environment on the
decomposition process. J Appl Polym Sci 1972, 16:191–202.
28. Day M, Wiles DM: Photochemical degradation of poly (ethylene
terephthalate): III Determination of decomposition products and
reaction mechanism. J Appl Polym Sci 1972, 16:203–215.
29. O’Donnell B, White JR, Holding SR: Molecular weight measurement in
weathered polymers. J Appl Polym Sci 1994, 52:1607–1618.
30. Grossetête T, Rivaton A, Gardette JL, Hoyle CE, Ziemer M, Fagerburg DR,
Clauberg H: Photochemical degradation of poly (ethylene terephthalate)-
modified copolymer. Polymer 2000, 41:3541–3554.
31. Zhang R, Chen H, Cao H, Huang CM, Mallon PE, Li Y: Degradation of
polymer coating systems studied by positron annihilation spectroscopy.
IV. Oxygen effect of UV irradiation. J Polym Sci B Polym Phys 2001,
39:2035–2047.
32. Christiaens FJ, Chardon A, Fourtanier A, Frederick JE: Standard ultraviolet
daylight for nonextreme exposure conditions. Photochem Photobiol 2005,
81:874–878.
33. Kollias N, Ruvolo E, Sayre RM: The value of the ratio of UVA to UVB in
sunlight. Photochem Photobiol 2011, 87:1474–1475.
34. Allen N, Rivalle G, Edge M, Roberts I, Fagerburg DR: Characterisation
and identification of fluorescent hydroxylated terephthalate species
in the thermal and UV degradation of poly (ethylene-co-1, 4-
cyclohexanedimethylene terephthalate) (PECT). Polym Degrad Sta bil
2000, 67:325–334.
35. Eastman: US Patents. 2014. http://patft.uspto.gov/netacgi/nph-Parser?
Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.
html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=8299204&OS=8299204&RS=
8299204, accessed on January 10
th
, 2014.
doi:10.1186/1476-069X-13-41
Cite this article as: Bittner et al.:Estrogenic chemicals often leach from
BPA-free plastic products that are replacements for BPA-containing
polycarbonate products. Environmental Health 2014 13:41.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit
Bittner et al. Environmental Health 2014, 13:41 Page 14 of 14
http://www.ehjournal.net/content/13/1/41
