Most Plastic Products Release Estrogenic Chemicals: A Potential Health Problem That Can Be Solved

Abstract

Chemicals having estrogenic activity (EA) reportedly cause many adverse health effects, especially at low (picomolar to nanomolar) doses in fetal and juvenile mammals.
We sought to determine whether commercially available plastic resins and products, including baby bottles and other products advertised as bisphenol A (BPA) free, release chemicals having EA.
We used a roboticized MCF-7 cell proliferation assay, which is very sensitive, accurate, and repeatable, to quantify the EA of chemicals leached into saline or ethanol extracts of many types of commercially available plastic materials, some exposed to common-use stresses (microwaving, ultraviolet radiation, and/or autoclaving).
Almost all commercially available plastic products we sampled--independent of the type of resin, product, or retail source--leached chemicals having reliably detectable EA, including those advertised as BPA free. In some cases, BPA-free products released chemicals having more EA than did BPA-containing products.
Many plastic products are mischaracterized as being EA free if extracted with only one solvent and not exposed to common-use stresses. However, we can identify existing compounds, or have developed, monomers, additives, or processing agents that have no detectable EA and have similar costs. Hence, our data suggest that EA-free plastic products exposed to common-use stresses and extracted by saline and ethanol solvents could be cost-effectively made on a commercial scale and thereby eliminate a potential health risk posed by most currently available plastic products that leach chemicals having EA into food products.

Full text

Environmental Health Perspectives
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989
Research
Chemicals that mimic or antagonize the
actions of naturally occurring estrogens are
defined as having estrogenic activity (EA),
which is the most common form of endocrine
disruptor activity [Interagency Coordinating
Committee on the Validation of Alternative
Methods (ICCVAM) 2003, 2006; National
Research Council 1999]. Chemicals having
EA typically interact with one or more of the
classical nuclear estrogen receptor (ER) sub-
types: ERα, ERβ, or non classical membrane
or ER-related subtypes (Hewitt et al. 2005;
Matsushima et al. 2008; National Research
Council 1999). In mammals, chemicals hav-
ing EA can produce many health-related
problems, such as early puberty in females,
reduced sperm counts, altered functions of
reproductive organs, obesity, altered sex-
specific behaviors, and increased rates of some
breast, ovarian, testicular, and prostate cancers
(Della Seta et al. 2006; Gray 2008; Kabuto
et al. 2004; National Research Council
1999; Newbold et al. 2004; Patisaul et al.
2006, 2009). Fetal, newborn, and juvenile
mammals are especially sensitive to very low
(sometimes picomolar to nanomolar) doses of
chemicals having EA (Gray 2008; vom Saal
et al. 2005). Many of these effects observed in
mammals are also expected to be produced in
humans, because basic endocrine mechanisms
have been highly conserved across all classes
of vertebrates (Kavlock et al. 1996; National
Research Council 1999).
ermoplastics, which are used for many
items that contain food, are made by polymer-
izing a specific monomer or monomers in the
presence of catalysts into a high-molecular-
weight chain known as a thermo plastic poly-
mer [see Supplemental Material, Figure 1
(doi:10.1289/ehp.1003220)]. The resulting
polymer is mixed with small quantities of
various additives (anti oxidants, plasticizers,
clarifiers, etc.) and melted, mixed, extruded,
and pelletized to form a base thermo plastic
resin. Base resins are either used as is [e.g.,
bisphenol A (BPA)-based polycarbonate (PC),
non-BPA-based polypropylene (PP) copoly-
mer (PPCO), and non-BPA-based PP homo-
polymer (PPHO)] or, more commonly, mixed
with other resins, additives, colorants, and/or
extenders to form plastic compounds (e.g.,
polymer blends and pre colored polymers).
Plastic products are then made by using one
or more plastic compounds or resins to form
a finished plastic part that can be subjected to
finishing processes that may use inks, adhe-
sives, and so forth, to make a finished product.
As previously described (Begley et al. 1990,
2005; De Meulenaer and Huyghebaert 2004),
plastic resins and manufacturing protocols
[see Supplemental Material, Figure 1 (doi:10.
1289/ehp.1003220)] collectively use many
monomers and additives that may exhibit EA
because they have physico chemical properties,
often from an insufficiently hindered phenol
(HP) group, that enable them to bind to ERs
(see Supplemental Material, Table 1). Because
polymerization of monomers is rarely complete
and additives are not chemically part of the
polymeric structure, chemicals having EA can
leach from plastic products at very low (e.g.,
nano molar to pico molar) concentrations that
individually or in combination can produce
adverse effects, especially in fetal to juvenile
mammals. This leaching of monomers and
additives from a plastic item into its contents
is often accelerated if the product is exposed to
common-use stresses such as ultraviolet (UV)
radiation in sunlight, micro wave radiation,
and/or moist heat via boiling or dishwashing.
e exact chemical composition of almost any
commercially available plastic part is propri-
etary and not known. A single part may consist
of 5–30 chemicals, and a plastic item contain-
ing many parts (e.g., a baby bottle) may con-
sist of ≥ 100 chemicals, almost all of which
can leach from the product, especially when
stressed. Unless the selection of chemicals is
carefully controlled, some of those chemicals
will almost certainly have EA, and even when
using all materials that initially test EA free, the
stresses of manufacturing can change chemical
structures or create chemical reactions to con-
vert an EA-free chemical into one with EA.
Address correspondence to G.D. Bittner, CertiChem,
Inc., 11212 Metric Blvd., Suite 500, Austin, TX
78758 USA. Telephone: (512) 339-0550, ext. 201;
Fax: (512) 339-0551. E-mail: gbittner@certichem.
com
Supplemental Material is available online (doi:10.
1289/ehp.1003220 via http://dx.doi.org/).
This work was supported by National Institutes
of Health (NIH) grants R44 ES011469 (01-03)
and 1R43/44 ES014806 (01-03) to C.Z.Y.; R44
ES016964 (01-03) to S.I.Y.; and P30 CA051008
to V.C.J.
C.Z.Y. is employed by, and owns stock in,
CertiChem (CCi) and PlastiPure (PPi). S.I.Y. and
D.J.K. are employed by PPi. V.C.J. has no financial
interests in CCi or PPi, but he was principal inves-
tigator for a subcontract at Northwestern Medical
School to help develop the MCF-7 assay on NIH
grant P30 CA051008 awarded to CCi. G.D.B. owns
stock in, and is the founder and chief excutive officer
of CCi and the founder and chief scientific officer
of PPi. All authors had freedom to design, conduct,
interpret, and publish research uncompromised by
any controlling sponsor.
Received 16 November 2010; accepted 24 February
2011.
Most Plastic Products Release Estrogenic Chemicals: A Potential Health
Problem that Can Be Solved
Chun Z. Yang,1 Stuart I. Yaniger,2 V. Craig Jordan,3 Daniel J. Klein,2 and George D. Bittner1,2,4
1CertiChem Inc., Austin, Texas, USA; 2PlastiPure Inc., Austin, Texas, USA; 3Lombardi Comprehensive Cancer Center, Georgetown
University Medical Center, Washington, DC, USA; 4Neurobiology Section, School of Biology, University of Texas, Austin, Texas, USA
Background: Chemicals having estrogenic activity (EA) reportedly cause many adverse health
effects, especially at low (picomolar to nanomolar) doses in fetal and juvenile mammals.
oBjectives: We sought to determine whether commercially available plastic resins and products,
including baby bottles and other products advertised as bisphenol A (BPA) free, release chemicals
having EA.
Me t h o d s : We used a roboticized MCF-7 cell proliferation assay, which is very sensitive, accurate,
and repeatable, to quantify the EA of chemicals leached into saline or ethanol extracts of many types
of commercially available plastic materials, some exposed to common-use stresses (microwaving,
ultraviolet radiation, and/or autoclaving).
re s u l t s : Almost all commercially available plastic products we sampled—independent of the type
of resin, product, or retail source—leached chemicals having reliably detectable EA, including those
advertised as BPA free. In some cases, BPA-free products released chemicals having more EA than
did BPA-containing products.
conclusions: Many plastic products are mischaracterized as being EA free if extracted with only
one solvent and not exposed to common-use stresses. However, we can identify existing compounds,
or have developed, monomers, additives, or processing agents that have no detectable EA and have
similar costs. Hence, our data suggest that EA-free plastic products exposed to common-use stresses
and extracted by saline and ethanol solvents could be cost-effectively made on a commercial scale
and thereby eliminate a potential health risk posed by most currently available plastic products that
leach chemicals having EA into food products.
ke y words: bisphenol A, endocrine disruptor, endocrine-disrupting chemical, estrogen receptor
binding, estrogenic activity, plastic. Environ Health Perspect 119:989–996 (2011). doi:10.1289/
ehp.1003220 [Online 2 March 2011]

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Very few studies (Soto et al. 1991; Till
et al. 1982) have examined the extent to
which plastics that presumably do not con-
tain BPA nevertheless release other chemicals
having detectable EA. For example, a recent
comprehensive review [table on page 72 of
Gray (2008)] described polyethylene (PE),
PP, and PE terephthalate (PET) plastics as
being “‘OK’ for use with respect to release of
chemicals exhibiting EA.”
Here, we report that most of the > 500
commercially available plastic products that we
sampled—even those that are presumably BPA
free—release chemicals having detectable EA,
especially if they are assayed by more polar and
less polar solvents and exposed to common-use
stresses. at is, we show that, to reliably detect
such leachable chemicals having EA, unstressed
or stressed plastic resins or products should
be extracted with more polar (e.g., saline) and
less polar [e.g., ethanol (EtOH)] solutions and
exposed to common-use stresses (boiling water,
micro waving, and UV radiation).
Materials and Methods
We developed a sensitive and accurate roboti-
cized version of the MCF-7 cell prolifera tion
assay (E-SCREEN assay) that has been used
for decades to reliably assess EA and anti-EA
(Leusch et al. 2010; Soto et al. 1995) and is
currently undergoing validation for interna-
tional use by ICCVAM/NTP (National
Toxicology Program) Interagency Center for
the Evaluation of Alternative Toxicological
Methods (NICEATM). Chemicals with EA
bind to ERs (ERα, ERβ, or ER-related sub-
types) and activate the transcription of estrogen-
responsive genes, which leads to proliferation of
MCF-7 cells.
Detailed methods for the MCF-7 assay
are provided in Supplemental Material,
(doi:10.1289/ehp.1003220). In brief, plastic
resins or products were extracted using saline,
a more polar solvent, or EtOH, a less polar sol-
vent. Aliquots of the extracts were then diluted
four to eight times to produce up to eight test
concentrations. Each test chemical or extract
at each concentration was added in triplicate
or quadruplicate to 96-well plates containing
MCF-7 cells in EA-free culture media. After
6 days of exposure, the amount of DNA per
well, an indication of cell prolifera tion, was
assayed using a micro plate modification of the
Burton diphenylamine assay (Burton 1956;
Natarajan 1994).
The effect of a test chemical or extract
on proliferation was expressed as the %E2,
a percentage of the maximum DNA per
well produced by the maximum response to
17β-estradiol (E2; positive control) corrected
by the DNA response to the vehicle (nega tive)
control [see Supplemental Material, Equation 1
(doi:10.1289/ehp.1003220)]. For estrogenic test
chemicals, the concentration needed to obtain
half-maximum stimulation of cell proliferation
[half-maximal effective concentration (EC50),
a measure of binding affinity] was calculated
from best fits to dose–response data that meet a
well-defined set of criteria by Michaelis-Menton
kinetics. e estrogenicity of extracts was calcu-
lated as the relative maximum %E2 (%RME2;
a measure of response amplitude), a percentage
of the maximum DNA per well produced by
an extract at any dilution with respect to the
maximum DNA per well produced by E2 at
any dilution, corrected by the DNA response to
the vehicle (negative) control (see Supplemental
Material, Equation 2). If a test chemical had a
positive response (> 15% RME2) but an EC50
could be calculated because not all criteria were
met, then the estrogenicity of the test chemical
was characterized simply as EA positive or by its
%RME2.
e EA of a test chemical or extract was
considered detectable if it produced cell pro-
liferation > 15% of the maximum response
to E2 (> 15% RME2), which is > 3SDs
Figure 1. Results of MCF-7 assays shown as dilution response curves (%E2) for E2 (A), E2 and BPA (B), BHA (C), and %RME2 of extracts of plastic bags (D), a PC
bottle (E), and a BPA-free bottle made from PETG (F). Abbreviations: PETG, PET glycol-modified polyethylene terephthalate; VC, vehicle control. Dotted lines repre-
sent 3 SD from the response. In B–F, the negative control (1% EtOH or saline) equals 0% E2. The E2 standard (10–9 M) is the positive control diluted as indicated in
C–F. Each point plotted is the average of three or four replicates for each concentration whose SD is very small and falls within the space taken up by each data
point. In (A), E2 was dissolved in EtOH (standard extract) or concentrated 10× and rediluted to show that the EtOH concentration protocol has very little effect on
the EC50 of E2 (50% E2). The EC50 of E2 is approximately 1.3 × 10–13 M, and the threshold of detection (15% E2) is approximately 10–15 M. The maximum E2 response
was attained at 10–11 M and remained constant at higher E2 concentrations. (B) The EC50 of both E2 (as in A) and BPA is approximately 6.6 × 10–8 M, and threshold
detection is approximately 10–9 M, all suppressed by 10–8 M ICI. (C) BHA does not meet criteria needed for accurate calculation of EC50 [see Supplemental Material,
pp. 5–7 (doi:10.1289/ehp.1003220)]. EA is positive; its maximum response is about 50% E2 (i.e., 50% RME2) and is suppressed by 10–8 M ICI. In D, commercially avail-
able plastic bags were extracted by 100% EtOH. Commercially available PC (E) and BPA-free (F) bottles were extracted with saline or EtOH as indicated.
120
100
80
60
40
20
0
–20
120
100
80
60
40
20
0
–20
120
100
80
60
40
20
0
–20
120
100
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0
–20
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100
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0
–20
120
100
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60
40
20
0
–20
–20 –6.0 –5.5 –4.5
E2E2 and BPA BHA
Commercial bag extracts PC bottle extracts BPA-free bottle extracts
–3.5–5.0 –4.0 –3.0
1/12,500 1/12,500
1/2,500 1/500 1/100 1/2,500 1/500 1/100 1/12,500 1/2,500 1/500 1/100
–18 –16 –14 –14 –13 –12 –11 –10 –9 –8 –7 –6 –5 –4–15
Log (M) Log (M) Log (M)
Extraction dilution Extraction dilution Extraction dilution
%E2
%E2
%E2
%RME2
%RME2
%RME2
–12 –10 –8
BHA
3 SD of VC
BHA + ICI
E2
3 SD of VC
EtOH extract
EtOH extract + ICI
Saline extract
Saline extract + ICI
E2
3 SD of VC
EtOH extract
EtOH extract + ICI
Saline extract
Saline extract + ICI
E2
3 SD of VC
Freezer bag 1
Freezer bag 1 + ICI
Storage bag 1
Storage bag 1 + ICI
E2
E2 + ICI
BPA
BPA + ICI
3 SD of VC
E2 standard
E2 concentrated 10 ×
VC
3 SD of VC

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from the historic control baseline response
(about 10–15 M), which is a rather conserva-
tive measure of EA detectability. Stimulation
of MCF-7 proliferation induced by the test
chemical or extract was confirmed to be
estrogenic (compared with non specific) in
an EA confirmation study: If the stimulation
of MCF-7 proliferation by a test chemical or
extract was suppressed by coincuba tion with
a strong anti estrogen [ICI 182,780 (ICI) at
10–7 to 10–8 M], the EA of the test chemical
or extract was confirmed. Therefore, a test
chemical or extract was classified as not hav-
ing detectable EA if it did not induce MCF-7
cell proliferation or if it induced proliferation
that could not be inhibited by ICI.
Figure 1 shows typical MCF-7 responses
plotted as %E2. Figure 1A–C show responses to
some test chemicals: E2 (positive control), BPA,
and butylated hydroxy anisole (BHA; a com-
mon anti oxidant). Figure 1D–F show %RME2
responses to test extracts of plastic food bags,
PC bottles, and BPA-free baby bottles and their
ICI-suppressed responses, confirming their EA.
Some chemicals or products were also ana-
lyzed for anti-EA [for details, see Supplemental
Material, pp. 7–8 (doi:10.1289/ehp.1003220)].
Purchase and analyses of plastic products
in survey studies. For Tables 1 and 2, we pur-
chased 455 plastic products used to contain
foodstuffs from various commercial retailers
from 2005 through 2008. The rela tive fre-
quency of products having detectable EA did
not change with later compared with earlier
purchases. In some cases, we instructed under-
graduate students or employees to purchase
a mix of plastic items used to contain food-
stuffs from a given large retailer (Albertsons,
H-E-B, Randalls, Target, Wal-Mart, Trader
Joe’s, and Whole Foods) mainly in the Austin,
Texas, or Boston, Massachusetts, areas, some
of which market many “organic” products. In
other cases, we purchased products of a par-
ticular plastic type (e.g., PE- or PP-based con-
tainers). We recorded the retailer, resin type
[high-density PE (HDPE), PET, PC, PP, poly-
styrene (PS), poly lactic acid], and product type
(flexible packaging, food wrap, rigid packag-
ing, baby bottle component, deli containers,
plastic bags). In addition, because the contents
of some plastic items might have added or
extracted chemicals having EA from the plastic
containers before we purchased and tested the
products (Sax 2010), we recorded whether the
plastic items had contents or were empty when
purchased. For any plastic container having
contents, we thoroughly washed out the con-
tainer with distilled water before testing the
plastic. Except for PC-based items, none of
these products were known to contain BPA.
(Plastic products typically do not list their
chemical composition, which is proprietary
to the manufacturer.) Samples were chosen
in product areas where adverse health effects
might occur if the samples leached chemicals
having EA. Samples from each retailer gener-
ally included most of the product types listed
above. In addition to surveying commercially
available products, we tested plastic resins [e.g.,
PC, PET, glycol-modified PET (PETG)] that
were purchased from M. Holland Company
(Northbrook, IL) and individual chemicals
used to manufacture plastic products [e.g.,
BPA, BHA, butylated hydroxytoluene (BHT),
dimethyl terephthalate, etc.] that were pur-
chased in their purest form from Sigma-Aldrich
(St. Louis, MO).
Many plastic products have more than
one plastic part. For example, baby bottles
have 3–10 different plastic parts in vari-
ous combinations [bottle, nipple, anti colic
item(s), sealing ring(s), liner bag, cap, etc.],
each part typically having different and rather
unique combinations of 5–30 chemicals. Over
the course of this entire study, we assayed
> 100 component parts from > 20 different
baby bottles, including many advertised as
BPA free. Only some (13) of these compo-
nent parts were purchased for the initial survey
study (Tables 1 and 2).
Table 1. Percentage of unstressed plastic products having EA in at least one extract.
Extraction solvent
EtOH Concentrated EtOH Saline Any extract
Plastic product n%D n%D n%D n%D
Resin type
HDPE 13 69 11 55 18 56 30 70
PP 23 52 6 33 16 81 37 68
PET 30 40 17 94 34 76 57 75
PS 13 62 — — 16 38 28 50
PLA 10 70 1 100 8 100 11 91
PC 1 0 1 100 2 100 2 100
Product type
Flexible packaging 82 66 6 33 35 74 121 67
Food wrap 9 100 — — 9 78 9 100
Rigid packaging 57 56 18 67 31 45 83 64
Baby bottle component 13 69 — — 16 94 19 89
Deli containers 11 36 — — 7 7 16 44
Plastic bags 33 97 1 100 23 96 43 98
Product retailer
Large retailer 1 31 81 2 100 4 75 36 81
Large retailer 2 4 50 4 0 50 54 53 53
Large retailer 3 18 83 2 100 7 29 25 72
Large retailer 4 37 51 — — — — 37 51
Large retailer 5 20 50 3 100 4 100 23 70
Organic retailer 1 28 71 5 60 5 80 32 81
Organic retailer 2 33 88 1 100 10 80 35 89
Total for extract 308 68 51 73 214 69 455 72
Abbreviations: —, not tested; %D, percent detectable (extract produced cell proliferation > 15% RME2; see “Materials and
Methods”); n, total number of samples purchased (less than the sum of n values for individual extracts if some items were
tested by more than one extraction protocol); PLA, poly lactic acid. Data are percentages of samples for which EA was
detected using a standard or concentrated EtOH extract, a saline extract, or one or more such extracts (any extract). Some
individual items are listed in two or three categories (e.g., PET and baby bottles) but were counted only once for the extract
total. Baby bottle components comprised 11 bottles and 2 sealant ring components.
Table 2. Percentage of unstressed plastic products having detectable EA (> 15% RME2) in two extracts.
Extraction solvent
Category nEtOH only Saline only
Both EtOH
and saline
Either EtOH
or saline
HDPE 13 15 31 15 61
PET 21 19 29 52 100
PP 4 0 25 75 100
PLA 7 0 14 86 100
Bottles 38 13 34 42 89
Baby bottles 11 0 36 64 100
Rigid packaging 10 30 20 40 90
Food wrap 8 25 0 75 100
All products 102 17 21 54 92
PLA, poly lactic acid. Values shown are percent (%) of unstressed plastic items (n) having detectable EA (> 15%RME2)
only in an EtOH extract (and not in a saline extract), only in a standard saline extract (and not in an EtOH extract), in both
EtOH and saline extracts, or in either EtOH or saline extracts. The last column is the sum of the three previous columns.
“All products” is the total for each column when each product (n = 102) is only counted once (some products are listed
in two categories). The standard EtOH extract was used for most (n = 81) products and the concentrated EtOH extract
for the remainder (n = 21). If EA was detected in a saline or standard EtOH extract in survey studies such as those
reported in Table 1, other extracts often were not performed. A concentrated EtOH extract was usually used to generate
data shown in Tables 1 and 2 only if EA was not detected in a saline or standard EtOH extract. That is, samples listed for
concentrated EtOH in Table 1 and EtOH in Table 2 had a selection bias for not having detectable EA.

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Most of the samples (338 of 455) in the
survey study (Tables 1 and 2) were extracted
using only one extraction protocol. For the
remaining samples (n = 102), both saline and
EtOH extractions were used so that the effi-
cacy of each protocol could be directly com-
pared. We used a paired Student’s t-test to
test whether differences between pairs of sam-
ples were statistically significant (p < 0.05).
Protocols for common-use stresses of some
plastic items. Given that common-use stresses
can alter the complex chemical composition
of plastics and/or increase the rate of leaching
(Begley et al. 1990, 2005; De Meulenaer and
Huyghebaert 2004), for some resins or prod-
ucts, we examined how leaching of chemicals
having EA might be affected by exposure to
micro wave radiation, auto claving (moist heat),
and UV light. Additional plastic items, some
of which are described in Figure 2 and Table 3,
were purchased in 2008–2010 and subjected
to common-use stresses. In addition, we tested
a variety of resins (including PE- and PP-based
resins; Table 3), anti oxidants [see Supplemental
Material, Table 3 (doi:10.1289/ehp.1003220)],
and other additives or processing agents (see
Supplemental Material, Table 4) identified by
our laboratory as being free of detectable EA
and hence possibly suitable for use to produce
final products that would be EA free even after
exposure to common-use stresses.
We used the following stresses:
•Sampleswereplacedabout2 feet from a
254-nm fluorescent fixture for 24 hr, simu-
lating repeated UV stress by sunlight (e.g.,
water bottles) or UV sterilizers (e.g., baby
bottles and medical items)
•Samples wereautoclavedat 134°C for
8 min, simulating moist heat stress in an
automatic dishwasher
•Weheatedsamplesin a microwave 10times
for 2 min each, using a 1,000-W kitchen
microwave oven set to “high,” simulating heat
and microwave radiation stress to reusable
food containers.
Results
Release of chemicals having EA from unstressed
plastics. Tables 1 and 2 show the percentage
of samples in each category that had reliably
detectable EA (> 15% RME2) in our survey
of 455 commercially available plastic products.
[For the %RME2 and content status of indi-
vidual samples, as well as the average %RME2
for products classified by resins (HDPE, PP,
PET, PS, poly lactic acid, PC), product type
(flexible packaging, food wrap, rigid packag-
ing, baby bottle components, plastic bags), and
retailer (large retailers 1–5 and large organic
retailers 1 and 2), see Supplemental Material,
Table 5 (doi:10.1289/ehp.1003220).] For
example, 9 of 13 HDPE plastic products
extracted by our standard EtOH protocol
(69%) had detectable EA (Table 1), with a
%RME2 (mean ± SD) of 66% ± 25% (see
Supplemental Material, Table 5A). For PET
products extracted by saline, 26 of 34 (76%)
had detectable EA (Table 1) with a %RME2
of 64% ± 41% (see Supplemental Material,
Table 5C). We found no consistent corre-
lation between the percentage of items in a
product type with detectable EA and their
mean %RME2 (data not shown).
We found no significant difference
(p > 0.05) in the percentage of items with
detectable EA between those with contents
and those with no contents (76%, n = 160)
at the time of purchase based on the stan-
dard EtOH extraction protocol [67% vs.
70%; see Supplemental Material, Table 2A
(doi:10.1289/ehp.1003220)] , the stan-
dard saline protocol (62% vs. 75%; see
Supplemental Material, Table 2C), or all
extraction protocols combined (69% vs.
76%). Most important, items with no con-
tents in all categories exhibited detectable EA
in at least one protocol (see Supplemental
Material, Tables 2 and 5), including 78% of
items made from HDPE (n = 18), 57% from
PP (n = 14), and 100% from PET (n = 6).
Given all of these results, we present the data
for all items shown in Tables 1 and 2 without
regard to their content status.
Using different solvents increased the prob-
ability of detecting EA. Most (71%) unstressed
plastic items released chemicals with reliably
detectable EA in one or more extraction proto-
cols, independent of resin type, product type, or
retailer (Table 1). Results often differed between
saline and EtOH extracts of the same unstressed
plastic item, and EA was reliably detected most
frequently (92% of all items listed in Table 2)
when analyzed using both saline (more polar)
and EtOH (less polar) extracts. For exam-
ple, 15% of unstressed HDPE plastic items
leached chemicals with detectable EA into both
EtOH and saline extracts, 15% leached only
into EtOH, and 31% leached only into saline
(Table 2). at is, the leaching of a chemical
with EA was significantly (p < 0.01) more likely
to be detected if we used both polar and non-
polar solvents (61%) than if we used only one
solvent (30% for EtOH only or 45% for saline
Figure 2. Total EA released by some PC and BPA-free water bottles (W) and baby bottles (B). The leach-
ing of chemicals having EA (measured as %RME2; excluding caps, nipples, and other components) were
extracted using saline or EtOH as solvents and exposed to auto claving, micro waving, and/or UV light (see
“Materials and Methods” for details). BPA-free water bottles W1, W2, W3, and W4 are PETG, and W5 is
PET. BPA-free baby bottles B1 and B2 are polyethersulfone; B3 is PETG; and B4 and B5 are PP. Orange bars
indicate the data set for each individual product. The %RME2 for saline extracts is represented by solid
black lines and for EtOH as solid red lines. Symbols represent the %RME2 of chemicals released by each
assay of a product after an autoclaving stress, microwaving stress, and UV light stress (see figure key).
The dotted horizontal line at 15% RME2 is the rather conservative value below which EA was considered
non detectable (ND) for any assay. For some products shown (e.g., PC B1, BPA-free B4), if one solvent and/
or stress condition showed reliably detectable EA, other solvents and stress conditions were not subse-
quently tested. Some values plotted as 0% RME2 actually had slightly negative %RME2 values (–1% to
–7% RME2) due to cellular toxicity.
140
120
100
80
60
40
20
0
%RME2
B1W1W2W3
B2B1B2B3B4B5W1W2W3W4W5
Product
PC
Baby Water Baby Water
BPA-free
ND
Autoclave
Microwave
UV light

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only). We obtained similar results for all types
of plastic products (data not shown).
Assays of > 100 component parts from
> 20 different baby bottles, including many
advertised as BPA free, indicated that extracts
of at least one bottle component of each
baby bottle always had EA based on at least
one assay (some data shown in Table 2 and
Figure 2), as did at least one other component
part (data not shown).
Stresses increased the release of chemicals
having EA. Leaching of chemicals with EA was
increased by common stresses. For example,
one unstressed sample of an HDPE resin (P5
in Table 3) that had no detectable EA (i.e.,
RME2 < 15%) in two saline extracts and
two EtOH extracts released chemicals with
EA equivalent to 47% RME2 when extracted
using EtOH after the resin was stressed with
UV light. Similarly, two samples of low-density
PE resins (LDPE resins 1 and 2) and PETG
resins (PETG baby bottle and PETG resin 1)
that had no detectable EA before stressing sub-
sequently exhibited EA when stressed, espe-
cially by UV (Table 3). Samples (n > 10) of
products made from PETG resins advertised
as BPA free all released detectable EA when
stressed, especially by UV light. Similarly, 25%
of unstressed samples of PET and 50% of
unstressed PS products surveyed did not have
detectable EA in assays of EtOH and/or saline
extracts (Table 1). However, when stressed and
assayed using both saline and EtOH extracts,
all PET (n > 10) and PS (n > 10) products
released chemicals having detectable EA in at
least one extracting solvent (Table 3).
EA-containing and EA-free monomers.
Polymerization of monomers is rarely com-
plete, and unpolymerized monomers are
almost always released from polymer resins
(Begley et al. 1990, 2005; De Meulenaer and
Huyghebaert 2004). PE and PP polymers
are often used to manufacture flexible and/or
non transparent rigid products (Figure 3).
MCF-7 assays (n = 6) consistently showed
that extracts of “barefoot” (no additives) poly-
mers (e.g., LDPE resin P1 in Table 3) were
EA free, even when stressed. (PP-based poly-
mers require anti oxidants to prevent severe
degradation during their use in manufactur-
ing plastic products.) Furthermore, PE- and
PP-based resins containing appropriate addi-
tives to produce fit-for-use products could be
constructed that remained EA free (n > 100
assays of > 10 resins), even when exposed to
common-use stresses. Representative data
from several such resins (LDPE resin P1,
HDPE resin P2, PP homo polymer resin P3,
PP copolymer resin P4) are shown in Table 3.
Figure 3 also shows other monomers and
polymers that can or cannot be used to make
hard-and-clear (HC) plastics. For example,
HC PC plastics (n > 10) all released chemi-
cals having EA (e.g., PC baby bottle B1 and
PC water bottle W1 in Figure 2), almost cer-
tainly phenolics such as BPA (Figure 1B). e
di methyl terephthalate monomer used to make
PET and PETG plastics exhibited anti-EA
(n = 3 assays; data not shown; for anti-EA
assay protocol, see Supplemental Material
(doi:10.1289/ehp.1003220)]. Furthermore,
breakdown products of dimethyl terephthalate,
PET, and PETG resins probably contain and
release phenolic moieties that have EA that
account for some of the data for PET prod-
ucts in Tables 1 and 2. Polyethersulfone HC
products also consistently released chemicals
having EA or anti-EA, especially when stressed
with UV light (data not shown), possibly from
unreacted phenolic monomer residues or phe-
nolic stress-degradation products. In contrast,
some HC cyclic olefin polymer/cyclic olefin
copolymer polymers produced from saturated
cyclic olefin monomers contained no phenolics
and did not release chemicals having detectable
EA, even when stressed (Table 3).
Polymers that can be made EA free have
a similar cost compared with polymers made
from monomers that have EA. For example,
currently, clarified PP having no additives
that exhibit EA (even when stressed) that is
suitable for molding bottles costs approxi-
mately $1.20/lb. PP resins containing addi-
tives that have EA also cost about $1.20/lb.
Commodity resins such as PET, which are
made from monomers having EA and are
suitable for molding bottles, are priced at
approximately $1.28/lb (Plastics News 2011).
EA-containing and EA-free ad ditives.
Many additives are physically, but not
chemically, bound to a polymeric structure
and hence can almost always leach from the
polymer, especially when stressed (Begley et al.
1990, 2005; De Meulenaer and Huyghebaert
2004). Anti oxidants are the most critical class
of additives because they prevent or mini-
mize plastic degradation due to oxidation that
breaks polymer chains (chain scission) and/
or causes cross-links (Kattas et al. 2000). e
oldest and most common anti oxidants deemed
suitable for food contact belong to a chemical
class known as HPs (hindered phenols), such
as BHT and BHA, in large part because both
are inexpensive and assumed to be non toxic.
However, BHT (n = 4 assays) had reliably
detectable EA, as did BHA (n = 3 assays). [e
EC50 of BHT and BHA (Figure 1C) could
not be accurately calculated because both also
exhibited cellular toxicity at higher concentra-
tions (10–5 M).] Other commonly used HP
anti oxidants (n = 4/5) and organophosphines
(n = 6/7) also exhibited reliably detectable
EA, especially when exposed to moist heat,
which presumably causes hydrolysis (data not
shown). For example, proprietary anti oxidants
Phos (phosphate) OX 1 and HP AOX 2 had
no detectable EA, whereas HP AOX 1 and
Ph (bisphenol) AOX 1 had reliably detect-
able EA [see Supplemental Material, Table 3
(doi:10.1289/ehp.1003220)].
Many other additives (n > 50) with a
phenolic group had reliably detectable EA,
such as agents found in many base resins
[tris(nonylphenyl) phosphite, octyl phenol,
nonyl phenol, butyl benzene phthalate],
colorants (especially blues or greens with
Table 3. Representative %RME2 values for stressed resins or parts made from flexible or HC polymers.
Stress/extraction solvent
Microwave UV Autoclave
Sample type Saline EtOH Saline EtOH Saline EtOH
Flexible polymers
LDPE resin 1 5 7 0 4 4 30a
LDPE resin 2 3 7 26a3 –1 27a
PET water bottle 100a3 31a2 47a1
LDPE resin P1 2 3 0 0 4 5
HDPE resin P2 6 –4 2 –2 –1 –3
PPHO resin P3 0 –4 3 2 –6 –3
PPCO resin P4 3 7 –7 –6 –9 –3
HDPE resin P5 ND ND ND 47aND ND
HC polymers
Water bottle 1.1 3 23a71a17a–1 19a
Water bottle 1.2 4 21a57,a 69,a 98a48,a 39a8 23a
Water bottle 2.1 –7 –5 81a22a0 4
Water bottle 2.2 34a–2 80a12 –1 1
PETG baby bottle 0 –2 122a44a0 1
PETG resin 1 –8 17a61a111a0 15a
PS 1 4 3 17a45a76a0
COC 3 9 7 20a20a0 6
COC resin P18 4 1 9 11 1 –2
COC resin P19 6 2 6 –2 4 2
Abbreviations: COC, cyclic olefin copolymer; ND, not determined; PPCO, polypropylene copolymer; PPHO, polypropylene
homopolymer. Numerical values are %RME2 responses of extract for several different baby bottle and other component
parts. Resins designated with P (e.g., P1, P18) are EA-free formulations developed at PlastiPure. Resin P5 exhibited EA
when stressed. Multiple values for water bottle 1.2 under UV stress are data for repeated analyses.
aPlastic items leaching chemicals having detectable EA > 15% RME2.

Yang et al.
994
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Environmental Health Perspectives
phthalo cyanine groups), PS-based purge
compounds, and mold-release agents [see
Supplemental Material, Table 4 (doi:10.1289/
ehp.1003220)]. In contrast, many metal-
oxide–based inorganic pigments did not
exhibit EA. However, these EA-free pigments
are often mixed with dispersing agents and
carrier resins that have EA to produce colo-
rant master batch concentrates. Nevertheless,
we have identified resins, dispersants, pig-
ments, and anti oxidants that are approved by
the Food and Drug Administration for direct
food contact (see Supplemental Material,
Tables 3 and 4) to create colorant master-
batch concentrates (n > 100) that produce
even colorant dispersion into plastics and that
have no detectable EA, cellular toxicity, or
adverse processing effects, even when stressed.
Because additives comprise a small frac-
tion (typically 0.1–1% by weight) of plastic
resins and compounds and because plastic
resins and compounds using EA-free addi-
tives are processed during manufacture in a
nearly identical manner as conventional resins
and compounds containing chemicals with
EA, the replacement of additives having EA
with EA-free additives should have very lit-
tle impact on the cost of the final product.
Furthermore, EA-free additives have only a
slightly higher or no additional cost compared
with additives with EA, so that their cost
impact is very small or non existent.
Products currently marketed as BPA free
are not EA free. In response to market and
regulatory pressures to eliminate BPA in HC
plastics, BPA-free HC materials have recently
been introduced as replacements for PC res-
ins. PET and PETG are two such resins, but
HC plastic products made from these res-
ins leached chemicals that had detectable EA
(Tables 1–3, Figures 2 and 3), often in the
absence of exposure to common-use stresses.
Two popular brands of water bottles made
from a PETG resin now marketed as an HC
BPA-free replacement also released chemicals
having significant EA (W1, W2, W3, and
W4; Table 3, Figures 2 and 3), as did uncom-
pounded PETG resins (Table 3). Most PE/
PP-based plastic products were presumably
BPA free but never the less had readily detect-
able EA (Tables 1 and 2), almost certainly due
to one or more additives having EA. Many
components of BPA-free baby bottles had
reliably detectable EA (22–95% RME2) when
extracted in either saline or EtOH, including
the bottle, nipple, anti colic device, and liner
(data not shown).
In fact, all BPA-replacement resins
or products tested to date (n > 25) released
chemicals having reliably detectable EA (data
not shown), including polyethersulfone and
PETG, sometimes having more total EA
meas ured as %RME2 than many PC prod-
ucts when stressed. For example, the %RME2
released by various BPA-free baby and water
bottle component parts extracted by saline
or EtOH solutions and exposed to one or
more common-use stresses can be greater
than PC products under the same conditions
(Figure 2). UV stress, in particular, often leads
to the release of chemicals having greater EA
than BPA-containing HC plastics currently
sold. For example, saline extracts of BPA-free
baby bottle B3 (Figure 2) after exposure to
UV showed greater EA than did any of the PC
baby bottle extracts after any of the stresses.
Saline extracts from BPA-free baby bottle B1
after any of the stresses (microwave, autoclave,
or UV) showed greater EA than did the saline
extracts from PC baby bottle B2 after any of
the stresses. EtOH extracts from BPA-free
baby bottle B1 after UV stress showed greater
EA than extracts from PC baby bottle B1.
Saline extracts from BPA-free baby bottle B2
after microwave or autoclave stresses showed
greater EA than did saline extracts from PC
baby bottles B1 or B2 after any of the stresses.
Note also in Figure 2 that multiple extracts
of the same product using the same solvent/
stress combination typically gave rather simi-
lar %RME2 data, but different solvent/stress
combinations gave very different results, from
very high EA to non detectable EA. For exam-
ple, EtOH extracts from PC baby bottle B2
Figure 3. Properties of monomers and polymers used to make common resins.
Polymers Monomers Structures EA Toxicitya
Flexible polymers
Low-density polyethylene (LDPE),
linear low-density polyethylene
(LLDPE), high density poly-
ethylene (HDPE)
Ethylene
HH
HH
No No
Polypropylene homopolymer
(PPHO)
Propylene No No
HC polymersb
Copolymer using terephthalate
PETG
1,4-Cyclohexanedimethanol,
dimethyl terephthalatec
HO
OH
OO
OO
YesdNo
Polycarbonate (PC) Bisphenol A,e phosgene
HO
Cl Cl
OH
O
Yes Yes
Polyethylene terephthalate (PET) Dimethyl terephthalatee
OO
O O
YesdNo
Polystyrene (PS) Styrene YesdNo
Polypropylene copolymer (PPCO) Propylene, ethylene
HH
HH
No No
Cyclic olefin polymer (COP),
cyclic olefin copolymer (COC)
Ethylene, norbornene
HH
HH
No No
Polyacrylonitrile (PAN) Acrylonitrile
N
No Yes
Polyethersulfone (PES) 1,4-bis(4-Chlorophenyl)
sulfone, 1,4-dihydroxy-
benzenee
HO OH
Cl S Cl
O
O
YesdNo
aPolymer exhibits other toxic effects (e.g., cellular damage or carcinogenicity), or toxic chemicals (e.g., phosgene and
acrylonitrile) are used or produced during polymerization. bHC polymers generally have a glass transition temperature
(Tg) above room temperature and limited or no ability to crystallize. cMonomer has anti-EA in MCF-7 assays. dUnder cer-
tain conditions, degradation products exhibit EA. eMonomer has EA in MCF-7 assays.

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995
showed very high EA under all stress condi-
tions, whereas saline extracts of the same bottle
under the same stress conditions showed no
detectable EA. Hence, to reliably detect EA,
plastic resins or products must be extracted
with both polar and non polar solvents and
exposed to common-use stresses.
Discussion
Most plastic products release chemicals having
EA. Our data show that both more polar (e.g.,
saline) and less polar (e.g., EtOH) solvents
should be used to extract chemicals from plas-
tics because the use of only one solvent sig-
nificantly reduces the probability of detecting
chemicals having EA. The ability to detect
more polar and less polar chemicals having EA
is important because plastic containers may
hold either type of liquid or a liquid that is a
mixture of more polar and less polar solvents
(e.g., milk). When both more polar and less
polar solvents are used, most newly purchased
and unstressed plastic products release chemi-
cals having reliably detectable EA independent
of the type of resin used in their manufacture,
type of product, processing method, retail
source, and whether the product had contents
before testing. However, the lack of signifi-
cant difference in average percentage having
detectable EA between plastic items with and
without contents does not imply that the con-
tents do not affect the total EA or specific
chemicals having EA released by individual
plastic items.
Our data show that most monomers and
additives that are used to make many com-
mercially available plastic items exhibit EA.
Even when a “barefoot” polymer (no addi-
tives) such as PE or polyvinyl chloride does
not exhibit EA, commercial resins and prod-
ucts from these polymers often release chemi-
cals (almost certainly additives) having EA.
We found that exposure to one or more
common-use stresses often increases the leach-
ing of chemicals having EA. In fact, our data
suggest that almost all commercially available
plastic items would leach detectable amounts
of chemicals having EA once such items are
exposed to boiling water, sunlight (UV),
and/or microwaving. Our findings are con-
sistent with recently published reports that
PET products release chemicals having EA
(Wagner and Oehlmann 2009) and that dif-
ferent PET products leach different amounts
of EA. For example, different PET products
release different amounts of EA measured as
%E2 or %RME2 [see Supplemental Material,
Table 5C (doi:10.1289/ehp.1003220)],
almost certainly because different PET
copolymer manufacturers choose different
monomers, additive packages, and synthetic
processes to produce PET copolymer resins.
Our data are consistent with the hypothe-
ses that the presence of a phenolic moiety
is the best predictor of whether a chemical
exhibits EA and that benzene moieties often
probably convert to phenolic moieties when
the monomer and/or polymer is exposed to
one or more manufacturing or common-use
stresses. For example, although in theory most
organo phosphites (anti oxidants commonly
used with HPs to provide synergistic oxida-
tion protection) in their unaltered state should
not bind to ERs [see Supplemental Material,
Table 1 (doi:10.1289/ehp.1003220)], organo-
phos phites are hydrolytically unstable and
often produce phenols when exposed to water
(Kattas et al. 2000). Most organo phosphite
anti oxidants we tested exhibited detectable
EA (data not shown).
Likewise, various additives that are high-
molecular-weight HPs do not have EA, but
if exposed to moist heat they can under go
hydrolysis and produce lower-molecular-
weight phenolics that have EA. Therefore,
anti oxidants and other additives should be
tested for EA both in their original, unstressed
form and after stressing. We can identify
monomers and additives (anti oxidants, clari-
fiers, slip agents, colorants, inks, etc.) hav-
ing no detectable EA for use at all stages of
manufacturing processes to make flexible
non transparent or HC plastic items that are
EA free, even after exposure to common-use
stresses. All of our data suggest that, when
both are manufactured in comparable quanti-
ties, carefully formulated EA-free plastic prod-
ucts could have all the fit-for-use properties
of current EA-releasing products at minimal
additional cost.
BPA free is not EA free. Although most
items listed in Tables 1–3 would not be
expected to contain BPA, nevertheless almost
all stressed plastic items tested leached chemi-
cals having reliably detectable EA meas ured
as %RME2 if extracted with both more polar
and less polar solvents. In response to mar-
ket and regulatory pressures, BPA-free PET
or PETG resins and products have recently
been introduced as replacements for PC res-
ins. However, all such replacement resins and
products tested to date release chemicals hav-
ing EA (measured as %RME2), sometimes
having more EA than BPA-containing PC
resins or products, especially when stressed
by UV light (Figure 2, Table 3). Monomer
or polymer breakdown products that have EA
account for some of this EA, but the rest of the
measured EA is almost certainly due to release
of additives having EA in BPA-free products,
including the bottle and many component
parts of baby bottles advertised as BPA free.
Avoiding a potential health problem.
We recognize that we quantitatively meas-
ured EA relative to E2 (EC50 or %RME2)
using sensitive assay and extraction protocols.
Furthermore, it is almost impossible to gauge
how much EA anyone is exposed to, given
such unknowns as the number of chemicals
having EA, their relative EA, their release rate
under different conditions, and their meta-
bolic degradation products or half-lives in vivo.
In addition, the appropriate levels of EA in
males versus females at different life stages are
currently unknown. Nevertheless, a) in vitro
data over whelmingly show that exposures
to chemicals having EA (often in very low
doses) change the structure and function of
many human cell types (Gray 2008); b) many
in vitro and in vivo studies docu ment in detail
cellular/molecular/systemic mechanisms by
which chemicals having EA produce changes
in various cells, organs, and behaviors (Gray
2008); and c) recent epidemiological studies
(Gray 2008; Koch and Calafat 2009; Meeker
et al. 2009; Swan et al. 2005; Talsness et al.
2009; ompson et al. 2009) strongly suggest
that chemicals having EA produce meas urable
changes in the health of various human popu-
lations (e.g., on the offspring of mothers given
diethylstilbestrol, or sperm counts in Danish
males and other groups correlated with BPA
levels in body tissues).
Many scientists believe that it is not appro-
priate to bet our health and that of future
generations on an assumption that known
cellu lar effects of chemicals having EA released
from most plastics will have no severe adverse
health effects (Gray 2008; Talsness et al. 2009;
ompson et al. 2009). Because we can iden-
tify existing, relatively inexpensive monomers
and additives that do not exhibit EA, even
when stressed, we believe that plastics having
comparable physical properties but that do not
release chemicals having detectable EA could
be produced at minimal additional cost.
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