Estrogenic activity in dry food simulants: chemical migration from paperboard packaging
Vandermarken T1, Boonen I1, Gryspeirt C1, Van Den Houwe K2, Denison MS3, Goeyens L1, Van
Hoeck E2, Elskens M1
1

Department of Analytical, Environmental and Geo-Chemistry, Vrije Universiteit Brussel, Brussels,Belgium, 1050, tara.vandermarken@vub.be; 2Sciensano, Brussels, Belgium, 1050; 3Department ofEnvironmental Toxicology, University of California at Davis, David, USA, 95616
Introduction
Non-inert materials, such as paperboard, can transfer chemicals into the food which are also known as migratingchemicals. Recalls of products have occurred over the years, leading to image loss of the brand and financial
consequences1,2. NonylPhenols (NPs), BisPhenol A (BPA) and phthalates have been shown to migrate frompackaging material into food3,4,5,6,7. These chemicals are examples of Endocrine Disrupting Compounds (EDCs).
They interfere with endogenous hormone mechanisms and have been associated with many adverse healtheffects such as obesity, diabetes, fertility changes, prostate and breast cancer8,9.
The objective of this study was to use the Estrogen Responsive Elements Chemically Activated LUciferase geneeXpression (ERE-CALUX) bioassay to investigate estrogenic activities in paperboard and to evaluate the impactof different aspects of this material such as the use of recycled fibers, the presence of a coating and the use ofprinting inks. An ERE-CALUX has the advantage that all estrogenic chemicals in a sample extract (known andunknown) can be measured and that mixture effects are taken into account. Samples were tested before theycame into contact with dry food. A “worst case” scenario using a direct paperboard extraction was compared to amore realistic scenario in which extraction of the dry food simulant was applied. Estrogenic activities, from theERE-CALUX bioassay, were directly compared to molecule-specific analysis from instrumental methods.
Materials and methods
Paperboard samples were kindly donated by the packaging industry before they are folded and before they comeinto contact with food. Regulation (EU) 10/2011 describes how to test food packaging for compliance and whichfood simulant should be used. For this study, poly(2,6-diphenylene oxide) or Tenax® was used as theappropriate simulant. This type of experiment is referred to as “migration”, since it only involves substances that
migrated from the packaging into the food simulant. Migration experiments were only performed on printed
paperboard samples, since they show the highest potential of estrogenic migrating substances10. To obtain a“worst case” scenario, the paperboard samples were also directly extracted to determine their full estrogenic
potential. An overview of this sample setup is given in Figure 1.

Figure 1: Printed and unprinted samples from virgin fibers, coated recycled content fibers and non-coatedrecycled content fibers were prepared for extraction and migration experiments.

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Plasticizers, bis(2-ethylhexyl)phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl phthalate (BBP) and 1,2cyclohexane dicarboxylic acid diisononyl ester (DINCH), were quantified in all extraction and migration
samples with GC-MS. A phthalate cleanup procedure was performed on each subsample and the estrogenic
activities of all raw and cleaned extracts and migration experiments were determined.
The overall estrogen activity is analyzed using the ERE-CALUX11. Briefly, extracts are diluted and dosed in 10
different concentrations yielding a full dose-response curve fitted with the “non-linear 4 parameter Hill function”
to calculate an Effective Concentration on 50% of the curve (EC50). The Bioanalytical Equivalent concentration(BEQ), expressed in pg 17β-estradiol (E2) equivalents per square decimeter, is calculated as the ratio of the EC50of the standard and the sample curve.
Results and discussion:
The extraction samples are graphically presented in Figure 2, where printed and unprinted paperboard samples of
all 3 fibers were tested. All virgin samples had BEQs of < 1000 pg E2 eq./dm2 whereas recycled content fibers
had BEQs higher than the virgin paper samples (BEQs of < 3000pg E2 eq./dm2). A two factor ANOVA, withpaperboard and printing impact, confirmed this significant difference due to fiber origin, but no significant
difference was observed between printed and unprinted samples of the same fiber.

Figure 2: Bioanalytical equivalent concentrations (BEQ) without blank correction expressed in pg E2
equivalents per dm2 of extraction experiments samples with and without a phthalate cleanup procedure.
UP stands for unprinted and P for printed samples. Values are expressed as the mean ± standard
deviations of triplicate determinations.
The extractions and migrations of all printed samples are compared in Figure 3. As expected, the amount of
estrogenic compounds migrating to Tenax® is lower than the compounds present in the “worse-case” total
extract samples since these samples would contain all of the extractable compounds and not just the ones able tomigrate. Additionally, only the food contact side was in contact with Tenax® for migration experiments and not
the non-food contact side while both sides are simultaneously extracted for the “worst case” scenario.
The results for the plasticizers in extraction and migration experiment samples are presented in Table 1. It isclear that paperboard samples with recycled content fibers show the highest overall phthalate content. The
migration experiments for virgin fibers are not interpretable because of the high estrogenic activity of theTenax® blank samples. Recycled content printed fibers, coated and non-coated, show similar levels of DEHPand DBP for total extracts and migrations. BBP is not detected in the virgin fibers and its amount in recycledcontent fibers was lower than about 10% of the levels of DEHP and DINCH in the sample extracts. The mostabundantly present compound was DINCH, however, concentration-response analysis revealed that DINCH had
no estrogenic activity in the ERE-CALUX bioassay.

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Figure 3: Bioanalytical equivalent concentrations (BEQ) without blank correction expressed in pg E2
equivalents per dm2 of printed extraction and migration experiments with and without a phthalatecleanup procedure. Values are expressed as the mean ± standard deviations of triplicate determinations.

Sample

DEHP

DBP

BBP

(µg/dm2)(µg/dm2)(µg/dm2)

Totalphthalates(µg/dm2)

DINCH(µg/dm2)

V UP

1.2

Extractions
0.4

0

1.7

19.1

VP

1.9

1.3

0

3.3

33.7

RC UP

76.1

29.6

6.4

112

98.7

RC P

61.3

20.8

8.4

90.5

124

RNC UP

9740.8

10.1

148

146

RNC P

10446.211161157

ACN Blank

0.4

0.3

0.2

0.80Migrations
VP

3.3

3.4

0

6.5

0RC P

43

16.1

3.1

62.2

77.4

RNC P

1314618195

144Tenax® Blank 1

4.2

2.2

0.4

6.80Tenax® Blank 2

1.8

1.6

1.5

4.9

7.3

Table 1: GC-MS results for bis(2-ethylhexyl)phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl
phthalate (BBP) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) in µg/dm2 for all
migration and extraction paperboard samples and blanks for Tenax® and acetonitrile (ACN). Averagerelative standard deviations (RSD) of 18%, 10%, 11% and 10% for DEHP, DBP, BBP and DINCH
respectively. V stands for virgin fibers, RC for recycled content coated and RNC for recycled content noncoated samples. P for printed and UP for unprinted.

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To compare the migration results to current legislation, the values obtained here are expressed per kg simulant
and are multiplied by a factor 6, as described in Regulation (EU) 10/2011. The Specific Migration Limit (SML)
is currently only valid for migration experiments for plastic food contact materials, but is used here as areference value for paperboard migrations. The results for recycled content fibers with average Tenax® blank
subtraction are presented in Table 2. The DBP results suggest that there might be a problem with the specificmigration from recycled content non-coated, since the migration concentration is close to the limit set for plastic
food contact materials. Accordingly, it might be interesting to include these compounds in future monitoring.
DINCH, BBP and DEHP show values below the SML for the recycled content samples and thus would not be
expected to represent a risk.
SampleDEHP(mg/kg)DBP(mg/kg)BBP(mg/kg)

DINCH(mg/kg)

RC P

0.24

0.09

0.01

0.44

RNC P

0.77

0.26

0.10

0.84

SML
1.5
0.3
3060
Table 2: GC-MS results for bis(2-ethylhexyl)phthalate (DEHP), dibutyl phthalate (DBP), butyl benzyl
phthalate (BBP) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) in mg/kg Tenax® for all
migration paperboard samples with blank correction. RC stands for recycled content coated, RNC forrecycled content non-coated and P for printed samples. The Specific Migration Limit (SML) of plastic
food contact materials for each compound is indicated.
Acknowledgements:
The VM7LUC4E2 cell line was developed by the University of California-Davis (USA) with funding from a
Superfund Research Program grant (ES04699) from the National Institute of Environmental Health Sciences.
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