Österreichisches Forschungsinstitut
www.ofi.at
für Chemie und Technik
Austrian Research Institute for Chemistry & Technology
CONSTRUCTION
PLASTIC PRODUCTS
PHARMA
BIOENERGY
SURFACE TECHNOLOGY
CERTIFICATION
Locations & Scope of Work
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Location
1030 Vienna, Arsenal
Location
1110 Vienna, Brehmstraße
Applied Polymer Technology
Surface Technology
Building & Construction
Sports technology
starting summer 2010:
Packaging
Pharma & Medical Devices
Bio energy
Food & Feed analysis
New location
at TFZ Wr. Neustadt
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Our Focus
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Polymer Technology
Processing & application of polymers,
elastomers, paints, coatings and adhesives
Building & Construction
Building materials, structural monitoring,
dehumidification of masonry, sports technology
Pharma & Packaging
Trace analysis, stability studies,
packaging development; food & feed analysis
Biomass fuels
Manufacturing method, quality assurance,
plant design
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Agenda
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What can you expect?
 Current discussion
 Definition – what are EDCs?
 Assay methods and first results
 What can we do?
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Studies on BPA
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NTP (NIH) USA 09/2008: At current exposure of the U.S.
population:
 Developmental toxicity to fetuses / infants : „some concern for
adverse effects (brain, behavior, prostate)“
Federal Office of Public Health (Switzerland) (2009): „No risk to
consumers“
 Problem of evasion to other / worse characterized ingredients
Statement of the Endocrine Society 06/2009: „EDCs are a
significant concern to public health“
Statement of the BfR 10/2009: „No health risk. No risk to infants
and young children.“
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Actual situation
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 EDCs in contact with food – plastic, paper, laminates, coated
metal cans...
 Effects of EDCs in packaging :
• Potential danger (but not proven)
• Currently concentration on a few substances
• Large number of potential EDCs in packaging
 Need for action:
• Analysis (Bioanalytsis; chemical analysis)
• Toxicological studies
• There are no comprehensive studies on EDCs in
packaging
• Reduction of the EDC burden of packaging
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Definition ED
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Endocrine Disruptors (EDCs)
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Definition:
EPA
Exogenous substances that act like hormones and
disrupt the physiologic function of the endocrine
system
European comission
Exogenous substances that act like hormones,
disrupt the physiologic function of the endocrine
system and cause adverse health effects.
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Effects on the environment
Change of sex organs
Purple snail (TBT)
Alligators (DDT, Dicofol)
Accumulation during food chain
Example. PCB:
Polar bear: 3 billion times the concentration
originally found in water
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EDCs in plastic
Monomers, stabilizers, plasticizers, antioxidants,
contaminants ...
Bisphenol A
Alkylphenols (Nonylphenol)
Phthalates
Problem: migration out of the plastic into the food
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Why estrogens?
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Xenoestrogens
 Act like the primary female sex hormone estrogen
 Estrogen is involved in the regulation of many sensitive
developing steps and metabolism functions in both men and
women
 A disfunction of these mechanisms can cause reproductive
problems, developmental disorders or cancer
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German study:
high estrogenic activity in mineral water
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 Authors assume that plastic
bottles are the source
 BUT: other reasons possible
 Highly contraversal study
 Direct analysis of the plastic bottles necessary (PET-Bottle,
screw cap)
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Selection of bioassays
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• Yeast-Bioassays (YES / YAS)
• Aspergillus Screen (highly sensitive
reportersystem)
• Cell cultures (breast cancer-, prostate cancer
celllines) – E-Screen
• Reproductiontests with Potamopyrgus
antipodarum
• ...
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Bioassays
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Reporter gene assays:
•
YES – Yeast Estrogen Screen (2 different
strains)
•
Aspergillus nidulans - Bioassay
Principle
•
Cloned gene for human estrogen receptor
•
Binding of estrogen or endocrine disruptor (e.g. plastic
additive) activates receptor
•
Expression of ß-Galaktosidase
•
Conversion of CPRG (yellow) => CPR (red)
•
Photometric measurement
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Effects of estrogens
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Reportergen-Assay
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Extraction vs. migration
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Total extraction
•
ASE – Accelerated Solvent Extraction
Food simulants
•
Water
•
3% Acetic acid
•
50% Ethanol
•
Isooctane
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17ß-Estradiol standard curve
Limit of detection: 10 pM
Limit of quantification: 20 pM
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First results
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Materials
total
negative positive
(pg EEQ* / g sample)
Limit of detection
[pg EEQ/g sample]
PP
Yeast strain 1
23
22
1
Yeast strain 2
23
23
0
400
120
HDPE
Yeast strain 1
13
12
1
2000
Yeast strain 2
13
12
1
1000
PET
6
Yeast strain 1
19
17
2
10 / 50
Yeast strain 2
19
14
5
50 - 80
*EEQ… 17ß-estradiolequivalent
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Plasticadditives
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Positive HDPE-sample:

Bisphenol A: ca. 20 - 40 µg / g sample

Benzylbutylphthalat: ca. 200 - 400 µg / g sample
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Plasticsamples
 12 PET-bottles and PET-bottles preforms
 4 PET-foils
 13 screw caps
 3 recycling flakes
 6 positive samples, relatively low activities
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Results
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Interpretation
 Estrogen active substances can be found in plastics
 Low activity, but synthetical estrogens have a higher risk
potential than natural estrogens
 Experiments with food simulants: no activity found
 In vitro - studies give no information on actual effects in
humans
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Why bioassays ?
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Advantages
 Measurement of known and unknown substances
 Integration of synergistic effects (mixing effects)
 Evaluation of the complete package
 Simple screening tool
 Evaluation of toxicological effects possible
 No direct hints on in vivo effects
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Bioassays
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Disadvantages
 False negative results possible
 No conclusion about possible in vivo effects
• only show: hormone binding to receptor
 Technical problems (solubilities / extracting agents / food
simulants ...)
 No information of which substances are causing the
hormone activity
 Combination with chemical analytic
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Chemical Analysis
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Chemical analysis
• Development of testing methods for endocrine
disruptors in packaging / food
• Goal: Development of a multimethode for the 50 most
frequent EDCs
• GC/MS
• LC/MSn
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Next step: E-screen
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 In-vitro bioassay
 Human breast cancer cells (MCF 7 cells)
 No genetical modification
 Reproduction of the cancer cell line dependents on
estrogens
 Reproduction of the cells (proliferation) is determined in
comparison to a negative control and to estrogen
standards
 high sensitivity (ca. 1 pmol/l)
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COIN
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 COIN program line „structure"
 Funding authority: FFG on behalf of bmvit and BMWFJ
 Dimension: national
 Goals of the program
• Development and improvement of key competences and functions
• for providers of application-oriented F&E&I-expertise
• particularly towards the KMU
 Runtime: 01.09.2010 until 30.08.2014 (48 months)
 Volume of the project: 1,8 Mio.€
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Goal:
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Establishment of new bioanalytical methods
 Characterization of the total hormone burden of food
contact materials (plastics, coated metal packaging,
paper)
 Bio-Assay-Battery
 Sensitivity, standardization and high-throughput-analytic
 Chemical detection for the 50 most important EDCs in the
ppb-range (preferable GC/MS)
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Advisory board
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International Scientific Advisory Board
• Toxicologists
• Food / Packaging analysts
• Reference laboratory
• Authorities
Project Advisory Committee
• Project partners (institutions)
• Participating companies
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Partners / Participating Companies
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The following companies already joined the project :

Plastics Europe Deutschland

Tetra Holdings GmbH

NÖM

MAM Babyartikel GmbH

Verein für Konsumenteninformation (VKI)

REWE

Teich AG

ALPLA

SIG Combibloc
The following companies have expressed their interest:

Greiner Packaging

EREMA

Miraplast

MM Karton
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- Hotline
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For any questions please contact:
DI Dr. Johannes Bergmair
(DW 976, E-Mail: johannes.bergmair@ofi.at)
ofi Research Institute
Brehmstraße 14A
1110 Vienna
+43-(0)1-798 16 01 -DW
+43-(0)1-798 16 01- 480
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