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Research unit
FSVO
Project number
4.20.02
Project title
Validation and implementation of thin-layer chromatography based methods for identifying (geno)toxicants in broad assessment of food contact materials and drinking water

Texts for this project

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Key words
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Short description
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Project aims
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Abstract
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Transfer and application
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URL-addresses
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CategoryText
Key words
(German)

Lebensmittelverpackung, Trinkwasser, Hochleistungs-Dünnschichtchromatographie, Östrogenität, Genotoxizität

Key words
(English)

food packaging, drinking water, high-performance thin layer chromatography, estrogenicity, genotoxicity

Short description
(German)

Lebensmittelkontaktmaterialien (FCM) und Trinkwasser können mit toxischen Stoffen, z.B. unabsichtlich eingebrachten Substanzen (NIAS) oder Pestiziden, belastet sein. Zur Identifizierung dieser Stoffe in Nahrungsmitteln sind sensitive Methoden erforderlich, welche eine fundierte Risikobewertung, Minderung bestimmter chemischer Gefahren und ein Reagieren auf Notfälle ermöglichen. In einem ersten FSVO-Projekt verknüpften wir die Entwicklung von Biotests und hochauflösender Massenspektrometrie (HRMS/MS) mit Hochleistungsdünnschichtchromatographie (HPTLC). Zusammen mit Projektpartnern streben wir jetzt ein breites Screening von FCM, Wasser und weiteren relevanten Materialien mit HPTLC-Biotests an, um unbekannte Substanzen für die Identifikation mittels HRMS/MS zu priorisieren. Wir werden Tests für vielfältige Probenmaterialien validieren und Methoden für Gentoxizität bezüglich ihrer Empfindlichkeit evaluieren. Die resultierenden Daten werden eine routinemässige Anwendung von HPTLC-Biotests als Monitore für Verbraucherprodukte ermöglichen.

Short description
(English)

Food contact materials (FCM) and drinking water are subject to potentially toxic contamination from, for example, non-intentionally added substances (NIAS) or pesticides, respectively. Tools for identifying these substances in consumer products are necessary for sound risk assessment, mitigation of chemical hazards, and preventing and responding to health emergencies. In an initial project funded by the FSVO, we advanced the development of bioassays and liquid chromatography - high resolution mass spectrometry (LC-HRMS/MS) on high performance thin-layer chromatography (HPTLC) for analysis of FCM. We observed that these are sensitive methods for detecting effects in complex mixtures. Now, the illustration and validation of HPTLC assays for broad sample matrices relevant to FSVO will provide needed data for routine use of HPTLC bioassays as monitors of consumer products. We aim to validate HPTLC-bioassay for genotoxicity and in collaboration with project partners, perform broad bioassay screening of FCM, water, and relevant materials to prioritize samples for substance identification with LC-HRMS/MS.

Project aims
(English)

Our overarching goal is to identify toxic chemicals in FCM and water as basic data for risk analysis. We aim to characterize in vitro bioactivity of diverse FCM and prioritize substances for identification. We will leverage successes developing (bio)analytical methods in the current FSVO-funded project, and will expand and apply the methods to a broad range of FCM and water samples. Specifically, we aim to (1) further develop and validate methods for evaluating genotoxicity of FCM and water samples on HPTLC plates, (2) screen a diversity of samples for primarily genotoxicity, and secondarily, estrogenicity with (HPTLC) bioassays, and (3) use the bioassay results to prioritize materials for non-target analysis with LC-HRMS/MS.

Abstract
(German)
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Abstract
(English)
Related documents
Transfer and application
(German)

A2) Further development and validation of the methodology of thin layer chromatography coupled with umuC assay for genotoxicity and YES for hormonal effects for the identification of bioactive trace substances in consumer articles and drinking water.

  • Robustness of HPTLC-umuC has been improved: positive control responses are consistent over time
  • Limits of detection for known genotoxicants were evaluated in comparison to regulatory thresholds, including thresholds of toxicological concern (TTC)
  • Depending on assumptions of food contact and laboratory conditions, many chemicals can be detected below stringent TTC
  • Potency of genotoxicants varies by many orders of magnitude, making it impractical to detect all substances at TTC levels
  • HPTLC-bioassays are the most sensitive versions of assays investigated in this study

A3) Data base for bioactivities in samples from consumer articles and in drinking water/raw water samples.

More than 90 samples of food packaging, drinking water, and related materials were provided by project partners. The HPTLC-bioassay parameters and results remain at the Ecotox Centre. They may be accessed to investigate what kinds of samples are bioactive, and under what conditions.

A4) Identification of bioactive trace substances in consumer goods and drinking water, which may require a detailed risk assessment.

Paperboard example from KLZH:

  • Genotoxic substances were detected in samples of paperboard (unprinted and printed)
  • The unknown genotoxic substances were investigated with non-target chemical analysis (i.e. toxicant identification)
  • One compound was identified with the help of custom suspect lists developed in this project: 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT)
  • Migrates of unprinted and printed paperboard did not elicit genotoxic response in HPTLC-umuC

    Drinking water example from IWB collaboration:

  • Detected bioactive substance(s) in extracts of drinking water source water
  • Inconclusive genotoxicity results were observed in the Salmonella reverse mutation assay (Ames)
  • It was decided, in collaboration with IWB, that chemical analysis toward identification of the responsible substance(s) was not feasible because this sample type is available only in limited volume.

 

B1) In this project, there is cooperation with the cantonal enforcement (KL SG, KL ZH) as well as with drinking water suppliers (IWB, Basel), which provide the researchers with corresponding sample material. This could lead to starting points for the application of the methodology in food and drinking water control.

  • samples were received from KLZH, KLSG, IWB, ZWV, SQTS
  • employees were hosted from KLZH and SQTS at the Ecotox Centre to learn HPTLC techniques and perform experiments

There is continued interest from the cantons and industrial partners to implement HPTLC-bioassays in their own laboratories, or have a contract laboratory available. Interaction with the Food Packaging Forum (FPF). The list of suspect chemicals was guided in part by the FCCdb, created by the Food Packaging Forum, and conversations with its authors. The FCCdb contains known ingredients in packaging manufacturing.

B2, B3) Within the framework of this project, bioactive substances could be identified for which risk management measures may be necessary after detailed risk assessment.

  • CMIT was identified in printed paperboard.
  • CMIT was previously not known by cantonal authorities to be present in these paperboard samples
  • BfR guidelines limit amount of CMIT that can be in extracts of paper products
  • Other bioactive compounds have been investigated but remain unidentified
  • Demonstration that HPTLC fractionation consistently reduces the number of chemicals by >95%, compared to the unfractionated sample

C1) Results should be presented in knowledge transfer and at a national conference.

Presentations:

Alan J. Bergmann, Teresa Mairinger, Daniel Olbrich, Eszter Simon, Juliane Hollender, Andreas Schönborn, Etienne Vermeirssen. June 2020. Sensitive detection of toxic chemicals in food packaging. Webinar hosted by Food Packaging Forum. Zürich, Switzerland.

Alan J. Bergmann, Andrea Schifferli, Nadine Bramaz, Kasia Aturi, Eszter Simon, Juliane Hollender, Andreas Schönborn, Inge Werner, Etienne Vermeirssen. Toward identifying genotoxicants in paper products with an HPTLC based bioassay. Presentation to Department of Environmental Chemistry, Eawag. December 2020. Dübendorf, Switzerland.

Alan J. Bergmann, Kasia Aturi, Daniel Olbrich, Eszter Simon, Juliane Hollender, Andreas Schönborn, Etienne Vermeirssen. February 2021. Sensitive detection of toxic chemicals in food packaging. Knowledge transfer webinar hosted by Swiss Federal Food Safety and Veterinary Office. Bern, Switzerland.

Alan J. Bergmann, Andrea Schifferli, Nadine Bramaz, Kasia Aturi, Eszter Simon, Juliane Hollender, Andreas Schönborn, Inge Werner, Etienne Vermeirssen. HPTLC-bioassays, a review. Plus updates in ToxSISTEM: toward identification of toxicants in food packaging and drinking water. October 2021. Online seminar to Ecotox Centre. Dübendorf, Switzerland.

Alan J. Bergmann, Beat J. Brüschweiler, Eszter Simon, Gregor McCombie, Celine Muñoz, Maurus Biedermann, Juliane Hollender, Andreas Schönborn, Etienne Vermeirssen. HPTLC-bioassays for the detection and identification of toxic NIAS in food packaging. Abstract for poster presentation submitted to ILSI food packaging meeting. May 2022.

Anticipated: Seminar to EPFL Environmental Engineering Institute (planned March 2022)

Posters:

Alan J. Bergmann, Eszter Simon, Andrea Schifferli, Andreas Schönborn, Etienne Vermeirssen. Chemical safety of food packaging: (HPTLC-)bioassays for detection and identification of toxic substances. May 2020. Society for Environmental Toxicology and Chemistry, Europe. Online.

Alan J. Bergmann, Rebekka Merki, Vera Baumgartner, Heidi Moor, Etienne Vermeirssen, Thomas Gude. Challenging chemicals in genotoxicity assays: GLYMO as an example. Abstract for poster presentation submitted to ILSI food packaging meeting. May 2022.

Anticipated: Non-target analysis meeting:

Kasia Aturi, Alan Bergmann, Juliane Hollender. 2022. Novel nontarget pipeline deploying data mining for linking environmental exposures to toxic effects. Durham North Carolina, United States.

C2) The results of this project will be published in scientific journals.

Alan J. Bergmann, Eszter Simon, Milena Breitenbach, Gregor McCombie, Celine Muñoz, Maurus Biedermann, Andreas Schönborn, Etienne Vermeirssen. HPTLC genotoxicity assays are best option for detecting hazards in food packaging at TTC levels. To submit early 2022. Possibly: Food Additives and Contaminants A (Kapitel 2 des Schlussberichts).

Alan J. Bergmann, Eszter Simon, Milena Breitenbach, Gregor McCombie, Celine Muñoz, Maurus Biedermann, Andreas Schönborn, Etienne Vermeirssen. HPTLC genotoxicity assay for detecting hazards in food packaging at thresholds of toxicological concern (Kapitel 3 des Schlussberichts).

Alan J. Bergmann, Kasia Aturi, Andreas Schönborn, Juliane Hollander, Etienne Vermeirssen. HPTLC EDA: Toward identification of toxic substances in food packaging with HPTLC-bioassays. (Kapitel 4 des Schlussberichts).

URL-addresses
(German)