8 th BioDetectors. Applications of bioassays to prioritize chemical food safety issues. Maricel Marin-Kuan
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1 8 th BioDetectors Applications of bioassays to prioritize chemical food safety issues Maricel Marin-Kuan
2 Hazard identification in food industry substances potentially used in packaging industry Co packersco packersco packers (adapted from U.S. EPA) substances potentially coming from environmental, agricultural practices and process-related contamination Diverse sources of potential food contamination M. Marin Kuan Chemical Food Safety NRC p. 2
3 Hazard identification: Which are the tools available? % activation compared to E 2 30nM Chemistry Toxicology Docking QSAR In vitro 2) log Transform In vivo Zearalenone (Z2125) ZAL ZAL ZOL -ZOL Estradiol Read-across concentration ( log M) Analytics Bioassays In silico M. Marin Kuan Chemical Food Safety NRC p. 3
4 Knowing the sources: e.g. dealing with food packaging Expected/known Intentionally Added Substances IAS Regulatory toxicology data Safe level of exposure Compliance Risk Assessment principles Unexpected/unknown Non-Intentionally Added Substances NIAS: Impurities in ingredients Reaction intermediates/products Decomposition products Many? Not characterized? Food packaging safety evaluation No harmonized & standardized procedures Traditional toxicology-based methodology requiring full characterization of all substances neither practical nor feasible M. Marin Kuan Chemical Food Safety NRC p. 4
5 Multidisciplinary approach for packaging safety Packaging safety Composition Applications Suppliers Early warning Migration study: Food simulant(s) Existing and new EU/Nestlé compliance: IAS and known NIAS (target analysis) unknown NIAS (non-target analysis) Compliance In vitro bioassays Analytical chem. In silico toxicology Hazard identification Characteris. NIAS Specifications QA low Level of concern Suitable high M. Marin Kuan Chemical Food Safety NRC Management actions: Mitigation Risk assessment: Quantification/exposure Computational toxicology/ttc Not suitable Rejection p. 5 Risk assessment Risk management
6 In vitro tools for the biodetection of potential food active compounds
7 I. Sound Biological Targets in toxicology Reactive toxicity Non-specific toxicity Effect-based toxicity Mitochondria Image adapted from: National Human Genome Research Institute M. Marin Kuan Chemical Food Safety NRC p. 7
8 II. Nestlé BioDetection battery of tests Endpoints Platform/method Bioassays Endocrine activity Genotoxicity Cytotoxicity ToxInsight CALUX ELISA ToxInsight ToxInsight Bluescreen p53 ToxInsight Estrogen & androgen receptor redistribution assays Estrogen, androgen, AhR, thyroid, PPARg2 receptor activation H295R Steroidogenesis assay (hormone production) Histone gh2ax phophorylation Micronucleus assay Gadd45 expression, Ames test (mutagenicity) Organelle health (mitochondrial markers) Cell proliferation (DNA, Br-deoxyuridine incorporation) and cell death (apoptosis, caspase 3, p53) Xenometrix Speed, convenience, high content, robustness, relevance Ability to cover several mechanisms/modes of action Cytotoxicity screening: protein synthesis, lysosomial and nuclear membrane integrity biomarkers M. Marin Kuan Chemical Food Safety NRC p. 8
9 Case studies: Application of the biodetection tool battery
10 Case study 1: plastic material in development Identifying responsible compounds Material bioactivity: Migration simulation assay In vitro battery: toxicity endocrine genotoxicity Estrogenic activity Analytical profile Information on material: ingredients, recipes Verify in vitro: effect(s) dose-response Comparison with analytical data: concordance Adapted CALUX test List substances substance with known endocrine activity: 4-nonylphenol Confirmation in vitro In silico screen for alerts M. Marin Kuan Chemical Food Safety NRC p. 10
11 Case study 2: presence of azaarenes in food Comparing toxicity to known analogues Case study: Background: PAH-nitro derivatives: Structural and sources similarities with PAHs but less well characterized Very limited information on levels in food and exposure (e.g. oils) Toxicological data:» Limited toxicological data» Evidences suggest stronger toxicity as compare to PAH» Further information is needed to define the toxicity of PANHs as compared to PAHs M. Marin Kuan Chemical Food Safety NRC p. 11
12 Known PAHs mode of action Reference compound: Benzo(a)pyrene Genotoxic after metabolic activation: Protein and DNAadducts formation Potent Aryl hydrocarbon Receptor (AhR) activator Other nuclear receptors also involved (e.g. ER) M. Marin Kuan Chemical Food Safety NRC p. 12
13 5 rings 4 rings 3 rings Tested Compounds CONTROL compound M. Marin Kuan Chemical Food Safety NRC p. 13
14 Data summary Weak activator Stronger activator EC 50 LPC (lowest (+) concentration) CD 40% M. Marin Kuan Chemical Food Safety NRC p. 14
15 General summary Reference compound: BaP Results in line with the literature: cytotoxic, genotoxic and nuclear receptors activator: AhR, ER and anti-ar Confirm the role of metabolic activation Battery of bioassays works appropriately Receptor-mediated effect AhR: Most sensitive parameter (2-3 orders of magnitude) Correlation between AhR activity and number of rings Decreased activation of ER receptor with the number of rings (less potent AhR) Anti-AR effect with all compounds (except Dibenz(ch)acridine) Other parameters No correlation between AhR activation and other parameters No consistent trends between PAHs vs PANHs regarding cytotoxicity and genotoxicity potency Some evidences of higher genotoxicity and cytotoxicity induction by the 5-rings PAHs and its derivatives No good consistency across genotoxicity tests M. Marin Kuan Chemical Food Safety NRC p. 15
16 Conclusions case study 2 Compared to PAHs, azaarenes are likely to have similar toxicological profile However, they may not bring additional risk burden since exposure seems much lower than for PAHs M. Marin Kuan Chemical Food Safety NRC p. 16
17 Cell culture-based in vitro battery summary Receptor-mediated effect model: CALUX model is a sensitive test for receptor activation studies Need to address the role of metabolic activation on nuclear receptors activation parameter Genotoxicity Optimization of the genotoxicity assessment is needed to address consistently: sensitivity specificity metabolic activation optimal cell model Data interpretation for decision making: cut-off values and/or trigger values needed In vitro toxicology issues for decision making, need to be address through a multidisciplinary approach M. Marin Kuan Chemical Food Safety NRC p. 17
18 Acknowledgements Chemical Food Safety Group Nestlé Research Center: Benoît Schilter Myriam Coulet Master student: Fanny Minetto from the University of Brest (France) Julie Mollergues Dominique Piguet Helia Latado Patrick Serrant Claudine Bezençon BioDetection System (BDS Amsterdam) Harrie Besselink Peter Behnisch M. Marin Kuan Chemical Food Safety NRC p. 18
19 THANK YOU FOR YOUR ATTENTION Questions or Comments? M. Marin Kuan Chemical Food Safety NRC p. 19
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