The 11th BioDetectors conference took place on September 13-14, 2018, in Aachen, Germany. The event was hosted by RWTH Aachen University and sponsored by BioDetectionSystems (BDS). Several presentations focused on the use of bioassays in food contact materials (FCMs) analysis, while others discussed applications in environmental studies.
Amaury Patin and Karma Fussel from the Nestlé Research Centre, Lausanne, Switzerland, discussed how Nestlé uses bioassays to assess the safety of food packaging, among other applications. Patin discussed an example of 1,4,7-trioxacyclotridecane-8,13-dione (also called AADEG cyclic ester, CAS 6607-34-7), a compound found to migrate from polyurethane-based adhesives at about 30 ppb. After in vitro digestion, 60-80% of a starting amount remained in cyclic form, while the rest was hydrolyzed into linear form. The linear form was found to cause a stronger activation of estrogen receptor alpha (ERα), while the cyclic form was active in the aryl hydrocarbon receptor (AhR) assay for dioxin-like compounds and in the NF-E2 related factor (Nrf2) assay detecting oxidative stress. Further characterization and risk assessment are ongoing.
For the most part, the endocrine assays included in the Nestlé’s pipeline focus on the measurement of (anti)estrogenic and (anti)androgenic activities only. For these assays, Nestlé developed metabolic activation methods and approaches to differentiate true receptor inhibition from cytotoxicity-mediated effects. Fussel informed that these assays have now been automatized to allow for higher throughput analysis at Nestlé.
In 2017, a review by scientists of the Food Packaging Forum (FPF) highlighted the need to examine effects mediated by receptors other than ER or androgen receptor (AR) (FPF reported), such as thyroid receptors (TRs) (FPF reported) or peroxisome proliferator-activated receptors (PPARs) (FPF reported). Recently, Nestlé used PPAR gamma (PPARγ) assay to test bisphenol A (BPA, CAS 80-05-7) and several BPA analogs. The findings were reported in an article published in the peer-reviewed journal Toxicology In Vitro on August 20, 2018. All tested bisphenols showed little or no agonistic activity, but exhibited a “reasonable” antagonism of PPARγ. The authors concluded that “it seems probable that there are additional obesogenic effects of these chemicals which would not be detected by this assay.”
Elisa Mayrhofer from the Austrian Research Institute for Chemistry and Technology (OFI) and Bernhard Rainer from the University of Applied Sciences, both Vienna, Austria, reported on the progress of the currently ongoing MigraTox project (FPF reported). Among the 114 FCM samples analyzed so far, 25% were cytotoxic, 32% were ER-active, and about 10% each showed anti-ER, anti-AR, and p53 activity. Furthermore, 11% of samples were positive and 13% equivocal in the Ames assay for directly acting mutagens.
Vera Baumgartner from the Swiss Quality Testing Services (SQTS) introduced the ongoing work on the optimization of a new analytical method based on high performance thin-layer chromatography (HPTLC) coupled with bioassays, the so-called “planar” assays. A planar assay with yeast estrogen screen (planarYES) has been shown to be more sensitive than the conventional YES assay. Using planarYES, Alan Bergmann from the Oekotox Centre, Switzerland, detected estrogenic activity in several food cans; identification of the active substances is currently ongoing. Overall, Baumgartner characterized planar assays as an “excellent approach to combine physical and chemical detection.”
BDS is a company commercializing CALUX bioassays. CALUX stands for Chemical Activated Luciferase gene eXpression. These assays are based on modified cell lines containing a luciferase reporter gene controlled by a promoter being made responsive to specific chemicals, such as estrogens, androgens, or aryl hydrocarbons. The presence of targeted chemicals leads to an increase in luciferase production, which can be detected with a quantifiable light reaction. Several ongoing projects at BDS pursue further assay optimization or development of new assays. For example, the predictivity of the assays for estrogenic and anti-androgenic activity has been recently improved by complementing them with a system mimicking hepatic metabolism in vivo. Berenice Collet presented a novel suite of CALUX bioassays for thyroid receptor β and transthyretin transporter disrupting activities.
The project ReSolve, presented by Barbara Van Vugt-Lussenburg from BDS, uses CALUX bioassays to assess the safety of novel bio-based solvents derived from non-food carbohydrates, aimed to replace toluene (CAS 108-88-3) and N‑methyl-2-pyrrolidone. BDS scientists also used CALUX bioassays to assess safety of bio-based plastics. In this project, migrates from food-grade polyethylene (PE) and from brightly colored flexible polyethylene terephthalate (PET) were found to be estrogenic. The authors concluded that “the panel of human-cell based reporter gene bioassays is a useful tool to evaluate the safety of packaging materials and plastic additives.”
Dusserre, C., et al. (2018). “Using bisphenol A and its analogs to address the feasibility and usefulness of the CALUX-PPARγ assay to identify chemicals with obesogenic potential.” Toxicology in Vitro 53:208-221.
Marin-Kuan, M., et al. (2017). “Differentiating true androgen receptor inhibition from cytotoxicity-mediated reduction of reporter-gene transactivation in-vitro.” Toxicology in Vitro 45:359-365.
Mollergues, J., et al. (2017). “Incorporation of a metabolizing system in biodetection assays for endocrine active substances.” ALTEX 34: 389-398.
Schoenborn, A., et al. (2017). “Unprecedented sensitivity of the planar yeast estrogen screen by using a spray-on technology.” Journal of Chromatography A 1530:185-191.
Van der Linden, S., et al. (2014). “Development of a panel of high-throughput reporter-gene assays to detect genotoxicity and oxidative stress.” Mutation Research/Genetic Toxicology and Environmental Mutagenesis 760:23-32.
Van Vugt, B., et al. (2017). “Effect-based safety assessment of bio-based chemicals: a case study on bio-based plastics.” Poster (pdf).
Van Vugt-Lussenburg, B., et al. (2018). “Incorporation of metabolic enzymes to improve predictivity of reporter gene assay results for estrogenic and anti-androgenic activity.” Reproductive Toxicology 75: 40-48.