Dose Addition in the Induction of Craniofacial Malformations in Zebrafish Embryos Exposed to a Complex Mixture of Food-Relevant Chemicals with Dissimilar Modes of Action

Abstract

Background: Humans are exposed to combinations of chemicals. In cumulative risk assessment (CRA), regulatory bodies such as the European Food Safety Authority consider dose addition as a default and sufficiently conservative approach. The principle of dose addition was confirmed previously for inducing craniofacial malformations in zebrafish embryos in binary mixtures of chemicals with either similar or dissimilar modes of action (MOAs). Objectives: In this study, we explored a workflow to select and experimentally test multiple compounds as a complex mixture with each of the compounds at or below its no observed adverse effect level (NOAEL), in the same zebrafish embryo model. Methods: Selection of candidate compounds that potentially induce craniofacial malformations was done using in silico methods—structural similarity, molecular docking, and quantitative structure–activity relationships—applied to a database of chemicals relevant for oral exposure in humans via food (EuroMix inventory, ). A final subselection was made manually to represent different regulatory fields (e.g., food additives, industrial chemicals, plant protection products), different chemical families, and different MOAs. Results: A final selection of eight compounds was examined in the zebrafish embryo model, and craniofacial malformations were observed in embryos exposed to each of the compounds, thus confirming the developmental toxicity as predicted by the in silico methods. When exposed to a mixture of the eight compounds, each at its NOAEL, substantial craniofacial malformations were observed; according to a dose–response analysis, even embryos exposed to a 7-fold dilution of this mixture still exhibited a slight abnormal phenotype. The cumulative effect of the compounds in the mixture was in accordance with dose addition (added doses of the individual compounds after adjustment for relative potencies), despite different MOAs of the compounds involved. Discussion: This case study of a complex mixture inducing craniofacial malformations in zebrafish embryos shows that dose addition can adequately predicted the cumulative effect of a mixture of multiple substances at low doses, irrespective of the (expected) MOA. The applied workflow may be useful as an approach for CRA in general. https://doi.org/10.1289/EHP9888

Invited Perspective: How Relevant Are Mode-of-Action Considerations for the Assessment and Prediction of Mixture Effects?

“…The work by Van der Ven et al. 1 supports the idea of the default application of dose addition, even to mixtures viewed as acting dissimilarly, as advocated by the European Food Safety Authority. 13 Regulators need broader criteria for building common assessment groups based on common adverse outcomes, regardless of perceived mechanisms.…”

“…However, the well-designed study by Van der Ven et al. 1 of a mixture of chemicals producing craniofacial malformations in zebrafish embryos calls the relevance of such distinctions into question.…”

“…Van der Ven et al. 1 assessed the accuracy of dose addition with a mixture of eight chemicals that produce malformations by diverse mechanisms. By carefully distinguishing major MOAs and placing them in pathway networks, the authors identified informative gene markers and tested their expression.…”

“…Van der Ven et al. 1 concluded that dose addition does not depend on the MOA of the components in the mixture. Work with chemical mixtures in fish led van Leeuwen 7 to a comparable insight in 1995.…”

Retrospective cumulative dietary risk assessment of craniofacial alterations by residues of pesticides

“…Results of a range of bioassays investigating the liver steatosis, craniofacial alterations and endocrine‐related effects were in agreement with the dose‐addition model. As it has been shown, the analyses of mixture effects revealed additivity for all the different combinations of MoA and endpoints that were tested (Zoupa et al, 2020 ; Luckert et al, 2019 ; Lichtenstein et al, 2020 ; van Oostrom et al., 2020 ; Moretto et al, 2013 ; Metruccio et al, 2020 ; Van Der Ven et al, 2022 ). Thus, the dose‐addition model was found to be adequate in the tested cases, in mixtures containing substances eliciting the common adverse effect through both similar and dissimilar modes of action.…”