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, $n=\mathrm{1,598}$). 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

中文翻译：

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暴露于具有不同作用模式的食品相关化学物质的复杂混合物中的斑马鱼胚胎诱导颅面畸形的剂量添加

 抽象的

 背景：

人类暴露于化学物质的组合中。在累积风险评估（CRA）中，欧洲食品安全局等监管机构将剂量添加视为默认且足够保守的方法。先前已证实剂量添加的原理可在具有相似或不同作用模式（MOA）的化学物质二元混合物中诱导斑马鱼胚胎颅面畸形。

 目标：

在这项研究中，我们探索了一个工作流程，在同一斑马鱼胚胎模型中选择并实验测试多种化合物作为复杂混合物，每种化合物均处于或低于其未观察到的不良影响水平 (NOAEL)。

 方法：

使用计算机方法（结构相似性、分子对接和定量结构-活性关系）选择可能诱发颅面畸形的候选化合物，并将其应用于人类通过食物口服暴露相关的化学物质数据库（EuroMix inventory，$\mathrm{<span\; data-immersive-translate-walked="14d299ec-f53b-4c3e-a99e-180a98c9ad4d">n</span>}<span\; data-immersive-translate-walked="14d299ec-f53b-4c3e-a99e-180a98c9ad4d">=</span>\mathrm{<span\; data-immersive-translate-walked="14d299ec-f53b-4c3e-a99e-180a98c9ad4d">1,598</span>}$ ）。最终的子选择是手动进行的，以代表不同的监管领域（例如食品添加剂、工业化学品、植物保护产品）、不同的化学品家族和不同的 MOA。

 结果：

在斑马鱼胚胎模型中检查了最终选择的八种化合物，并在暴露于每种化合物的胚胎中观察到颅面畸形，从而证实了通过计算机方法预测的发育毒性。当暴露于八种化合物的混合物（每种化合物均处于其 NOAEL 水平）时，观察到明显的颅面畸形；根据剂量反应分析，即使胚胎暴露于该混合物的 7 倍稀释液中，仍然表现出轻微的异常表型。尽管所涉及的化合物的作用机制不同，但混合物中化合物的累积效应与剂量添加一致（调整相对效力后单个化合物的添加剂量）。

 讨论：

这一复杂混合物诱发斑马鱼胚胎颅面畸形的案例研究表明，无论（预期）MOA如何，剂量添加都可以充分预测低剂量多种物质混合物的累积效应。所应用的工作流程通常可以作为 CRA 的一种方法。 https://doi.org/10.1289/EHP9888