Evaluating the Endocrine‐Disrupting and Oxidative Stress Potential of a 50‐Component Human‐Relevant Complex Chemical Mixture Using In Vitro Tests

Humans are chronically exposed to mixtures of environmental contaminants. Exposure to endocrine‐disrupting chemicals (EDCs) contributes to increased health impairment observed globally. This study aimed to evaluate the endocrine‐disruptive and oxidative stress potential of a human‐relevant, complex chemical mixture in vitro. By testing chemical class subgroup mixtures, the identity of toxicological drivers and mixture additivity could be investigated. A 50‐component mixture was compiled based on Swedish human blood concentrations (xHBC), consisting of six subgroup mixtures: polychlorinated biphenyls (PCBs) and 2,3,7,8‐tetrachlorodibenzo‐ p ‐dioxin (PCB mixture), brominated flame retardants (BFR mixture), per‐ and polyfluoroalkyl substances (PFAS mixture), pesticide mixture, synthetic phenolic contaminants (phenol mixture), and phthalate mixture. These were tested in four chemically activated luciferase gene expression (CALUX) assays: dioxin responsive (DR‐), estrogen receptor α (ER α ‐), androgen receptor. (AR‐), and nuclear factor erythroid 2‐related factor 2 (Nrf2)‐CALUX, along with an adipocyte cell assay. The total mixture caused significant agonistic activity in DR‐ and ER‐, and antagonistic activity in AR‐CALUX at 0.1–15 xHBC, depending on the assay. Mixture additivity was assessed in ER α ‐, DR‐, and anti‐AR‐CALUX using subgroup mixtures and the concentration addition (CA) model. The total mixture followed the CA model in ER α ‐, anti‐AR‐ and DR‐CALUX. The toxicological drivers of these activities were mainly the PCB and phenol mixture. A significant increase in differentiated adipocytes was observed at 100 xHBC. These results raise concerns regarding potential health effects on the endocrine system. The additive effects at human‐relevant concentrations observed in this study motivate considering mixtures in regulatory contexts to protect the well‐being of future generations.