Advancing chemical mixture toxicity assessment using advanced in vitro NAMs: current status and future perspectives

Area of Science:

• Toxicology
• In vitro toxicology
• Chemical mixtures

Background:

• Real-world chemical exposures predominantly involve mixtures, contrasting with traditional single-substance toxicological assessments.
• New approach methodologies (NAMs) are gaining traction for more human-relevant and mechanistically informative toxicity testing.
• The application of advanced in vitro NAMs in chemical mixture toxicity assessment requires structured examination.

Purpose of the Study:

• To evaluate the current application of advanced in vitro NAMs in chemical mixture toxicity assessment.
• To consider the potential value of these advanced in vitro NAMs for regulatory translation.
• To identify how advanced in vitro NAMs address key challenges in mixture toxicity assessment.

Main Methods:

• A systematic literature search of PubMed for studies published between 2021 and 2025.
• Inclusion of 70 advanced in vitro NAM-based studies out of 353 identified studies on chemical mixture toxicity.
• Categorization of advanced in vitro NAMs, including route-of-exposure-relevant models, Omics, 3D models, stem cell-based models, and HTS/HCI approaches.

Main Results:

• Route-of-exposure-relevant models were the most frequently applied advanced in vitro NAM category.
• Advanced in vitro NAMs were used to assess overall mixture effects, compare predicted vs. observed responses, and identify toxicity drivers.
• Studies addressed diverse mixtures like air pollutants, PFAS, phthalates, pesticides, and consumer product chemicals.

Conclusions:

• Advanced in vitro NAMs are valuable tools for addressing inherent challenges in mixture toxicity assessment, such as defining relevant mixtures and interpreting non-additive effects.
• Their future regulatory value depends on technological advancement and integration into fit-for-purpose testing strategies.
• These NAMs can effectively generate decision-relevant evidence for specific mixture assessment needs and regulatory contexts.