Effect biomarkers can be used to elucidate relationships between exposure to environmental chemicals and their mixtures with associated health outcomes, but they are often underused, as underlying biological mechanisms are not understood. We aim to provide an overview of available effect biomarkers for monitoring chemical exposures in the general and occupational populations, and highlight their potential in monitoring humans exposed to chemical mixtures. We also discuss the role of the adverse outcome pathway (AOP) framework and physiologically based kinetic and dynamic (PBK/D) modelling to strengthen the understanding of the biological mechanism of effect biomarkers, and in particular for use in regulatory risk assessments. An interdisciplinary network of experts from the European chapter of the International Society for Exposure Science (ISES Europe) and the Organization for Economic Co-operation and Development (OECD) Occupational Biomonitoring activity of Working Parties of Hazard and Exposure Assessment group worked together to map the conventional framework of biomarkers and provided recommendations for their systematic use. We summarized the key aspects of this work here, and discussed these in three parts. Part I, we inventory available effect biomarkers and promising new biomarkers for the general population based on the H2020 Human Biomonitoring for Europe (HBM4EU) initiative. Part II, we provide an overview AOP and PBK/D modelling use that improved the selection and interpretation of effect biomarkers. Part III, we describe the collected expertise from the OECD Occupational Biomonitoring subtask effect biomarkers in prioritizing relevant mode of actions (MoAs) and suitable effect biomarkers. Furthermore, we propose a tiered risk assessment approach for occupational biomonitoring. Several effect biomarkers, especially for use in occupational settings, are validated. They offer a direct assessment of the overall health risks associated with exposure to chemicals, chemical mixtures and their transformation products. Promising novel effect biomarkers are emerging for biomonitoring of the general population. Efforts are being dedicated to prioritizing molecular and biochemical effect biomarkers that can provide a causal link in exposure-health outcome associations. This mechanistic approach has great potential in improving human health risk assessment. New techniques such as in silico methods (e.g. QSAR, PBK/D modelling) as well as 'omics data will aid this process. Our multidisciplinary review represents a starting point for enhancing the identification of effect biomarkers and their mechanistic pathways following the AOP framework. This may help in prioritizing the effect biomarker implementation as well as defining threshold limits for chemical mixtures in a more structured way. Several ex vivo biomarkers have been proposed to evaluate combined effects including genotoxicity and xeno-estrogenicity. There is a regulatory need to derive effect-based trigger values using the increasing mechanistic knowledge coming from the AOP framework to address adverse health effects due to exposure to chemical mixtures. Such a mechanistic strategy would reduce the fragmentation observed in different regulations. It could also stimulate a harmonized use of effect biomarkers in a more comparable way, in particular for risk assessments to chemical mixtures.

Recent advances in analytical chemistry have allowed a greater possibility of using quantitative approaches for measuring human exposure to chemicals. One of these approaches is biomonitoring (BM), which provides unequivocal evidence that both exposure and uptake of a chemical have taken place. BM has been a longstanding practice in occupational health for several reasons. BM integrates exposure from all routes. It can help identify unintentional and unexpected exposures and assess the effectiveness of existing risk-management measures. BM also provides relevant information to support policy development by delivering better evidence of workers’ exposure to chemical substances, even within the framework of the present regulations. Thus, BM can allow for both the evaluation of the impact of regulation and identification of further needs for new or improved regulation. However, despite all these well-recognized advantages, BM is currently an underused exposure assessment tool. This paper provides an overview of the key aspects to be considered when using BM in the context of occupational health interventions. Additionally, this paper describes the potential of BM as an exposure assessment tool, distinguishing the role of BM in exposure assessment and health surveillance and clarifies ethical and communication aspects to guarantee that general data protection regulations are followed. In addition, actions and research needs are identified (particularly with reference to the European situation), which aim to encourage the increased use of BM as an exposure assessment tool.

Biomonitoring has been widely used in assessing exposures in both occupational and public health complementing chemical risk assessments because it measures the concentrations of chemical substances in human body fluids (e.g., urine and blood). Biomonitoring considers all routes and sources of exposure. An occupational biomonitoring guidance document has been elaborated (OECD Occupational Biomonitoring Guidance) within the OECD framework and specifically, the Working Parties on Exposure and Hazard Assessment by scientific experts from 40 institutes and organizations representing 15 countries. The guidance provides practical information for assessing chemical exposures in occupational settings including the three common routes of exposure: inhalation, skin absorption and ingestion due to hand to mouth contact. The elaborated stepwise approach for conducting biomonitoring is tailored for occupational health professionals, scientists, risk assessors, and regulators. It includes methods for selecting appropriate biomarkers, devising sampling strategies, and assessing laboratories for validated analytical methods for the biomarker of interest, and ensuring timely feedback of results. Furthermore, it describes procedures for setting up efficient biomonitoring programs based on the Similar Exposure Group (SEG) approaches. Derived health-based human exposure biomarker assessment values called Occupational Biomonitoring Levels (OBLs) are proposed for use in occupational exposure and risk assessment. It also helps with the interpretation of biomonitoring results routinely collected and procedures for communicating biomonitoring results at individual, collective, and workplace levels. Ethical considerations associated with biomonitoring are also discussed. The ultimate goal of this biomonitoring approach is to promote harmonized application and interpretation of biomarkers as well as evidence-based occupational risk management measures.

Derivation of occupational biomonitoring levels (OBLs) is needed to effectively utilize biomonitoring for assessing exposures to chemical substances, and consequently, implement risk reduction measures to reduce health risks among workers. OBLs are the appropriate option for chemical substances that can be absorbed through the skin. This methodology for derivation of OBLs has been developed in collaboration with scientific and regulatory experts from more than 40 institutes in 15 countries within the Organization for Economic Cooperation and Development (OECD) framework. This manuscript provides a summary of the guidance on derivation of OBLs destined for scientists, risk assessors, and regulators who are tasked with establishing OBLs for regulatory purposes and implementing occupational biomonitoring programs. The derivation methodology follows a tiered approach based on the strength of evidence and quality of the data that we have labeled level of confidence. The tiered approach serves as a practical framework in occupational health risk assessment and management. We distinguish between four OBL levels depending on the strength of scientific evidence and confidence level: health-based derivation of OBL based on robust epidemiological data showing causal exposure-health effect relationship and Provisional OBL (POBL) based on robust toxicological animal data showing dose-response relationship as well as two assessment values which are not health based: reference levels in the general population (Reference OBL or (ROBL)), and Technical achievable OBL or (TOBL). Four case studies illustrating the derivation methods for OBLs and POBLs are also provided. Using this state-of-the-art approach (OECD guidance document no. 370) will lead to a harmonized derivation of OBLs and subsequently to evidence-based risk management measures.

The international initiative titled: "Using Adverse Outcome Pathways (AOP) to address combined exposures to chemicals with relevant effect-biomarkers", was coordinated by the OECD's Working Parties on Exposure Assessment (WPEA) and Hazard Assessment (WPHA). It brought together over 90 experts from 25 countries to advance the regulatory and scientific use of effect biomarkers for assessing exposures to chemical mixtures and their associated biological and health-relevant effects. Effect biomarkers provide biologically integrated information that can complement traditional single-substance approaches by supporting the interpretation of combined chemical exposures and associated biological responses. While the official OECD Guidance targets primarily regulatory audiences, this article communicates the project's rationale and methodology to academic, industrial, and regulatory communities. Assessing chemical mixture exposures through effect biomarkers overcomes the limitations of traditional single-substance approaches that have guided regulatory frameworks for decades. Effect biomarkers, anchored in AOPs, offer biologically integrated assessments resulting from both known and unknown exposures integrating all exposure routes. Effect biomarkers are powerful in identifying biologically relevant responses, enabling targeted health assessments of vulnerable populations within human biomonitoring programs. This OECD project has produced a harmonised framework for deriving effect-based mixture threshold levels, applicable to both Occupational Biomonitoring Effect Levels (OBELs) for human health and Effect-Based Trigger values (EBTs) for environmental assessments. The framework establishes biologically anchored trigger values that help identify risks from exposures to complex chemical mixtures and can guide appropriate risk management measures. The derivation was guided by four core selection criteria and additional quality principles, ensuring biological relevance to regulatory-defined adversity, quantifiability, robustness, sensitivity, and interpretability for risk assessment. A four-tiered system (Tiers 0-3) reflects increasing toxicological and methodological certainty and relevance, with thresholds informing risk management decisions based on observed changes in biomarkers. Seven case studies demonstrate the framework's applicability across key hazard classes (genotoxicity/carcinogenicity, endocrine disruption, neurotoxicity, and reproductive toxicity), thus supporting harmonised group- and individual-level assessments and broader integration of effect biomarkers into occupational and environmental health assessments.