This article reviews the current legislative requirements for risk assessment of combined exposure to multiple chemicals via multiple exposure routes, focusing on human health and particularly on foodrelated chemicals. The aim is to identify regulatory needs and current approaches for this type of risk assessment as well as challenges of the implementation of appropriate and harmonized guidance at international level. It provides an overview of the current legal requirements in the European Union (EU), the United States and Canada. Substantial differences were identified in the legal requirements for risk assessment of combined exposure to multiple chemicals and its implementation between EU and non-EU countries and across several regulatory sectors. Frameworks currently proposed and in use for assessing risks from combined exposure to multiple chemicals via multiple routes and different durations of exposure are summarized. In order to avoid significant discrepancies between regulatory sectors or countries, the approach for assessing risks of combined exposure should be based on similar principles for all types of chemicals. OECD and EFSA identified the development of harmonized methodologies for combined exposure to multiple chemicals as a key priority area. The Horizon 2020 project "EuroMix" aims to contribute to the further development of internationally harmonized approaches for such risk assessments by the development of an integrated test strategy using in vitro and in silico tests verified for chemical mixtures based on more appropriate data on potential combined effects. These approaches and testing strategies should be integrated in a scientifically based weight of evidence approach to account for complexity and uncertainty, to improve risk assessment.

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A human intoxication incident attributed to pesticide abuse was investigated using cutting-edge analytical methodologies. An LC-ESI-MS/MS method, based on a hybrid solid-phase extraction protocol (hybrid-SPE), was applied for the detection and quantification of several pesticides and metabolites in human biological fluids. Concomitantly, an UHPLC-HRMS method was applied to investigate potential metabolites, assisted by a complementary GC-MS method to elucidate the presence of plausible pesticides co-formulants. The LC-ESI-MS/MS method exhibited acceptable mean recoveries at the lower limit of quantification (LLOQ) and three additional levels, varying from 85 to 106% for all analytes and matrices. In serum, urine, and gastric fluid samples, the suspect compounds, namely chlorpyrifos and myclobutanil, predominated. Gastric fluid samples contained the highest concentrations of chlorpyrifos (39,800 ng/mL) and myclobutanil (18,800 ng/mL), while the neonicotinoid imidacloprid was also quantified, below 30 ng/mL. Notwithstanding, the UHPLC-HRMS analysis unveiled several metabolites of chlorpyrifos and myclobutanil. In parallel, GC-MS analysis, corroborated the presence of several co-formulants in gastric fluid samples, exemplified by m- and o-xylene, and cyclohexanone. Overall, three analytical methods were implemented to elucidate the chemical causality of a human intoxication incident. The presence of suspected active substances, one additional, and several metabolites and co-formulants were documented.

Based on the “Human in vitro dermal absorption datasets” published as supporting information to the revised EFSA Guidance on Dermal Absorption, in silico models for prediction of absorption across the skin have been evaluated. For this evaluation, a systematic literature search and review was performed, identifying 288 publications describing mathematical models for prediction of dermal absorption. Eleven models potentially relevant to the regulatory assessment of pesticides and which cover a range of approaches were selected for in depth evaluation. This included three mixture models taking into account physicochemical properties of the co‐formulants such as polar surface area, hydrogen bonding or octanol‐water partition coefficients. Additional data on the pesticidal active substances and information on the composition of some of the formulations covered in the dermal absorption dataset were gathered, as these were required as input parameters for the selected models. The models were implemented with settings reflecting as much as possible realistic exposure scenarios and the experimental conditions under which the measured data were obtained. As the majority of the models predicted either maximum flux or the permeation coefficient, further combination with a model achieving translation into percentage absorption was required. This was done with and without consideration of the lag time. Only one model directly predicted percentage dermal absorption, which was a spreadsheet‐based single substance model taking into consideration several skin parameters, experimental conditions, various physicochemical properties of the active substance and the type of vehicle. Statistical analysis of model predictions revealed overall low concordance with measured values, thereby limiting regulatory acceptance. Additional analysis was performed on the results of two mixture models and the above mentioned complex single substance model which showed moderate correlation between predicted and measured data. Options to improve model performance were discussed and Bayesian random effects modelling was explored to adjust predicted percentage dermal absorption to measured data as was model combination. When taking into account observed uncertainties of predictions, one of the models may provide a Tier 2 tool to estimate dermal absorption value in the absence of adequate experimental data when the predicted values are in the range of 10 to 70%. However, further work is needed to better understand the effect of co‐formulants on dermal absorption of pesticides and to improve model predicitivity.

This paper reviews key elements in the assessment of human health effects from combined exposure to multiple chemicals taking into consideration current knowledge and challenges to identify areas where scientific advancement is mostly needed and proposes a decision-making scheme on the basis of existing methods and tools. The assumption of dose addition and estimation of the hazard index (HI) is considered as a starting point in component-based risk assessments. When, based on the generic HI approach, an unacceptable risk is identified, more specific risk assessment options may be implemented sequentially or in parallel depending on problem formulation, characteristics of the chemical group under assessment, exposure levels, data availability and resources. For prospective risk assessments, the reference point index/margin of exposure (RPI/MOET) (Option 1) or modified RPI/normalized MOET (mRPI/nMOET) (Option 2) approaches may be implemented focusing on the specific mixture effect. Relative potency factors (RPFs) may also be used in the RPI approach since a common uncertainty factor for each mixture component is introduced in the assessment. Increased specificity in the risk assessment may also be achieved when exposure of selected population groups is considered (Option 3/exposure). For retrospective risk assessments, human biomonitoring data available for vulnerable population groups (Option 3/susceptibility) may present more focused scenarios for consideration in human health risk management decisions. In data-poor situations, the option of using the mixture assessment factor (MAF) is proposed (Option 4), where an additional uncertainty factor is applied on each mixture component prior to estimating the HI. The magnitude of the MAF may be determined by the number of mixture components, their individual potencies and their proportions in the mixture, as previously reported. It is acknowledged that implementation of currently available methods and tools for human health risk assessment from combined exposure to multiple chemicals by risk assessors will be enhanced by ongoing scientific developments on new approach methodologies (NAMs), integrated approaches to testing and assessment (IATA), uncertainty analysis tools, data sharing platforms, risk assessment software as well as guideline development to meet legislative requirements.