Evaluating the Toxicity of Mixtures

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Evaluating the Toxicity of Mixtures

Risk assessment of chemical mixtures whether for human health and/or environmental risk is extremely challenging. The evaluation of not just multiple components but their numerous combinations requires rigorous science in a landscape where the momentum is shifting from a substance-by-substance approach to a more mechanistic “Mode of Action” approach to determine adverse effect(s) from exposure to mixtures using Adverse Outcome Pathways (AOP’s).

Prioritising the evaluation of mixtures and their components is based upon human or environmental exposure, and applies to:

  • Intentional mixtures: The composition of such mixtures and the hazardous properties and classification of the constituents is generally known, i.e. manufactured formulated products.

  • Unintentional mixtures: The composition can either be known (i.e., an effluent) or it can be unknown.

  • Coincidental mixtures: Their composition is unknown and can vary in both space and time, i.e. multiple substances from multiple and varying sources,

In a recent conference on “The Toxicology of Mixtures and the Application of Cumulative Risk Assessment” (Regulatory Science Associates, 2019), current approaches to the assessment of pesticide mixtures were discussed based on EFSA’s EU guidance for classifying mixtures into Cumulative Assessment Groups (CAGs), and the longer established US-EPA pesticide guidance. Testing policies were considered appropriate but the science was not judged to be where it should be based on concerns such as “do we really understand where mixture toxicity arises from based on unrealistic dosing practices”. Mixture testing of pesticides in the EU is primarily driven by concerns over dietary risk following human consumption. With many mixtures tested at high doses, are these levels representative of human exposure? Would testing of lower doses (with a significant dose margin) reflect a more realistic assessment of exposure?

Ongoing collaborations to address the different aspects of mixtures are being undertaken by five EC funded projects, namely, EDC-MixRiskEuroMixEU-ToxRisk, HBM4EU and SOLUTIONS, their aim to maximise the impact of work on mixtures and to enhance chemical safety. 

Furthermore, EFSA has also recently published guidance on the “Genotoxicity of Chemical Mixtures.” In summary, a mixture where not all components are known must be characterised as fully as possible, and the identity and stability of the key components clearly demonstrated to support the marketed mixture. For chemically “fully defined” mixtures, a component‐based approach is recommended using all available data, read-across and QSAR considerations of genotoxic potential.

  • If one or more chemical substances within a mixture are assessed as genotoxic in vivo, via a relevant route, the mixture raises genotoxic concern.

  • If all components of a chemically “fully” defined mixture are confirmed as non- genotoxic, the mixture raises no genotoxic concern.

  • Where any individual component indicates a genotoxic concern, but with inconclusive data, additional data should be sought to complete an assessment.

  • If a mixture contains a substantial fraction of non-chemically identified substances, it is recommended that the chemically identified substances are first assessed individually for potential genotoxicity using all available information.

  • If none of the identified chemical substances in a mixture raises a concern over genotoxicity, the unidentified fraction should be evaluated. Should this not be feasible, the whole mixture should be tested following guidance for individual chemical substances (EFSA, 20112017):


For further advice on this or any other chemical regulatory concern, please contact

Genotoxicity assessment of chemical mixtures, EFSA Journal 2019;17(1):5519.

Position Paper (2018): Preventing risks for people and environment from hazardous chemical mixtures.

Regulatory assessment of chemical mixtures: Requirements, current approaches and future perspectives (2016).

Something from nothing? (2018) Ensuring the safety of chemical mixtures.

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