Chemical mixtures

In the context of human biomonitoring, the term mixtures refers to the common occurrence of chemical xenobiotic substances in the body. In other words, any combination of chemical substances or their metabolites circulating in the human body at a given time. There is no broadly accepted operational definition of mixtures.

In principle, every single substance, once it enters the body, will exhibit its health effects in interaction with a person’s genetic makeup and acquired characteristics, and in concert with all other xenobiotic substances from previous and simultaneous exposures. These combined and/or simultaneous exposures may arise either involuntarily or voluntary, through different exposures routes from ambient environments, indoor and occupational environments, diet, consumer products, medication, medical or voluntary implants, recreational drugs, performance enhancing drugs, tattoo ink, etc. These mixtures present a challenge to experimental and observational science, to chemical risk assessment and to chemical risk management.

The European Commission (EC) communication on “The combination effects of chemicals – Chemical mixtures” (EC, 2012) was published in response to a request from the European Parliament for the Commission to consider the extent to which the existing legislation “adequately addresses risks from exposure to multiple chemicals from different sources and pathways, and on this basis to consider appropriate modifications, guidelines and assessment methods”.

In the communication, various types of mixtures are described as follows:

• Intentional mixtures: these are manufactured formulated products that are marketed as such. The composition of such mixtures and the hazardous properties and classification of the constituents is generally known;
• Mixtures originating from a single source: also known as ‘unintentional mixtures’ these are the result of discharges to the environment during the production, transport, use or disposal of goods, often containing a mixture of chemical substances. The composition can either be known (for example an effluent) or it can be unknown; and
• Mixtures of chemicals originating from multiple sources and through multiple pathways: also known as “coincidental mixtures” these relate to multiple substances from multiple and varying sources.  Their composition is unknown and can vary in both space and time.

HBM4EU explores how human biomonitoring can contribute to both the science and regulation of human exposure to mixtures. The focus falls on mixtures of chemicals with exposure routes through the environment, food, occupation and/or consumer products.

In terms of the contribution that human biomonitoring can make to better understanding mixtures, the Commission’s 2012 communication states that “there is a need to better understand human and environmental exposures, both through the use of monitoring and modelling”.  A 2014 report of the German Environment Agency on mixtures in the environment states that human biomonitoring data could be used not only to detect single substances for potential prioritisation, but also be analysed to determine whether certain substances occur in combination or are correlated in concentration to each other. This would enable the identification of priority mixtures (UBA, 2014).

Researchers in the US have developed a method for identifying mixtures most prevalent in humans, by applying frequent itemset mining to biomonitoring data from the National Health and Nutrition Examination Survey (NHANES). They identified 90 chemical combinations, consisting of relatively few chemicals, that occur in at least 30% of the US population (Kapraun et al, 2017).

Since mixtures in the body are comprised of a wide range of chemical substances,  all classes of hazardous properties are potentially involved. This poses the challenge of understanding where antagonism, addition or synergies in effects come into play, based on modes of action.

The EU Chemicals legislation is based predominantly on assessments carried out on individual substances. Since humans and their environments are exposed to a wide variety of substances, there is increasing concern in the general public about the potential adverse effects of the interactions between those substances when present simultaneously in a mixture.

In a 2011 opinion on Toxicity and Assessment of Chemical Mixtures, the non-food scientific committees of the European Commission, including the Scientific Committee on Consumer Safety (SCCS), the Scientific Committee on Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging and Newly Identified Health Risks (SCENIHR), concluded that under certain conditions, chemicals will act jointly in a way that the overall level of toxicity is affected. Chemicals with common modes of action act jointly to produce combination effects that are larger than the effects of each mixture component applied singly.  These effects can be described by dose/concentration addition. For chemicals with different modes of action, the opinion found no available robust evidence that exposure to a mixture of such substances is of health or environmental concern if the individual chemicals are present at or below their zero effect levels.

The committees found that interactions (including antagonism, potentiation, and synergies) usually occur at medium or high dose levels (relative to the lowest effect levels). At low exposure levels, they are either unlikely to occur or are toxicologically insignificant.

Dealing with mixtures in research poses specific challenges (Kortenkamp 2007, Kortenkamp et al, 2009). In toxicological research working mechanisms, mode of action and adverse outcome pathways can be studied in detail. However, typically only a few permutations of possible mixtures can be assessed.

This does not do justice to the wide array of substance to what populations are exposed to. On the other hand, observational studies in humans may capture these multiple substance, but often fall short in characterizing the dynamics of exposure and ADME characteristics (absorption, distribution, metabolism, and excretion) and typically cannot document mechanisms and causality.

Developments in modern techniques such as in sensor technologies, and in epigenomics, transcriptomics, metabolomics, as well as development in biostatistics now allow more in depth research of multiple exposures, body burdens and their effects in humans (Woodruff 2011, Lenters 2015, Agier 2016). To optimise the benefits from these developments new forms of cooperation between traditionally separated research communities and projects need to be build. HBM4EU provides an excellent opportunity to build these alliances.

A central problem in the discussion on mixtures is the virtual absence of adequate exposure data. In many human biomonitoring projects, as well as in cohort studies and biobank studies, groups of pollutants have been studied that include multiple individual substances. Yet reporting is typically restricted to distributions and central tendency measures of single compounds or groups of compounds. Common rationales for grouping chemicals include:

• chemical families – such as phthalates, bisphenols, dioxins, poly chlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAH) or volatile organic compounds (VOC)
• exposure routes – such as food or household dust
• use categories – such as plasticisers, flame retardants, pesticides
• supposed working mechanisms – such as endocrine disruption, carcinogenicity or neurotoxicity

In a few cases, studies report the distribution of an indicator of cumulative body burdens in individuals. However, this only summarizes body burdens for the groups mentioned above.

It is therefore largely unknown whether specific profiles of high exposures across multiple groups of chemical mixtures exist. For example, we do not know whether individuals with a high body burden of PCBs, also exhibit high body burdens of pesticides, flame retardants, per- and poly-fluorinated compounds and mycotoxins.

Meaningful indicators to capture such profiles need to be developed for mixtures in the broader meaning of the word. Aggregated mixture indicators could then be used to characterise exposure profiles of concern, and to identify potential hotspots or risk groups based on existing data and data from new studies. As such tools become available, existing data merit re-evaluation from a mixtures perspective.

Dealing with mixtures poses substantial regulatory challenges. In order to get an overview of scientific knowledge and regulatory approaches, in 2007 the European Commission contracted a study to review the current scientific knowledge and regulatory approaches. The study was completed in 2009 and produce a report entitled State of the Art Report on Mixture Toxicity (Kortenkamp et al, 2009)

In their 2011 opinion on Toxicity and Assessment of Chemical Mixtures, the non-food scientific committees of the European Commission argue that in view of the almost infinite number of possible combinations of chemicals, some form of selection criteria are required to allow a focus on mixtures of potential concern. They note that regarding the assessment of chemical mixtures, a major knowledge gap is the lack of exposure data and the rather limited number of chemicals for which there is sufficient information on their mode of action. HBM4EU is directly addressing the gaps in exposure data on chemical mixtures.

In May 2012, the Commission reported to the Council in its Communication from the Commission on Combination effects of Chemicals (Chemical mixtures). Through this Communication, the Commission launched a process to ensure that risks associated with chemical mixtures are properly understood and assessed. The report recognizes that while EU laws set strict limits for the amounts of particular chemicals allowed in food, water, air and manufactured products, the potentially toxic effects of these chemicals in combination are rarely examined. It recognises that “within the framework of EU legislation, there is no mechanism for a systematic, comprehensive and integrated assessment of mixture effects taking into account different routes of exposure and different product types”.

The Communication states that the Commission will identify priority mixtures to be assessed and ensure that the different strands of EU legislation deliver consistent risk assessments for such priority mixtures. The Commission will also tackle some of the data and knowledge gaps to improve understanding of the mixtures to which people and the environment are exposed.

The Commission’s approach to tackling mixtures draws heavily on the opinion of the three non-food scientific Committees, Toxicity and Assessment of Chemical Mixtures, as well as on the State of the Art Report on Mixture Toxicity.

In 2013, the EU’s 7^th Environmental Action Programme set the goal by 2020 of ensuring that the combination effects of chemicals are effectively addressed in all relevant Union legislation, as part of a new strategy for a non-toxic environment.

In 2014, the European Commission’s Joint Research Centre (JRC) produced a report entitled Assessment of mixtures – Review of Regulatory Requirements and Guidance (JRC, 2014). The report concludes that “while many pieces of EU legislation are in place to protect humans and the environment against adverse effects of chemicals including mixtures, in many cases it remains unclear how this is to be carried out and only few explicitly consider (real life) exposure to mixtures. In cases where mixtures are considered, the assessment is frequently limited to some well-known components. Several mathematical models and approaches have been developed to assess the toxicity of mixtures, but their routine application is hampered by considerable information gaps”.

The legislative status of mixtures varies depending on the type of mixture:

• Intentional mixtures: these are formulated products that marketed as such and so are covered by requirements under CLP, the authorisation of plant protection products as well as rules governing the composition of cosmetics and approval of medicinal products. For intentional mixtures, composition is well known and assessments are based on the properties of the constituents supplemented, where appropriate (but often in exceptional circumstances), by tests carried out on the entire product.
• Mixtures originating from a single source: also known as ‘unintentional mixtures’. Where composition is known (or can be identified) assessments can be made based on knowledge of the constituents and where the composition is unknown an assessment can in principle be based on tests carried out on the whole mixture but, in practice, there are very few examples of EU legislation specifically requiring the assessment or testing of whole mixtures. The Water Framework Directive and waste legislation have elements that require consideration of effects in combination.
• Mixtures of chemicals originating from multiple sources and through multiple pathways: also known as “coincidental mixtures”.  There is only a limited coverage of issues in EU legislation and these are limited in their scope.

Risk assessment for mixtures

Current approaches to chemical risk assessment mainly focuses on exposure to individual chemicals, mostly considering only a single source, while “real life” exposure comprises multiple chemicals from different sources and routes. Mixture risk assessment is often confounded by gaps in exposure and hazard data. At the same time, there is the potential to improve mixtures risk assessment through novel alternative tools and a need for guidance that harmonises approaches across different legislative sectors (Kienzler et al, 2016).

A framework for the risk assessment of combined exposure to multiple chemicals was developed at a 2009 WHO/IPCS workshop (Meek et al., 2011). This framework describes a general approach for the risk assessment of combined exposure to multiple chemicals, adaptable to specific needs. However, its use is often hampered by data gaps on exposure as well as hazard.

Several different guidance documents on risk assessments for mixtures have been published recently, each focusing on a specific group of compounds or type of assessment. Examples include the guidance on aquatic risk assessment under REACH (Bunke et al., 2013), the assessment of mixture effects of biocides (ECHA, 2014) and guidance produced by EFSA on mixtures of pesticides.

In the context of the REACH Regulation (No 1907/2006), guidance has been developed concerning the assessment of multiple sources of exposure to a single substance and in specific cases to the assessment of several closely related and similarly acting substances, such as different salts of the same metal or a number of closely related derivatives of organic substances (see for example ECHA, 2016, section E.3.5).  Whilst this gives some scope to assess possible adverse effects associated with known combinations, it does not address possible concerns associated with exposure to unknown mixtures.

Regarding mixtures of pesticides, Regulation 396/2005/EC on maximum residue levels of pesticides in or on food and feed of plant and animal origin highlights the importance of further work to develop a methodology to take into account cumulative and synergistic effects. The regulation notes that “In view of human exposure to combinations of active substances and their cumulative and possible aggregate and synergistic effects on human health, maximum residue levels should be set after consultation of the European Food Safety Authority”.

In response to this mandate, the European Food Safety Authority (EFSA) has undertaken a programme of work on mixtures risk assessment (see EFSA’s webpages on mixtures). EFSA’s activities in the area of mixtures have included:

• approaches for assessing combined exposure to multiple pesticides in bees (Spurgeon, 2016);
• a scientific opinion on the identification of pesticides for inclusion in cumulative risk assessment groups on the basis of their toxicological profile (EFSA, 2014); and
• guidance on probabilistic modelling of dietary exposure, applied to pesticide residue mixtures from the triazole group (Boon et al., 2015).

In addition, under an ongoing initiative called MixTox, EFSA has established a working group of experts to develop guidance aimed at harmonising methodologies for assessing human and ecological risks resulting from exposure to multiple chemicals. EFSA is undertaking or supporting several research activities to contribute to the development of this guidance:

• Data collection and analysis of combined toxicity of multiple chemicals  of relevance to EFSA in the area of human, animal and environmental toxicology.
• Development of tools for modelling (tiered approaches, population dynamics, human variability) for human and ecological risk assessment for single and multiple chemicals.

JRC has also been active in the field of risk assessment for mixtures, producing a 2015 survey and literature review on scientific methodologies for the assessment of combined effects of chemicals (Bopp et al 2015). The report reviews alternative tools for assessing the hazard of chemical Mixtures, with a focus on the adverse outcome pathway (AOP) concept, in vitro methods, omics techniques, in silico approaches such as quantitative structure activity relationships (QSARs) and read-across, toxicokinetic and dynamic energy budget (DEB) modelling, and on integrated approaches to testing and assessment (IATA).

In 2016, JRC published a review of case studies on the human and environmental risk assessment of chemical mixtures (Bopp et al, 2016). In all case studies, assessments were made on the basis of concentration addition, mainly applying the Hazard Index. The review found evidence for combined exposure to chemicals regulated under different legislation, as well as evidence that such chemicals can elicit similar effects or have a similar mode of action. The review concludes that mixture risk assessment across regulatory sectors should therefore be further investigated.

Risk management of mixtures

Being formulated products, the chemical composition of intentional mixtures is generally well known. Further, owing to legislation that applies to individual chemicals, the physicochemical, toxicological and ecotoxicological properties of the substances making up a mixture are also known.  The first level of regulation is via the REACH Regulation (No 1907/2006) and the classification, labelling and packaging of substances and mixtures regulation (CLP Regulation (EC) No 1272/2008). A brief overview of the coverage of intentional mixtures under REACH and CLP is available for download here: Intentional mixtures under REACH and CLP.

In contrast to the intentional mixtures that are marketed as products, managing the risks of unintentional and coincidental mixtures is much more challenging. Owing to the complexities of substance identity and variations including in composition, sources, pathways, receiving environment and individuals, there are relatively few legislative instruments that address unintentional or coincidental mixtures. Unintentional and coincidental mixtures are addressed to some extent under health and safety legislation, environmental legislation and consumer legislation.

Occupational health and safety legislation

The Chemical Agents Directive 98/24/EC outlines the minimum requirements for the protection of workers health and safety arising, or likely to arise, from the effects of chemical agents in the workplace or the use of chemical agents at work. The Directive sets out the obligation for employers to carry out an assessment of the risk to health and safety arising from the presence of chemical agents and specific protection and prevention measures. The Directive specifies maximum levels for individual substances and explicitly refers to chemical agents in combination, including both unintentional and coincidental mixtures.

Consumer legislation

Regarding food contaminants, with the exception of dioxins and dioxin-like compounds, Regulation 315/93/EEC on contaminants in food does not take mixture toxicity or combination effects. Regarding food contact materials, while Regulation 1935/2004/EC on materials and articles intended to come into contact with food considers cumulative effects, these are undefined and mixture toxicity is not considered.

Regulation 396/2005/EC on maximum residue levels of pesticides in or on food and feed of plant and animal origin acknowledges the need for cumulative and mixture toxicity assessment when establishing maximum residue levels and identifies a role for EFSA, as discussed in the section on risk assessment for mixtures above.

Environmental legislation

The Water Framework Directive (WFD) 2000/60/EC establishes a framework for Community action in the field of water policy with the aim of achieving ‘good ecological status’ of water bodies across the community. Although it does not explicitly identify combination effects of chemicals, the requirement for water bodies to achieve good ecological status as well as good chemical status requires a focus not only on the concentrations of individual chemicals but also on their effects on the aquatic environment in combination.

The Marine Strategy Framework Directive 2008/56/EC establishes a framework to achieve good environmental status in the marine environment by 2020. The Directive indicates that the analysis of pressures and impacts on water status should consider cumulative and mixture effects.  However, how this should be achieved is not specified.

Directive 2010/75/EU on industrial emissions lays down the rules on integrated prevention and control of pollution from industrial activities. It aims to prevent, or where this is not possible, to reduce emissions to air, water and land and to prevent the generation of waste. With the exception of dioxins and furans, however, mixture and combination effects are not considered.

The Environmental Impact Assessment Directive 2011/92/EU sets out requirements for the environmental impact assessment of large scale public and private projects. There is a requirement to consider and estimate emissions of pollutants and assess cumulative effects of impacts. Chemical mixture toxicity is not, however, specifically addressed.

The overarching objective of the mixture activities in HBM4EU is to improve the efficacy of HBM to inform science, policy/regulatory actions and societal debate with respect to dealing with mixtures. Some underlying questions include:

• What is the information need of regulatory bodies and stakeholders?
• What are common HBM mixture patterns in the European population?
• Can we identify hotspots or risk groups with high mixture exposures?
• Which sources & pathways contribute most to HBM mixture values?
• Which effect markers can we use to assess health risks of mixtures?
• What action perspectives are available to reduce mixture levels?

The more specific objectives are:

• Develop summary indicators to describe the exposure and body burdens of mixtures with an emphasis on defining priority mixtures and drivers of mixture toxicity.
• Re-evaluate existing HBM mixture data to identify real-life exposure patterns to mixtures.
• Collect new HBM mixture data in selected European countries.
• Further develop and apply practical approaches to assess the potential health risks and impacts of mixtures.
• Inform policy makers, stakeholders and the public at large about mixture exposures, possible health risks and perspectives for action.

Five EC funded H2020 and FP7 research projects, namely EDC-MixRisk, EuroMix, EU-ToxRisk, HBM4EU and SOLUTIONS, are working together to address different aspects of the impacts of mixtures on human health and the environment, including also research activities at the European Food Safety Authority (EFSA) and the Joint Research Centre (JRC).

Through this collaboration, synergies, knowledge exchange and the ability to exchange and use methods and data will be promoted.   A joint workshop ‘Advancing the Assessment of Chemical Mixtures and their Risks for Human Health and the Environment’ was be held 29-30 May 2018 at the Joint Research Centre in Ispra, Italy.

Participants discussed the current state of knowledge and identified and prioritised areas for future policy and research needs.

To express concern and raise awareness on hazardous chemical mixtures and combined exposure, the Coordinators and representatives of the above mentioned research projects sent a position paper in April 2018 to the Director‐Generals of DG Environment, DG Research and Innovation and DG Health and Food Safety.

The position paper, entitled “Preventing risks for people and environment from hazardous chemical mixtures”, calls for the uptake of existing methods as well as the development of new approaches, methodologies and tools. It proposes 12 key actions and recommendations to better address combined effects and overcome remaining gaps in chemical mixture research and policy making. It also provides some ideas from the perspective of these research projects as input to preparations for the next Framework Programme, Horizon Europe.

Link to the Position Paper 

In the interest of transparency and accountability, HBM4EU invites interested stakeholders to submit comments on the scoping document.

All submitted comments will be made available for download on this webpage and will be taken into consideration by the HBM4EU consortium, where possible.

Please click here to submit your comments.

Please click here to access the Substance Report.

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