EUROPEAN COMMISSION Brussels, 14.10.2020 SWD(2020) 250 final COMMISSION STAFF WORKING DOCUMENT Progress report on the assessment and management of combined exposures to multiple chemicals (chemical mixtures) and associated risks Accompanying the document COMMUNICATION FROM THE COMMISSION TO THE EUROPEAN PARLIAMENT, THE COUNCIL, THE EUROPEAN ECONOMIC AND SOCIAL COMMITTEE AND THE COMMITTEE OF THE REGIONS



Chemicals Strategy for Sustainability



Towards a Toxic-Free Environment



{COM(2020) 667 final} - {SWD(2020) 225 final} - {SWD(2020) 247 final} - {SWD(2020) 248 final} - {SWD(2020) 249 final} - {SWD(2020) 251 final}



Contents 1. INTRODUCTION 2. WHY ARE MIXTURES A CONCERN? 3. REGULATORY FRAMEWORKS AND REQUIREMENTS REGARDING MIXTURES 3.1 Intentional mixtures 3.2 Unintentional mixtures 3.3 Grouping approaches 3.4 Aggregate Exposure 3.5 Conclusions on existing legal requirements for intentional and unintentional mixtures, grouping and aggregate exposure 4. PROGRESS ON GUIDANCE AND REGULATORY ASSESSMENT 5. CASES OF REGULATORY ASSESSMENT OF MIXTURES 6. INTEGRATED AND CO-ORDINATED ASSESSMENT OF MIXTURES ACROSS DIFFERENT PIECES OF LEGISLATION 6.1 Interface between different pieces of legislation 6.2 Case studies on priority mixtures 7. CLOSING THE KNOWLEDGE GAPS – RECENT ACHIEVEMENTS 7.1 Effect, exposure and hazard data 7.2 Knowledge on mode of action, grouping, interactions and drivers of mixture toxicity 7.3 Remaining knowledge gaps 7.4 Future research and development priorities

8. INTERNATIONAL ACTIVITIES 9. POSSIBLE WAYS FORWARD TO ADDRESS CHALLENGES IN THE REGULATORY CONTEXT 10. CONCLUSIONS



1.INTRODUCTION Humans as well as other organisms are exposed to a great variety of chemicals occurring in combinations also referred to as ‘mixtures’. Scientific evidence of the strengthened toxicity of such mixtures is mounting. Real-life examples of such mixtures are the different chemicals appearing simultaneously or sequentially in e.g. human blood or in a water body. The total risk related to the exposure to a combination of chemicals typically exceeds the risk related to the exposure to each of the individual chemicals in the mixture on their own, at their respective concentration in the mixture . Therefore, exposure to a mixture can give rise to adverse health and environmental effects, even at levels of exposure which are considered ‘safe’ for the individual chemicals on their own. Science demonstrates that in the majority of the cases, the toxicity of the mixture can be assessed by adding up the toxicities of the individual chemicals in the mixture. This is called the ‘concentration addition model’. In relatively rare cases, so called ‘interactions’ are observed, where the toxicity of the mixture is stronger or weaker than what would be expected from applying concentration addition . A distinction can be made between intentional and unintentional mixtures. Intentional mixtures are intentionally manufactured and often have a known composition and are contained in one product. Typical examples are products like paint, glue and detergents. Unintentional mixtures arise spontaneously or coincidentally, for example in air, water, soil, as well as in humans and other organisms as a result of co-exposure. They arise whenever chemicals are used on their own, as components in different intentional mixtures or in objects (i.e. complex products). Hence, an unintentional mixture typically includes chemicals from a variety of sources. Their compositions are usually more or less unknown and may vary over time and in space. Due to the very large number of possible combinations of chemicals, the risk assessment and management of unintentional mixtures (sometimes also called ‘coincidental mixtures’ or ‘combination of chemicals’) represents a particular scientific and regulatory challenge. Regarding EU policy, already the White Paper for a Future Chemicals Policy from 2001 highlighted exposure to mixtures of chemicals as a research priority to cover knowledge gaps and achieve the goals set out. In 2009, the Environment Council adopted Conclusions on the ‘combination effects of known chemicals’, which recognised the difficulties and deficiencies surrounding the regulation of mixtures. It also invited the Commission ‘to assess how and whether relevant existing Community 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, and report back to the Council by early 2012 at the latest’. The Commission responded to the Council through a Communication in 2012 , drawing upon the joint opinion of three EU Scientific Committees and taking into account the comprehensive State of the Art Report on Mixture Toxicity . In the Communication, the Commission reviewed the scientific knowledge and the regulatory requirements regarding the assessment of mixtures and committed itself to several actions and to presenting a report reviewing the progress and experience associated with the actions in the Communication. The EU 7th Environment Action Program stressed the need for appropriate regulatory approaches to address combination effects of chemicals. Recent resolutions of the European Parliament , , , , the Conclusions of the Environment Council on the chemicals policy adopted in June 2019 , and the Commission Communication on endocrine disruptors adopted in November 2018 , all confirmed the need to better take into account the combined effects of different chemicals for the protection of human health and the environment. Further, the Commission identified the combination effects of chemicals as a target action to be addressed in a Chemicals Strategy for Sustainability as part of the European Green Deal . The purpose of the present report is to describe the progress made since 2012 on the assessment of mixtures (combinations of chemicals) – both intentional and unintentional – to deliver on the follow-up action announced in the Communication and to provide background information and evidence base to actions announced in the Chemicals Strategy for Sustainability. This report presents the relevant legal requirements, guidance documents, methodologies and knowledge base as well as the remaining challenges. It also provides an overview of suggestions made by Member States, scientists and other stakeholders on possible ways forward. Box 1. Terminology , , , , ·Mixture: (or chemical mixture) in this report means any set of chemicals to which an organism may be jointly exposed, and which may potentially cause an adverse combination effect, regardless of sources and exposure routes. ·Intentional mixture: a mixture or solution which is composed of two or more substances (Article 3(2) REACH, Article 2(8) CLP), normally referring to a formulated product (mixture), manufactured and marketed as such, or the result of intentional use of different products (mixtures) together, where the composition is normally known. ·Unintentional mixture: mixtures of chemicals co-occurring in environmental media (water, soil, air), biota, feed, food, or human tissues as a result of releases from various sources and through multiple routes of exposure (the synonym term ‘coincidental mixture’ is sometimes used). Unintentional mixtures include degradation and transformation products of chemicals released into the environment. The composition of unintentional mixtures is often unknown and varying in time and space. ·Priority mixture: a chemical combination/mixture) of high concern to human or environmental health, which is a priority to identify and further assess for risk reduction. ·Combined exposure: simultaneous or sequential exposure to multiple substances via single or multiple pathways/routes (the synonym ‘cumulative exposure’ is sometimes used)

·Aggregate exposure: exposure to a single substance from multiple sources and via multiple pathways/routes. ·Combination effect (sometimes referred to as ‘cumulative’ or ‘mixture effect’): (eco)toxicological effect on an organism arising from exposure to a chemical mixture. Type and strength of the effect will vary depending on the composition of the mixture and the level of exposure. ·Sources: places of release of chemicals (e.g. industrial installations, diffuse sources such as products). ·Routes of exposure: ways of entering the organism (dermal, oral, inhalation, normally referring to human health). ·Compartment: (environmental) media where chemicals are taken up (air, water, sediment, soil, food/feed). ·Exposure pathway: includes fate and transport processes by which chemicals move from the original point of release through the environment, and the routes/interaction(s) through which populations or individuals are exposed (oral/ingestion, inhalation, dermal) via various media (water, sediment, soil, air, food/feed, products). ·Mode of action (MOA): the way a chemical exerts its biological effects. A common mode of action is assumed to involve one or several key events between the chemical and its biological target(s), leading to an (eco)toxicological effect. ·Interaction: an event where the joint effect of different chemicals in a mixture causes either an enhanced or weaker effect than that is expected based on the application of models such as concentration addition or independent action to predict the toxicity of the mixture. ·Synergism: an interaction between chemicals that enhances the toxicological effect beyond that is expected based on the toxicity of the individual components in the mixture. ·Antagonism: an interaction between chemicals that weakens the toxicological effect compared to what is expected based on the toxicity of the individual components in the mixture. ·Whole mixture testing: an approach where a sample or chosen media containing a mixture of chemicals is tested experimentally for one or several toxicological endpoints. ·Component-based approaches: methods for calculating mixture toxicity based on information on the toxicity and exposure to the individual compounds in the mixture. The two common models for mixture assessment are concentration (or dose) addition, and independent action. ·Concentration (or dose) addition: method/model for calculating mixture toxicity, based on the toxicity and concentration of the individual substances and under the assumption that they do not interact and have similar mode(s) of action. ·Independent action: assumes that mixture components contribute to a common endpoint via dissimilar and fully independent sequences of events, from an initial interaction with different molecular target sites to different diseases or different adverse effects seen at the level of individuals or populations. Consequently, the individual effects can be considered to be independent events in probabilistic sense. ·Toxicological Equivalence Factor (TEF): the toxicity weighted summation of the relevant mixture components. The TEF is in some regulations referred to as the ‘Summation Method’ and may be used to assess if the sum exceeds a pre-defined effect threshold. ·Default safety factor: a factor (sometimes referred to as default uncertainty factor), normally applied in the hazard and risk assessment of chemicals. Depending on the regulation different uncertainty factors may be applied. It may be used in the extrapolation from experimental animal studies to potential harmful effects on humans. Uncertainty factors may also be applied when choosing the part of the population that represents the exposure used in a study, as input to the risk assessment, as well as for the allocation of exposure to different sources. Various factors are also used in deriving predicted no-effect concentrations (PNECs) in environmental risk assessment. ·Mixture Assessment Factor (MAF): sometimes also called ‘Mixture Allocation Factor’, is an additional safety factor that can be applied in the risk assessment of single chemicals, in order to generically cover for combined exposure without performing a mixture-specific assessment. The possible use of MAFs in risk assessment has long been discussed, but the approach has so far not been applied for regulatory purposes at EU level. 2. WHY ARE MIXTURES A CONCERN? Chemicals normally appear as mixtures. They are intentionally blended to form products, such as paints, cosmetics, pesticides and biocides, as well as more or less complex materials like plastics and textiles. Chemicals may also be used together (e.g. for plant protection purposes or when different biocidal products are combined) and unintentionally emitted in combination and/or sequentially (e.g. as discharges during their manufacturing and use as well as from waste management and sewage treatment plants). Hence, humans, biota and environmental compartments are constantly exposed to such mixtures, often of unknown composition, from various sources and via different pathways. The occurrence of combination effects of chemicals is long known and has accordingly been an area of scientific research, and to some extent subject to regulatory action . A well-known example is the interaction between different pharmaceuticals when a patient is treated with two or more medicines simultaneously.

It is widely documented that the combined exposure to multiple chemicals can trigger stronger (or occasionally weaker) (eco)toxicological effects than exposure to individual chemicals alone , , . Even exposures at concentrations regarded as safe (i.e. where no effects are expected) for the individual chemical can result in adverse (eco)toxicological effects when several chemicals occur together in a mixture . Current regulatory approaches to the risk assessment of single chemicals normally involves establishing a level of exposure considered reasonably safe in relation to the inherent hazardous properties of the particular chemical , . When extrapolating from animal test data to a safe level of exposure for humans or organisms in the environment, safety margins are applied. The purpose is to take account of differences in sensitivity between animals and humans, i.e. between different species as well as between individuals. In the model for humans applied under REACH, these safety margins add up to a default safety (or uncertainty) factor of 100, commonly used in single chemicals risk assessment. Similar factors of different magnitude are used to establish safe levels of exposure in environmental hazard and risk assessment. However, these default uncertainty factors do not take into account the effect of combined exposures on humans or the environment . A large body of research in the EU and internationally over the past decades, including laboratory experiments, human and environmental monitoring and epidemiological studies provides a growing body of evidence clearly showing that exposure to mixtures of chemicals of anthropogenic origin is the norm in the human and natural environment. Further, research shows that combination effects do occur, in real life exposure situations as well as in experimental studies involving realistic exposure levels (see cases in Box 2). Box 2: Growing evidence of health and environmental concern relating to exposure to unintentional mixtures Since 2012, the body of evidence indicating that exposure to mixtures is a cause of health and environmental concern has grown considerably. Several studies show that mixtures are a cause of concern at real-life exposure levels and can be linked to effects through epidemiological and experimental means. Below are some selected examples from recent studies. JRC review of mixture risk case studies A 2016 report from the Commission’s Joint Research Centre reviews 21 case studies on mixture risk assessment conducted since 2014. The case studies include human health and environmental risk assessments and cover several classes of chemical compounds (e.g. pesticides, phthalates, parabens, polybrominated diphenyl ethers (PBDEs), pharmaceuticals, food contact materials, dioxin-like compounds and anti-androgenic chemicals). All selected studies involved testing of either real samples or realistic artificial samples. The reviewed case studies included unintentional mixtures like contaminants in breast milk, chemicals in indoor air and in environmental media, such as surface water, ground-water and drinking water. Several of the case studies indicated that exposure to the assessed mixtures posed concerns, especially regarding vulnerable populations. The review thus clearly demonstrates that unintentional chemical mixtures, also across chemical classes and legislative sectors, need to be better addressed. Aspects needing further attention include possible interactions/synergism, bioaccumulation and effects of metabolites appearing in mixtures as well as data gaps hampering a more refined assessment. Exposure of children, unborn children and pregnant women to endocrine disruptors and neurotoxic substances In 2017, the Danish Environmental Protection Agency published a report on the exposure of children under the age of three, unborn children and pregnant women to selected endocrine disrupting and neurotoxic chemical substances. The study included 37 endocrine disrupting chemicals (e.g. phthalates, perfluoro-substances, bisphenols and UV filters) and 39 neurotoxic chemicals (e.g. heavy metals, bisphenol A, PCBs, dioxins and perfluoro-substances), of which seven substances were relevant for both endpoints. Data regarding exposure via food, drinking water, indoor and outdoor environment, cosmetics and various consumer products were combined with human biomonitoring data to estimate exposure levels. The overall risk associated with the combined exposure was calculated through a concentration addition approach, establishing risk quotients (RQtotal) for the mixtures. The results of the Danish study indicate that the overall exposure of children under 3 years old to mixtures of endocrine disrupting chemicals is of concern (RQtotal >2) with respect to all three types of disruptive effects (anti-androgenic, estrogenic and thyroid hormone disrupting) already at average exposure levels. Similarly, for pregnant women and unborn children there were indications of possible concern (RQtotal≈1) already at average levels of exposure to combinations of endocrine disruptors. Also considering the exposure to combinations of neurotoxic substances, results indicate concern regarding children under 3 years old (RQtotal 61.1) as well as pregnant women and unborn children (RQtotal 7.9). Environmental risks of pesticide mixtures in inland waters A study by Gustavsson et al. from 2017 (part of the EU-funded SOLUTIONS project), evaluates the environmental risks associated with complex pesticide mixtures occurring in freshwater ecosystems. It is based on 1308 individual samples collected between 2002 and 2013 from rivers and streams in an agricultural area in southern Sweden, monitoring for 141 different pesticides. The study demonstrated that pesticides are present as mixtures, with up to 53 pesticides found in the same sample, with most commonly eight pesticides per sample. These findings correspond with multiple studies demonstrating the presence of complex pesticide mixtures in surface waters in other areas. Evaluation of the eco-toxicological effects of the pesticides mixtures, applying a statistical method (the Kaplan-Meier method), and based on an assessment using concentration addition and the water quality objectives established for the individual pesticides, shows that the environmental risk exceeded acceptable levels in 73% of the samples. Further, no clear time-trend in the level of risk was detected, but risk appears to remain relatively constant, despite regulatory interventions. A conclusion is that current single-substance risk assessment and mitigation is insufficient to manage risks related to the total and overall use of pesticides. A limited number of pesticides explains the largest share of toxicity of the mixture (i.e. so-called risk drivers). However, the composition of the mixtures of pesticides varies considerably between samples. Hence, 83 of the 141 monitored pesticides has to be included in the assessment to account for 95% of the risk at all sites and years. This highlights the need for continuous monitoring as a component of risk assessment and mitigation. The study also shows that pesticides, in particular insecticides, even if they are only present at concentrations close to detection limits, still contribute to the overall toxicity of the mixture. Further, the component-based approaches applied in the study do not take account of the whole picture, including e.g. interactions. Therefore, the authors suggest complementary approaches, including effect-based monitoring and in-situ experiments. Prenatal exposure to chemical mixtures linked to impaired sexual development, neurodevelopment and metabolism in children The EDC-MixRisk research project (funded through the EU research programme Horizon 2020) studied health and development of early life-exposure to complex mixtures of endocrine disrupting chemicals (EDC) through a combination of approaches. Baseline chemicals exposure data were derived from a pregnancy cohort (SELMA). Out of 54 potential endocrine disruptors analysed in blood and urine from the 2,300 pregnant women, 41 (75%) were found above detection levels in a majority of the samples. The researchers used epidemiological and statistical methods to identify which of these chemicals (i.e. in mixtures) are associated with adverse health outcomes in children, with regard to sexual development, neurodevelopment and metabolism/growth. The identified mixtures were tested in experimental models to uncover mechanisms and pathways behind the health outcomes and the analysed dose-response relationships. Hence, the EDC-MixRisk project demonstrated that exposure to the identified mixtures caused effects and dysfunctions in cell and animal models at levels similar to those measured in the SELMA cohort. Observations in the experimental studies included morphological changes to reproductive organs, interference with thyroid hormone signalling, changes in gene expression associated with autism spectrum disorder and intellectual disability in humans, behavioural changes as well as changes in fat cell differentiation and birth weight. Researchers could also link some of the experimentally induced biomarkers to adverse health outcomes in the exposed children of the cohort. Finally, the human biomonitoring data from the SELMA cohort and the experimental data were compared and analysed using new statistical/analytical approaches developed as part of EDC-MixRisk. Results showed that the approaches developed in the project, comparing and integrating human biomonitoring data and experimental models were more sensitive (i.e. indicated risk for adverse effects at lower exposure levels) than traditional additivity or single substance risk assessment approaches, at least for some endpoints. It also showed that the regulatory guideline values for most of the assessed chemicals seem to be insufficiently protective against combined exposures to multiple substances. Box 3: The joint statement of 2011 by the Scientific Committee on Health and Environmental Risks, the Scientific Committee on Emerging and Newly Identified Health Risks and the Scientific Committee on Consumer Safety

Regarding impacts of mixtures on human health, the Scientific Committees concluded that, under certain conditions, chemicals in a mixture with a similar mode of action may act jointly and produce a combination effect that is larger than that from each component of the mixture used singly, and hence represent a health concern. No robust evidence was available at the time of the drafting of the opinion for substances with different modes of action to indicate that exposure to such mixtures of chemicals is a health concern if the individual chemicals are present at or below their estimated zero-effect levels. The Committees concluded on the possible health concerns of such mixtures, that ‘if the intended level of protection is achieved for each individual substance, the level of concern for mixtures of dissimilarly acting substances should be assumed to be negligible’. However, for the ecological effects of chemical mixtures, the Scientific Committees concluded that not only exposure to mixtures of chemicals with similar modes of action was a concern. Combinations of dissimilarly acting substances should also be considered as a possible concern, even if the exposure to all the substances involved is at a level where no effects are predicted for the individual substances (i.e. below the individual Predicted No-Effect Concentrations - PNECs). 3.REGULATORY FRAMEWORKS AND REQUIREMENTS REGARDING MIXTURES The Commission Communication of 2012 concluded that current EU legislation does not provide for a comprehensive and integrated assessment of the combination effects (the Communication uses the synonym term cumulative effects) of different chemicals taking into account different routes of exposure. It also concluded that ‘where a mixture of concern is identified and where such a mixture contains chemical substances regulated under different pieces of EU legislation, no mechanism currently exists for promoting an integrated and coordinated assessment across the different pieces of legislation’ (the latter aspect is further described in section 6). In 2014, the European Commission Joint Research Centre (JRC) published a Report reviewing the existing regulatory requirements for the assessment of mixtures in current EU chemicals-related legislation. The Report investigated such requirements in selected pieces of the three main categories of chemicals-related EU legislation: ·legislation focused on chemical substances and/or intentional/commercial mixtures, normally involving prospective (pre-marketing) assessment, e.g. industrial and consumer chemicals, pesticides, biocides, food and feed additives, pharmaceuticals and cosmetics (12 legislative pieces), ·legislation mainly oriented towards (or specific to) the use of chemicals in products, normally not involving prospective (pre-marketing assessment), e.g. food contact materials and toys (2 legislative pieces), and ·legislation focused on chemical emissions from certain activities, e.g. industrial emissions and the presence of pollutants in food/feed, at work place and in certain environmental media, e.g. air, water (13 legislative pieces). Most pieces of legislation in the first category include some provisions for the assessment of the exposure to and risks from intentional/commercial mixtures, for which the added chemicals are normally well-known and subject to a prospective risk assessment (see below: Intentional mixtures). However, none of the pieces of legislations in this category includes any requirements for the assessment of unintentional mixtures, except for Plant Protection Products and Biocidal Product Regulation in which cumulative and synergistic effects are covered more generally without making a distinction whether they refer to intentional and unintentional mixtures (see below: Intentional and Unintentional mixtures). Regarding legislation in the second category, a specific provision regarding the assessment of intentional mixtures exists in the Toy Safety Directive . The directive considers any toy to be a mixture as regards the presence of substances which are carcinogenic, mutagenic and, toxic to reproduction (CMR). CMRs are prohibited by default in toys, but by derogation may be present in a toy up to the generic or specific concentration limit (whichever is stricter), specified in the CLP Regulation. There is however no reference to the assessment of unintentional mixtures in any piece of the analysed products-focused legislation. The emission and pollution focused legislation mainly looks at the environmental exposure and sometimes risk resulting from the use of chemicals, including to unintentional contaminants present in these chemicals as well as breakdown and transformation products that might form. In line with this, it does not include any requirements for the prospective assessment of single chemicals or intentional mixtures . However, some of the pieces of legislation in this category include legal requirements for the assessment of unintentional mixtures (see below: Unintentional mixtures). The legislation focused on protection of workers from chemicals at work place contains explicit requirement to assess risks of unintentional mixture present at the work place such chemicals in combination (see below: Unintentional mixtures). Of the 27 pieces of chemicals-related legislation assessed, 10 do not include any explicit reference to the assessment of either intentional or unintentional mixtures , . However, mixtures are sometimes considered on an ad hoc basis also under these pieces of legislation, particularly focussing on groups of substances. There are examples of such cases, for instance in the area of plastics materials regulation for food contact materials , but this is the exception rather than the rule. The regulatory requirements for mixtures have not changed in any significant respect since 2012. Hence, chemicals-related legislation is still largely focused on the risk assessment and management of single chemicals. The background to this predominantly substance-by-substance approach to risk assessment is that many pieces of legislation were developed to provide marketing and use provisions for chemicals used for specific purposes, and to define the responsibilities of individual economic actors involved (i.e. producers, importers and users). The prevailing need to better address chemical mixtures has however resulted in the development of common approaches to assess related hazards, exposures and risks. Several guidance documents for such assessments have also been developed. However, to what extent these approaches are actually applied in the absence of specific legal requirements is not known. (see examples in section 4). 3.1 Intentional mixtures Various kinds of references to the assessment of intentional mixtures appear in 11 legislative acts out of the 12 pieces of legislation focused on chemical substances and/or intentional/commercial mixtures assessed. Eight of these refer to such assessment for both human health and the environment and three for only human health . These delimitations usually coincide with the overall scope of the legislation regarding health and the environment, e.g. environment is out of scope of the Cosmetics Regulation, which refers to REACH regarding environmental risks. An example of legislation requiring assessment of mixtures with regard to both human health and the environment is the regulation on biocidal products. The regulation on plant protection products considers effects on human health both related to dietary exposure (food, feed, drinking water) and to non-dietary exposure (workers, residents, bystanders). Biocidal and plant protection products are normally intentional mixtures, which apart from the active substance(s) might include solvents, synergists (adjuvants), safeners and surfactants. The Plant Protection Products (PPP) Regulation covers both the approval of the individual substances used in plant protection products and the formulated products (i.e. intentional mixtures). Active substances used in PPPs are approved in a procedure at the EU level. This normally involves testing and risk assessment of the individual substances, and in some cases testing of the whole formulation for the purpose of environmental risk assessment for particular organism groups. The authorisation also includes the requirement to assess maximum residue levels (MRL) of pesticides in food and feed with regard to human health. MRLs are set based on scientific advice of the European Food Safety Authority (EFSA) according to Regulation (EC) No. 396/2005. Formulated plant protection products, in which an approved active substances is used together with other (formulating) substances, are authorised at the Member State level. The whole formulations are generally assessed only for acute toxicity to humans and for organisms in the environment that might come into direct contact with the product, but Member States have the possibility to require submission of any other information they consider necessary. Data to be provided are either from direct testing of the product itself (whole-mixtures approach), data on all components of the plant protection product, or in some cases the application of a non-animal test method.

The Biocidal Products (BPR) Regulation covers the approval of active substances and the authorisation of formulated biocidal products (i.e. intentional mixtures). In both cases the regulation requires ‘cumulative and synergistic effects’ to be taken into account under ‘realistic worst case conditions of use’ (Article 19(2)). It also requires that ‘for biocidal products that are intended to be authorised for use with other biocidal products, the risks to human health, animal health and the environment arising from the use of these product in combinations shall be assessed’ (Annex III Section I Point 8.5.4). Further, common principles for the evaluation of biocidal products, including several references to cumulative and synergistic effects, are set out in Annex VI. The risk assessment of the formulated biocidal products is normally based on data for the individual components addressing all relevant endpoints as required by the data requirements for biocides in Annex VI to the Biocidal Products Regulation. Guidance documents for human health and environmental risk assessment relating to biocidal products are available (see Section 4). Assessment of intentional mixtures is generally conducted in connection with the hazard based classification and labelling under the Classification, Labelling and Packaging (CLP) Regulation . This process relies firstly on test data on the whole mixture when such are available . Alternatively, the classification can be based on bridging principles, i.e. the use of data on a similar mixture. Finally, in the absence of information on the mixture itself, classification can be based on the toxicological properties of the ingredients in the mixture and application of the summation rule or concentration addition method. The REACH Regulation covers, in principle, all intentionally manufactured chemical substances, on their own, in mixtures or in products, unless they are specifically exempted under REACH due to being regulated by sector or product-related legislation (e.g. pharmaceuticals and food additives) or are radioactive substances or waste. It requires the producer/importer to register a chemical to be marketed or imported in quantities above 1 ton per year and in this context provide test data, hazard assessment and a risk assessment of the identified uses. REACH registration requirements apply to each of the individual chemicals in an intentional mixture, but not to the mixture itself. The registration must include a hazard assessment of the chemical and a risk assessment for all identified uses. The risk assessment must cover the use of a substance in a mixture if this is placed on the market and be documented in a chemical safety report. REACH is mainly focused on individual substances and contains no specific methodology for the assessment of intentional mixtures, beyond multi-constituent substances (MCSs) and so called UVCBs (see below). In principle, it is however possible to take into account combined effects of intentional mixtures, e.g. during the assessment of substances for restriction and authorisation (this is also true for intentional mixtures, see section 5). Further, REACH obliges the supplier of a mixture to provide the downstream user of that mixture with a safety data sheet (SDS), if the mixture is classified as hazardous. The SDS contains (eco)toxicological information and a chemical safety assessment for the mixture, if one has been performed. Under several pieces of legislation (e.g. CLP, REACH, biocides, cosmetics), special consideration is given to impurities in a substance or a constituent in a multi-constituent substance (MCS) as well as in the so-called UVCBs (Substances of Unknown or Variable composition, Complex reaction products or Biological materials). Although such substances can be seen as mixtures from a toxicological point of view, they are considered substances from a legal point of view (e.g. the REACH definition ). Hence, they are registered as one substance under REACH. Procedures for assessing such substances are described in the REACH and CLP guidance. The approach generally involves the testing of the substance itself, but might also involve methods for predicting the overall risk, based on information on the individual components, in particular for CMR properties. In conclusion, most pieces of legislation focusing on chemicals and intentional/commercial mixtures require a hazard and/or risk assessment of these. The scope and level of detail in these provisions vary, including whether human health and/or environment are considered. Methodologies and guidance documents are generally available, and assessments are usually performed through applying whole mixture approaches or based on information on the individual components in the mixture. 3.2 Unintentional mixtures The European Commission Joint Research Centre (JRC) report from 2014 analysed whether 12 pieces of legislation focused on chemical substances and intentional mixtures include requirements for the assessment of unintentional mixtures. Such requirements were found to be absent from all these pieces of legislation, except for the Plant Protection Products and the Biocidal Product Regulations which require the consideration of cumulative and synergistic effects (see Box 4). The Plant Protection Product Regulation include such a specific reference only regarding human health (dietary and non-dietary (for workers, residents, bystanders)), while the Biocidal Product Regulation regarding human health and the environment. The JRC report also analysed 15 further pieces of legislation (products, pollution, food, work-place and environmental-media related legislation) regarding requirements for the assessment of unintentional mixtures. Nine pieces of legislation have no reference to the assessment of unintentional mixtures, while six of them include some reference to such assessment. ·Two pieces of legislation mention the assessment of unintentional mixtures in relation to both human health and the environment in the operative text (Environmental Impact Assessment Directive, and Water Framework Directive) , . ·One piece of legislation mentions the assessment of unintentional mixtures in relation to only the environment in the operative text, while human health is only mentioned in a recital (Environmental Quality Standards Directive) . However, its consideration is implicit through the link the Water Framework Directive. ·Three pieces of legislation include such a reference only regarding human health (Maximum Pesticide Residue Levels Regulation, Protection of the Health of Workers Directive and Toy Safety Directive) , . The references to assessment of unintentional mixtures in products, pollution and environmental media related legislation are however in most cases vague and general. There is usually a lack of detailed and explicit provisions or guidance on when and how such assessment should be performed. As a result, unintentional mixtures are seldom subject to any regulatory assessment, and when this happens, it is on an ad hoc basis. The most explicit requirement to perform assessment of unintentional mixtures is in the Directive on the protection of the health and safety of workers from the risks related to chemical agents at work . It includes an obligation to assess the combined risks to workers of all hazardous chemical agents in the workplace (see Box 4). The regulation on the maximum residues levels of pesticides in food and feed (MRL Regulation) includes also a relatively clear general requirement for taking into account the possible presence of pesticide residues arising from sources other than current plant protection uses of active substances, and their known cumulative and synergistic effects (see Box 4). For three of the analysed pieces of legislation, the requirement to assess unintentional mixtures refers exclusively to particular groups of substances. This is true for the Industrial Emissions Directive , which requires assessment of mixtures of chemicals belonging to the groups Dioxins and Furans. The Food Contaminants Regulation and its guidance document requires the same for chemicals of the group poly-aromatic hydrocarbons (PAHs). The Ambient Air Quality and Cleaner Air for Europe Directive also assess PAHs as a group, using benzo(a)pyrene as a marker for the carcinogenic risk from the whole group of PAHs in the setting of target values. Waste and industrial emissions are unintentional mixtures with a more or less complex composition, which may exhibit considerable variability. Hence, applying the principles of mixtures assessment might be challenging, but relevant in these contexts. For emissions and wastes originating from e.g. particular industrial processes, the composition can be estimated or measured with some accuracy, while more mixed waste streams like household waste are more complicated. Under the Waste Framework Directive (2008/98/EC), specific waste streams are classified as hazardous or non-hazardous to ensure that waste management can be carried out without endangering human health or harming the environment by safely handling hazardous properties materials. The classification of waste is largely based on the hazard classes and criteria in the CLP Regulation. Given that waste generally constitutes mixtures of substances, mixture classification rules apply, and are described in Annex III of the Directive. For certain hazardous properties, direct testing of waste is also an option. Calculation and test methods to classify waste are used in conjunction with the so-called ‘List of Waste’ (Decision 2000/532/EC) . The list covers over 800 specific waste types and specifies the classification as hazardous and non-hazardous. Detailed guidance on how to characterise and classify waste according to the List of Waste has been issued by the Commission in its notice on technical guidance on the classification of waste (2018/C 124/01) .

Box 4: Legal requirements for the assessment of unintentional mixtures in the worker protection, pesticides and biocides legislation Directive on the protection of the health and safety of workers from the risks related to chemical agents at work Under this Directive, the employer has the responsibility to assess any risk to the safety and health of workers arising from the presence of hazardous chemical agents at the work place, taken into consideration the level, type and duration of exposure, and to set out the necessary preventive and protective measures. Article 4 specifically requires that: ‘In the case of activities involving exposure to several hazardous chemical agents, the risk shall be assessed on the basis of the risk presented by all such chemical agents in combination’. A Commission guidance document on the application of a simplified risk assessment methodology (the Homogeneous Exposure Group (HEG) methodology) is available. Pesticides legislation The Regulation on the Maximum Residues Levels (MRL) of pesticides in food and feed and the Regulation on the placing on the market of Plant Protection Products (PPP) have general requirements for the assessment of unintentional mixtures of pesticides. The setting of the MRL have to take into account: ‘Human exposure to combinations of active substances and their cumulative and possible aggregate and synergistic effects on human health’ (preamble point 6). ‘The possible presence of pesticide residues arising from sources other than current plant protection uses of active substances, and their known cumulative and synergistic effects, when the methods to assess such effects are available.’ (Article 14, (2) (b)). A plant protection product shall meet, according to the PPP Regulation, among others, the following requirements: ’It shall have no immediate or delayed harmful effect on human health, including that of vulnerable groups, or animal health, directly or through drinking water (taking into account substances resulting from water treatment), food, feed or air, or consequences in the workplace or through other indirect effects, taking into account known cumulative and synergistic effects where the scientific methods accepted by the Authority to assess such effects are available; or on groundwater;’ (Article 4(3) (b). Until now, cumulative risk assessment according to the requirements in these regulations has not been systematically performed for regulatory purposes, as the methodology is not yet available. The European Food Safety Agency (EFSA) has, however, taken several steps regarding the development of a methodology and presented a guidance document focussing on human health risks from dietary exposure to chemicals and on environmental risks of chemicals falling under its remit - i.e. pesticides, food and feed additives. EFSA has published the results of its two first pilot assessments on the risks posed to humans (effects on nervous system and thyroid) by residues of multiple pesticides in food. , Biocides legislation The Biocidal Products Regulation has provisions for the assessment of unintentional mixtures of biocides. Article 19 (2) requires that ‘the evaluation of whether a biocidal product fulfils the criteria for authorisation shall take into account cumulative and synergistic effects’. Article 8 (3) further specifies, that ‘where the evaluating competent authority considers that there are concerns for human health, animal health or the environment as a result of the cumulative effects from the use of biocidal products containing the same or different active substances, it shall document its concerns in accordance with the requirements of the relevant parts of Section II.3 of Annex XV to Regulation (EC) No 1907/2006 and include this as part of its conclusions. 3.3 Grouping approaches Assessing and managing chemicals in groups, rather than substance by substance, contributes to addressing risks from exposures to some unintentional mixtures , , , . Chemicals are usually grouped for regulatory purposes based on:

·Similarities in their chemical structure as a proxy for their similarity of toxicity; ·Common intrinsic properties such as toxicity/hazard classification, and exposure related properties, e.g. persistence, bioaccumulation and environmental mobility ·Common exposure routes and/or potential for emission/exposure, e.g. chemicals which workers are exposed to in occupational settings ·Common areas of use and/or technical functionalities, e.g., chemicals occurring in certain consumer products. Chemicals belonging to such groups may be more likely to occur together and hence be subject to co-exposure, or have similar toxicities and hence demonstrate cumulative effects. Thus, risk management of such groups of chemicals contributes to management of some unintentional mixtures. There are some examples of legal provisions applying grouping approaches in the assessment or regulation of certain chemicals together based on physical-chemical (and/or structural) similarities combined with common routes of exposure and/or potential for co-exposure. These provisions address some particular cases of exposure to specific mixtures and/or aggregate exposure, without requiring an actual mixture assessment. One example is the Drinking Water Directive (98/83/EC), which establishes standards for individual substances and groups of substances, like pesticides. In the context of the revision of the Directive, the possibility of requiring actual mixture assessments was discussed. It was however finally decided to keep the approach with lists of substances rather than move toward assessments of combined exposure for individual water supplies. The revised Directive will however require risk assessments and monitoring of two additional groups of substances: PFAS and haloacetic acids. The Groundwater Directive also includes a standard for total pesticides. The Industrial Emissions Directive (IED) (2010/75/EU) lacks detailed provisions for risk assessment of chemical pollution. The assessment may be based on knowledge produced in other contexts (e.g. under REACH). This can include grouping or concepts for the assessment of mixtures of structurally similar substances for the setting of limit values, e.g. toxic equivalency factors (TEF), which has been used for the dioxin-group. The same approach has been used for dioxins also under the Water Framework Directive. Addressing groups of substances is possible and is increasingly becoming normal practice under REACH, which contains specific provisions for grouping.. The grouping of similar substances is mentioned in connection with substance evaluation (Article 47). The testing and assessment requirements may be modified for a group of structurally similar substances, hence limiting the testing to one or a few substances, while predicting the properties of the other substances in the group based on these tests (Article 13; Annex I point 0.4 and; preamble of Annexes VII to X Annex XI). REACH Article 62(3) allows submitting applications for authorisations for groups of substances. There are several examples of authorisations and restrictions of groups of similar substances such as metal compounds, PAHs, phthalates and PFASs (see examples of restriction in Section 5). 3.4 Aggregate Exposure Aggregate exposure refers to the exposure to the same substance from different independent sources and/or via different pathways. It is related to and hence often considered in connection with unintentional chemical mixtures. Clear legal requirements for performing aggregate exposure assessments across different regulatory sectors currently exist only in some pieces of chemicals legislation (i.e. legislation focused on chemical substances and intentional mixtures), and is hence not normally and systematically taken into account. Under REACH, aggregate exposure may be assessed when authorities evaluate a substance or consider authorisation (REACH, Title VII). There are examples of restrictions under REACH that are based on aggregate exposure of particular chemicals, such as perfluorooctanoic acid and phthalates. The Cosmetics Regulation requires the consideration of aggregate exposure in the human health risk assessment of certain ingredients in cosmetics. According to the guidelines from the Scientific Committee on Consumer Safety (SCCS) , the evaluation of a cosmetic ingredient for the product safety report must take into account aggregate exposure of the ingredient from different types of cosmetic products. The Regulation on food contact materials also requires a consideration of different sources of exposure to the same substance or group of substance to which a specific migration limit (SML) is applied. Consequently, several substances regulated in particular in plastic food contact materials acknowledge this and include an allocation factor that further restricts the amount of substance permitted to migrate into food. Legislation oriented towards pollution and certain environmental media (e.g. water and air related pieces of legislation) normally consider the aggregate exposure to a substance, regardless of source. However, it does usually not consider exposure via all relevant environmental media or pathways. 3.5 Conclusions on existing legal requirements for intentional and unintentional mixtures, grouping and aggregate exposure Regarding intentional mixtures, there are requirements in place in most pieces of chemicals legislation (chemical substance and intentional/commercial mixture focused legislation) as well as in one piece of products-specific legislation, namely the Toy Safety Directive. Requirements on the assessment of unintentional mixtures are broadly absent in chemical substance and mixture focused legislation. The only exceptions, where requirements for the assessment of cumulative exposure to unintentional mixtures exist, is the directive on the protection of the health and safety of workers from the risks related to chemical agents at work, the regulation on the maximum residues levels of pesticides in food, the Plant Protection Products regulation and the Biocidal Product regulation. The requirements under the regulations on maximum residues levels and the Plant Protection Products are however not yet implemented as the methodology is still under development.

Requirements for the assessment of unintentional mixtures exist in a few additional pieces of legislation focused on products, pollution and environmental media. Out of the 15 assessed pieces of such legislation, six include a reference to the assessment of unintentional mixtures, regarding human health or the environment, or both. These references are however usually vague and general, with a lack of provisions and/or guidance for how to perform the assessment, and hence rarely implemented. In four of these pieces of legislation, the requirement only refers to particular groups of substances, e.g. dioxins or PAHs. A few pieces of legislation provide for grouping approaches in the assessment of certain functionally or structurally similar substances, without requiring an actual mixture assessment. Aggregate exposure to the same substance from different sources and/or via different routes of exposure can be considered in substance evaluation and when considering authorisation and restriction under REACH as well as in the regulations on cosmetics and biocides. It is not considered in any other pieces of chemicals or products related legislation. . However, aggregate exposure is normally considered in pollution and environmental media focused legislation, but usually limited to the particular environmental media or pathway in scope of the legislation. Overall, the progress on legal requirements for the assessment of unintentional mixtures has been limited since 2012. 4.PROGRESS ON GUIDANCE AND REGULATORY ASSESSMENT In the 2012 Communication (Section 5.3, paragraph 2), the Commission commits to coordinate the development of technical guidelines in order to promote a consistent approach for the assessment of priority mixtures across different pieces of EU legislation. Some overarching guidance documents to support consistent methodologies were recently developed. These are based on the framework for the assessment of combined exposures to multiple chemicals presented by WHO/IPCS in 2011 , and the methodology is summarized in the State of the Art Report in 2012. EFSA , likewise, published a comprehensive guidance document on mixture risk assessments in 2019, to support harmonised methodologies for the environment, animal and human health in the fields falling within EFSA’s competence. The Commission also contributed to the 2018 Organisation for Economic Co-operation and Development (OECD) report on risk assessment of combined exposure , which describes all essential steps to be considered, independent of the legislative framework, the complexity of the mixture (intentional or unintentional), the scope of the assessment, and the assessor. The approach for the assessment of chemical mixtures is in principle common to all guidance developed so far and applicable to both intentional and unintentional mixtures. It includes: ·Prioritisation methods and/or grouping approaches to select the components to be considered; ·Options to use (eco)toxicity and exposure data on the single components to predict mixture hazard and/or risks, through component-based approaches (Concentration Addition or Independent Action); ·Options to test the whole mixture as such and assess hazard and/or risk based on the test data; ·Tiered approaches to refine the assessment depending on the availability and quality of data and information on the mode of action. Further legislation-specific guidance documents for the assessment of mixtures have been developed for some pieces of EU legislation in recent years. Currently, such guidance is available for plant protection products , biocidal products , and veterinary pharmaceuticals , in all cases including references to the risk assessment of intentional mixtures regarding either human health or the environment. For human health assessment, guidance documents are also available for plant protection products and their residues with respect to Cumulative Assessment Groups (CAGs) , and probabilistic modelling as well as for biocidal products . Moreover, technical guidance on the classification of waste , which includes aspects of mixture assessment, was published in 2018 (see section 3). In conclusion, there has been some progress on the availability of both general and legislation specific guidance since 2012. All the legislation specific guidance documents presented refer to the assessment of intentional mixtures, except for the guidance on pesticide residues in food and feed and some elements in the guidance on classification of waste. 5.CASES OF REGULATORY ASSESSMENT OF MIXTURES The 2012 Communication concluded that methodologies for the identification of chemical mixtures of potential concern as well as for the assessment of chemical mixtures are available, although the extent of the assessment is limited by knowledge and data gaps. It also foresaw that information collected in the context of EU legislation, in particular REACH registration will contribute to reducing these gaps. In 2014, the Commission (JRC) conducted a survey on methodologies for assessing the combined effect of chemicals , answered by 58 experts in risk assessment from 21 countries, representing among others, authorities, academia and industry, with experience in mixture risk assessment, in most cases for regulatory purposes. According to responses to the survey, most of the practical experience with mixture assessments exists in the area of plant protection products and, as regards intentional mixtures in the context of CLP and REACH. Concentration addition appeared as the most commonly used component-based approach. Cases of mixture risk assessment based on combined exposure and combined effects for regulatory purposes are generally important benchmarks for future action. A recent case of risk management for mixtures is the restriction of the four phthalates DEHP, BBP, DBP, DIBP under REACH . The risk assessment of these, considering combined exposure, was based on biomonitoring data. The limit value of this restriction refers to the combined unintentional exposure to the four substances in articles. This is the first clear-cut example of a REACH restriction where the risk assessment considers combined exposure to structurally similar chemicals (i.e. in this report referred to as an unintentional mixture). A similar restriction is being introduced in food contact materials on the basis of the assessment of co-exposure to these phthalates by EFSA . There are also some cases where groups of substances were considered and restricted together as a group, without actually assessing the risk from their combined exposure. Instead, the joint restrictions were based on similarities in their chemical structure (e.g. lead compounds and poly-aromatic hydrocarbons), hazard profile (e.g. CMR in consumer mixtures), uses (e.g. CMR in textiles) and exposure pathways. Further examples include the restriction of the phthalates DINP , DIDP and DNOP in toys and childcare articles , and the ongoing restrictions of chemicals in tattoo inks and PFAS. Such restrictions can manage some risks related to combined exposure, but are also motivated by other aspects, e.g. the avoidance of regrettable substitution. In addition, some experience relating to the risk assessment of unintentional mixtures, probabilistic modelling, and grouping of chemicals in cumulative assessment groups (CAGs) has been gained under the Regulation on Maximum Residue Levels of Pesticides. The first CAGs for pesticides that have acute effects on the nervous system and chronic effects on the thyroid system were recently published (see also Box 4).

Further cases of combined exposure to chemicals considered in a regulatory context relate to the area of food contaminants. These were mineral oil saturated hydrocarbons , hexabromocyclododecanes , non-ortho polybrominated biphenyls , marine biotoxins , , ergot alkaloids , polyaromatic hydrocarbons , pyrrolizidine alkaloids and, more recently, PFAS . Lastly, lists of known synergists (chemicals interacting so that (eco)toxicological effects are enhanced in cases of co-exposure) are being compiled and are supposed to be included in the respective Annexes of the Biocides and Plant Protection Products Regulations, to better address and regulate these compounds. The findings in the JRC survey and other developments referred to above, demonstrate that knowledge, methods for the assessment of mixtures and experience in applying them are available in several areas. Regarding the MRL Regulation, knowledge and methodology (CAGs) are still being developed. However, as the development has proven to be extremely complex, CAGs are not yet applied for regulatory purposes under that Regulation. Hence, there is in several areas an established methodology in principle ready to apply in a more systematic manner, although often limited by factors such as data gaps and absence of specific guidance. Grouping approaches can contribute to addressing some unintentional mixtures, and have been applied much more frequently than actual assessments of mixtures, e.g. under REACH and in the area of food contaminants. 6.INTEGRATED AND CO-ORDINATED ASSESSMENT OF MIXTURES ACROSS DIFFERENT PIECES OF LEGISLATION 6.1 Interface between different pieces of legislation As already concluded in 2012 by the Commission, EU legislation does not provide for a comprehensive and integrated assessment of combined effects of different chemicals across the different pieces of legislation. In 2016 , the JRC reviewed 21 published case studies on risk assessments of real-life mixtures for human health and the environment, all conducted between 2014 and 2016. Assessed cases covered a range of different substance classes and media. All studies addressed unintentional mixtures (apart from one looking at aggregate exposure). Most of them were based on monitoring data, a few on modelled exposure. Seven of the cases involves chemicals across different classes and legislative sectors. Available risk assessment tools were successfully used in all the reviewed cases. The concept of Concentration Addition was applied as default to predict mixture toxicities, while the Maximum Cumulative Ratio approach was often used for prioritisation purposes and to decide whether only certain substances or a larger number of chemicals were responsible for the mixture risks. Several of the case studies identified potential risks from mixtures for human health and/or the environment (see Box 2). According to the JRC review, the relatively few cases where regulatory assessment of unintentional mixtures is actually performed (see also Section 3), are usually limited to mixtures consisting of chemicals that fall under the same particular piece of legislation. Thus, no systematic identification, of (unintentional) real-life priority mixtures is currently performed. The regulatory risk assessment or risk management of real-life unintentional mixtures is accordingly performed only rarely and on an ad hoc basis, while available scientific case studies are isolated examples. The 2016 JRC review concluded that ‘so far there is no prospective risk assessment concerning chemical substances related to various regulatory sectors and/or uses, and although numerous chemicals fall under several regulatory frameworks (biocides, pesticides, REACH...), the potential for co-exposure is hardly assessed or taken into account in their risk assessment.’ A related issue is how to address aggregate exposure to the same chemical when this is regulated under different pieces of legislation. An example is the restriction of four phthalates under REACH (see also Section 5). The exposure to one of these phthalates (DEHP) from food intake was identified as the major source of exposure according to the assessment. This part of the exposure, falling under the Food Contact Materials Regulation , or other sources of exposure such as environmental contamination or food contaminants was not addressed in the REACH restriction. Article 16(2) of the Water Framework Directive (WFD) specifies how risk assessments under existing legislation should be used as a basis for listing priority substances in surface waters. It also allows the use of a simplified risk-based assessment procedure taking into account evidence regarding the intrinsic hazard of the substance (aquatic toxicity and human toxicity via aquatic exposure routes), evidence from monitoring and other factors which may indicate widespread environmental contamination, such as production volumes or use patterns. Such risk-based assessments inevitably consider aggregate risk from different uses of the same substance, and in some cases also several substances if they are in a group for which the Environmental Quality Standard (EQS) is set for a marker substance or the sum of the concentrations of related substances. The listing of substances in the priority substances list under the WFD on the basis of such risk-based assessments should be taken into account when substances are approved/authorised or reviewed under several of the relevant pieces of sectoral legislation, in particular if the monitoring data subsequently obtained show that the EQS are still not being met. There are references to the WFD objectives in those pieces of legislation, and the obligation to consider the WFD findings was further specified in Article 7a of the EQS Directive in 2013. The risk-based assessment under WFD does not consider exposure via other routes, e.g. air, nor combined effects from unrelated substances (although a quality standard for the broad grouping of “pesticides” is set under the Groundwater Directive). The above findings and examples highlight the remaining need to develop risk management measures and coordination across all relevant sectors and uses, when chemicals involved are regulated through different pieces of legislation. Such an approach would require mutual acceptance or alignment of procedures and decisions on substances and mixtures as well as improved co-operation, including the sharing of data between involved EU agencies and Member State authorities. The Commission has recently engaged in a number of evaluations of EU chemicals related legislation, which in several cases look at challenges related to combined exposure and assessment of mixtures, including mixtures consisting of chemicals from across regulatory areas. Evaluations looking at several pieces of legislation together, and the interplay between these, include the fitness check of most chemicals legislation except REACH , (covering over 40 pieces of legislation), the fitness check of the EU water legislation , , the evaluation of the EU legislation on plant protection products and pesticides residues as well as the fitness check of the EU legislation on endocrine disruptors . Evaluations of single pieces of legislation that consider how they address mixtures include those of the Occupational Safety and Hygiene (OSH) Directive and the Detergents Regulation . Several of these evaluations point to the need to develop the assessment of mixtures, including across regulatory areas. As such integrated assessment of chemical mixtures has not been achieved yet, further efforts are needed, including closing interface gaps between pieces of legislation, improve data availability, harmonise approaches and conduct further regulatory and scientific case studies on mixture assessment. 6.2 Case studies on priority mixtures In the 2012 Communication, the Commission committed to develop a consistent approach to the assessment of priority mixtures across the different pieces of EU legislation. The Commission referred to a set of criteria and methodologies that could be applied to identify chemical combinations/mixtures that are priorities for further assessment proposed by its Scientific Committees SCHER, SCENIHR and SCCS in 2012 . The criteria proposed were intended to facilitate a focus on mixtures of potential concern. They included aspects such as: commercial mixtures with several components of concern; potential serious adverse effects of one or more chemicals at the likely exposure levels; likely large-scale and frequent exposure of humans or the environment; persistence in the body or the environment; information on potential interactions; prediction of similar modes of action; and mixtures containing one or more components without an effect threshold. An experimental laboratory study from 2014 evaluated the effects of realistic mixtures of 14-19 different chemicals falling under different pieces of legislation (pesticides, industrial chemicals, biocides and pharmaceuticals) which may jointly occur in surface waters. These were analysed in concentrations based on their respective individual environmental quality standard (EQS) in 35 different standardised in vitro and in vivo test systems. Although the individual chemicals were all present at concentrations considered safe under current legislative practices, clear adverse effects of the mixtures were demonstrated in a number of organisms, including fish, frogs, crustaceans, algae and micro-organisms. Further, some EU-funded research projects have identified risks of mixtures to the environment, on the basis of available monitoring/exposure modelling methodology and hazard data (SOLUTIONS) , or to human health, based on measurements of human blood samples and in vitro and in vivo test systems (EDC-MixRisk) (see two cases described in Box 2). The selection of cases for the assessment of mixtures is usually driven by the availability of data on toxicity and exposure. Therefore, a limited selection of well-studied chemicals tend to be overrepresented in current case studies, which are hence unlikely to be representative of human and environmental exposures. To circumvent this, a more systematic analysis of cases of mixture risk assessments is warranted. In conclusion, the assessment of unintentional chemical mixtures across regulatory areas as well as assessments to identify priority mixtures are rare and only performed on an ad hoc basis. Reasons for this include lack of regulatory requirements and coordination between sectors and regulatory bodies as well as absent or incomplete toxicity and exposure data.

7.CLOSING THE KNOWLEDGE GAPS – RECENT ACHIEVEMENTS To address remaining knowledge gaps, the 2012 Communication set out that the Commission would work further for a better understanding of the chemicals mixtures to which human populations and the environment are actually exposed. This includes reviewing data on toxicity and exposure collected under EU legislation or generated in research projects. The Communication also committed to the creation of a platform for chemical monitoring data, e.g. to help link exposure and epidemiological data on humans and the environment. Further, the Communication also set out how to address some of the other knowledge gaps, in particular: (i) The mode of action of chemicals; (ii) Criteria for grouping chemicals into categories or assessment groups; (iii) Predicting interactions; and (iv) Identifying chemical substances that are the main drivers of mixture toxicity. Below is a brief summary of progress in these areas. The Communication also concluded that the assessment of chemical mixtures must be carried out with respect to the principle of reducing, refining and replacing testing on vertebrate animals. To address the identified gaps, the Commission has among other activities funded several research projects, most of them under its Research and Innovation Program Horizon 2020. The outcome of these projects was recently summarised in two scientific publications , and is further described below. Some of the findings from two of these research projects are also described in Box 2. 7.1 Effect, exposure and hazard data The Commission developed the Information Platform for Chemical Monitoring (IPCHEM) in response to the major lack of monitoring and epidemiological data to understand the co-exposures of humans, animals and the environment to chemicals, as identified in the 2012 Communication. IPCHEM was launched in a demonstration version in 2014 and in a full, publicly available version in 2015 (see Box5). Recent EU-funded research projects also provide some comprehensive data sets on epidemiological data (e.g. EDCMixRisk, EuroMix, HBM4EU, all funded via Horizon 2020) and chemical monitoring and co-exposure modelling data for the environment (e.g. SOLUTIONS funded under the 7th Framework Programme for research and innovation). An important step forward regarding the availability of chemicals toxicity data is the finalisation of the three major rounds of registration of chemicals on the EU market under REACH. However, significantgaps remain in this area (cf. below, Remaining knowledge gaps). Currently, databases of the respective European agencies working in the chemicals field provide data on exposure, uses, compositions, substance properties and hazards for regulatory purposes under different frameworks. These include the ECHA databases for REACH and biocides, the EFSA data warehouse, the EEA databases on water as well as data from Member States. Moreover, the EU is also continuously contributing to international activities to bring hazard databases together, such as the OECD eChemPortal and OECD activities towards a ‘Global Chemicals Knowledge Base’ . The improved exchange of available data across regulatory areas and bodies involved is also crucial for the risk assessment of mixtures. To explore the possibility of enhancing the transparency and exchange of data, the Commission has launched a feasibility study on a common open platform for chemical safety data based on a request from the European Parliament . Box 5. The European Commission Information Platform for Chemical Monitoring (IPCHEM) The 2012 Communication highlighted the need to improve the understanding of human and environmental exposure to chemical mixtures. One of the operational follow-ups set out in response to this need was to promote a more coherent approach to the generation, collection, storage and use of chemicals monitoring data, through the creation of a platform for such data. The Commission developed the Information Platform for Chemical Monitoring (IPCHEM) in direct response to this. The overall purpose of IPCHEM is to help identify links between chemical exposure, including co-exposure, and the impact on human and ecosystem health. The objectives of the platform include improving access to monitoring data for policy makers and scientists, to host such data and to make it available at a defined quality (regarding spatial, temporal, methodological and metrological traceability). IPCHEM also aims at improving data quality and comparability, thereby facilitating risk assessment practices under EU policies. Other areas where the platform can be useful includes evaluation of policy effectiveness, the design of future monitoring programmes and for the analysing of collected data. IPCHEM provides a single access point for a wide range of chemical monitoring data, originating from European Commission bodies and Member States, as well as international and national organisations and the research community. An example is the EU-funded human biomonitoring project HBM4EU , which shares collected data through the platform. IPCHEM is cross-linked with other reference information systems (e.g. the OECD eChem Portal, ECHA-REACH info card, PubChem), thus also facilitating the search for chemicals hazard data relevant to the environment and human health. IPCHEM is structured into four thematic modules: environmental monitoring, human biomonitoring, food and feed, products and indoor air. From the platform, it is possible to search by chemical substance, environmental media and country. IPCHEM also includes a search tool for joint-occurrences of multiple chemicals at a particular location, which is particularly useful in relation to mixtures. At present, IPCHEM provides access to over 450 million data records, and continues to grow. The main challenges include harmonising the data and ensure their quality. To address these, specific rules and procedures are implemented, and guidance is provided to data providers as well as end users. The Commission in 2017 arranged a workshop specifically aimed at enhancing the use of IPCHEM for the assessment of mixtures. The resulting report identifies the need for a tighter collaboration and data sharing between research projects, scientific committees, Commission services and EU agencies. To enable this, there is a need to find solutions for limiting the access to restricted data to specific user groups. This requires legal, privacy or contractual obligations, as well as streamlining the efforts to harmonise the management and sharing of data between the different EU institutions involved.

7.2 Knowledge on mode of action, grouping, interactions and drivers of mixture toxicity The areas mentioned in this sub-section relate to some of the knowledge gaps highlighted in the 2012 Communication (see above) and progress related to these. Improved knowledge in these areas is important to understand and assess the risks related to mixture toxicity, including what methodologies to apply. It is also essential to manage data gaps and to prioritise which chemicals to consider in mixtures assessments. Information on the mode of action (MOA) of chemicals is used to predict possible adverse outcomes, to group chemicals based on their properties, or to close toxicological knowledge gaps via read-across approaches. Some improvements for an easier identification of the mode of action of chemicals have been made by using computer-based methods (sometimes called in silico methods, e.g. QSAR), as well as in vitro approaches and the Adverse Outcome Pathway concept . Grouping of chemicals into categories is important to decide which components to consider in a mixture assessment. The grouping may be based on (eco)toxicological properties, mode of actions, chemical structure or physicochemical properties, as well as the type of product, use or exposure scenarios,e.g. joint exposures (see also section 3.3). In 2018, the Commission (JRC) published a dedicated report on the MOA of existing priority substances under the Water Framework Directive and the possible use of effect-based methods in assessing chemical status. The report discusses how such methods could cover a wider range of chemical substances than currently included in the list by focusing more on their effects than on individual concentrations. The methods might be used in future for exploratory monitoring (e.g. for watch-list substances) or to capture better the true chemical status of surface waters based on appropriate trigger values for an adequate range of representative chemical-effect types or MOAs. Knowledge of MOA is important for linking exposure and eco-toxicological effects in the aquatic environment. Recent research in the area of pesticide residues has advanced the Cumulative Assessment Groups method, which is based on the chemicals involved having common target organs and effects (see also section 4). Developing such approaches can also help to assess and manage combined exposure, for instance to a group of structurally similar substances that are considered to have similar toxicological properties. This kind of approach can often be applied without performing an actual mixture assessment which involves the calculation of mixture toxicity (e.g. some REACH restrictions take this approach, see section 5). In addition to the specific usefulness of grouping approaches in connection with mixture assessment, they also contribute to the filling of data gaps (i.e. through the use of read-across and QSAR methods), to avoid regrettable substitution and to limit the need for animal testing . Several studies on interactions have been performed during the last decade. Higher or lower mixture toxicities than predicted by the usual additivity approach may occasionally occur due to interactions between the individual chemicals, for example if chemicals influence each other’s metabolism and thus (de)toxification processes in an organism. These phenomena are commonly referred to as synergisms or antagonisms. Several authors have reviewed the evidence of such interactions in real life mixtures. They concluded that interactions are rare at lower concentrations such as exposure via the environment. In observed cases, the interactions have also led to relatively small deviations, usually over or underestimations within a factor of four , compared to additivity-based predictions. A systematic literature review on the topic of prediction of interactions was finalised in 2019, through a contract study for the Commission . The review analysed 1220 eco-toxicological as well as mammalian/human health studies related to mixtures which were published since 2007. For a subset of 388 studies involving claims of interaction or providing indications of this, a quantitative reappraisal of the authors’ evaluation was conducted. The results indicate that relatively few of the studies claiming synergistic or antagonistic effects show a deviation of observed mixture effects of more than two-fold from concentration addition based predictions if re-evaluated. However, a small proportion of the reviewed experiments that indicated synergism was considered significant and of regulatory relevance. No clear trend in factors, including chemical composition, that tend to be involved in cases of synergism was detected. However, the review confirmed earlier indications that synergism is particularly frequent among pesticide combinations. A conclusion of the study is that the concentration addition concept can be applied as default for predictive assessment of chemical mixtures. This can be complemented on a case-by-case basis by specific considerations for mixtures where interactions are anticipated based on the components. The above finding confirms the recommendation by EFSA’s Scientific Committee in its guidance for risk assessment of mixtures, to use Concentration Addition for the assessment of chemicals with similar as well as dissimilar modes of action in the context of both human health and environmental risk assessment . Tools and criteria for prioritising chemicals and identifying the main drivers of mixture toxicity are also available. In recent EU-funded research projects, a selection of prioritising approaches were applied . The ‘Maximum Cumulative Ratio’ (MCR) , , approach is often used during regulatory and scientific assessments of intentional, as well as unintentional mixtures. In the context of human health assessment, information on Adverse Outcome Pathways and statistical and pharmacokinetic modelling is used. In the context of environmental assessment, the SOLUTIONS project has proposed the use of different lines of evidence based on ecological, effect-based and chemical monitoring to identify priority pollutants in surface waters . To reduce costs, improve efficiency and replace animal testing, alternative experimental and computational approaches have increasingly been introduced into (eco)toxicological assessments. In a 2014 Commission (JRC) survey on scientific methodologies for the combined effect of chemicals , experts confirmed that there is a high potential in further use of available novel tools and scientific methodologies for the assessment of chemical mixtures. Such methods include methods using e.g. cell cultures and biological molecules (in vitro), computer-based simulation methods (i.e. in silico) and modelling approaches , which can be used in integrated approaches. They allow meaningful information on individual mixture components or whole mixtures to be derived, enabling a better understanding of the underlying mechanisms of mixture effects and prediction of combined effects and risks. On the same theme, the EU-funded project EuroMix has proposed a tiered framework for the assessment of mixtures with a combination of in silico and in vitro tools. In conclusion, there has been considerable progress in the development of the calculation and monitoring methods for mixture assessment, as well as approaches to manage data gaps and methods for prioritising substances for assessment. These methodologies so far mainly appear to be used in the research context, less for regulatory purposes. This situation is a consequence of a limitation of the methodologies, which require detailed, sensitive and high quality data on both toxicity and exposure, which is rarely available. This will continue to hamper the certainty of mixture assessment (see 7.3 Remaining knowledge gaps), as detailed data needed will be lacking also in foreseebale future. One of the most significant scientific advancement in recent years, with direct regulatory implications, is that Concentration Addition can be used as the default method for the assessment of chemical mixtures with similar as well as dissimilar modes of action, in relation to risks to both human health and environment. 7.3 Remaining knowledge gaps Progress has been made on the development of assessment methodologies, test methods and modelling approaches, as well as on improving the quality of and access to knowledge on toxicity and exposure to chemicals, as described above. Despite this progress, significant knowledge and data gaps still remain in a number of areas, which will continue to hamper the assessment and risk management of mixtures. These knowledge and data gaps, and the challenges in closing them, are linked to the wide variety of chemicals from different sources occurring in mixtures to which humans and the environment are exposed. An important finding is that normally, a limited number of chemicals are responsible for the majority of the toxicity of a mixture. These chemicals are referred to as ‘drivers of toxicity’. However, the identities of these chemicals and how much they contribute to the overall toxicity (their potencies) vary greatly between various mixtures and exposure situations. Hence, it is not sufficient to gather data only on high volume or ‘priority chemicals’. Additional toxicity data will still be needed for a large number of substances to successfully assess mixtures, e.g. through calculation methods and in-silico modelling. The Communication of 2012 , drawing upon the joint opinion of three EU Scientific Committees and the State of the Art Report on Mixture Toxicity , highlighted that extensive knowledge gaps on toxicities and exposures to chemicals were limiting how accurately chemical mixtures could be risk assessed. The Communication mentioned that information to be collected in the context of EU legislation, in particular REACH, would contribute to reducing these uncertainties. Indeed, the overall quality of and access to toxicity data has improved considerably during the last decade. The registration under REACH of chemicals present on the market (i.e. phase-in chemicals), was finalised with the registration deadline in May 2018 . Hence, a total of 23,023 unique chemicals, placed on the EU market in quantities over 1 tonne per producer/importer and year, had been registered by June 2020. This number will continue to grow as new chemicals produced or imported to the EU are continuously registered. The REACH database is the most comprehensive available worldwide. Despite this progress, hazardous properties and occurrence is sufficiently well characterised to allow appropriate mixture risk assessment only for a fraction of all chemicals produced and used. A general challenge is that chemicals are often transformed to other forms along their life-cycles (e.g. during use, through degradation in the environment and upon uptake in living organisms). Further, in addition to the exposure to chemicals in current use, there is also exposure to legacy chemicals, still occurring in and released from products and waste, and accumulating in humans and the environment. It is not known exactly how many chemicals are produced and used worldwide, but estimates span from between 85,000 to over 140,000 , , . These chemicals can in principle occur in the human and natural environment. Another recent study shows that a total of approximately 350,000 individual chemicals and commercial/intentional mixtures are registered under different jurisdictions and traded globally. It should be noted that chemicals of relevance for assessment of unintentional mixtures and combination effects are not only the ones subject to e.g. registration under REACH, but all kinds of chemicals that can occur in the environment, including pesticides, biocides, pharmaceuticals and chemicals used and imported in different kinds of products.

In line with the above findings, several authors conclude that risk assessment of unintentional mixtures continue to be hampered by gaps in, or lack of access to, hazard and exposure data on the individual chemicals that are the components of mixtures , , , . For many chemicals known or suspected to appear in environmental media, information on uses, releases and toxicity is either absent, unavailable or of poor quality. This problem relates to chemicals across uses and regulatory areas. In particular hazard characterizations are frequently incomplete, especially for specific endpoints. This problem is associated with the type and level of information required under different pieces of legislation as well as the lack of alignment of such requirements across legislation , . An issue related to the REACH registration requirements is the usually low level of knowledge and/or availability of data on substances produced and used in smaller volumes (1-10 tonnes per producer/importer and year) . Such substances can represent a problem for mixture risk assessment as they despite being produced and used in low volumes can still appear as components of a mixture and thereby contribute to its toxicity. Further, some categories of chemicals including e.g. polymers and pharmaceuticals are exempted from registration under REACH. There are also issues related to a high degree of non-compliance and the low quality of registration dossiers under REACH , . Further, the publically available knowledge on occurrence and concentration of chemicals in articles and related exposure is insufficient. Overall, there is a lack of knowledge of the actual amounts of individual chemicals produced, how much is used for different purposes and how much is released into different environmental media. Case studies conducted as part of the SOLUTIONS research project illustrate the toxicity knowledge gaps. In one example, a total of 227 chemicals were monitored at different locations in the river Danube. For 27% of these chemicals, experimental toxicity data for acute toxicity was available, assessed through the most common bioassay (Daphnia magna) . For any other bioassay or test species and for chronic effects, data was considerably scarcer. Regarding exposure data, knowledge of the composition of the mixtures to which humans and wildlife are exposed and their effects, is often missing. Overall, the extent of human and environmental monitoring of chemicals is still insufficient to provide adequate knowledge on exposure and occurrence of chemicals in humans, biota and environmental media. Chemical monitoring also has its limitations. For instance, generally only chemicals suspected to be present are detected by targeted or suspect screening analyses. Hence, the chemicals not analysed, including breakdown products or metabolites, which can contribute to a mixture effects might be overlooked. In addition, monitoring data allow retrospective assessments only, while detection of rising levels and effects will be delayed by years or decades after the emissions took place. In order to prospectively model co-exposure to unintentional mixtures, more information on the types and quantities of chemical uses is needed. Examples of ongoing initiatives at the EU level that will contribute to improved chemicals exposure knowledge and data access includes HBM4EU and IPCHEM (see Section 7.1). Approaches that can contribute to filling toxicity data gaps include grouping and modelling methodologies. The 2018 EEA report ‘Chemicals in European Waters – Knowledge developments’ provides an overview of recent findings, challenges and remaining knowledge gaps related to the assessment of chemicals mixtures in the aquatic environment. These include knowledge of the biological actions of pollutants and, information needed to establish the use of effect-based monitoring methods under e.g. the Water Framework Directive. Activities to investigate the feasibility and potential implementations of effect-based monitoring method and tools under the Common Implementation Strategy for the Water Framework Directive started in 2012. The work to provide robust methodologies and to identify threshold values (trigger values) for methods developed is still ongoing, and has resulted in one scientific paper and one report , while another report will soon be published. 7.4 Future research and development priorities Several authors and projects have presented priorities and recommendations on future research and development needs. The project coordinators of the recent EU research projects (SOLUTIONS, HBM4EU, EDC-MixRisk, EuroMix) highlighted mixtures as a high priority for further research in a position paper in 2018 . The paper pointed out 12 key issues to be urgently tackled by scientists, regulators and industry. Their conclusions confirm the lack of data and difficulty to access available mixture exposure and hazard data for the components in a mixture (see Section 7.3), while also highlighting the need for validated and standardised test methods, for knowledge on the effects on vulnerable populations and on combined effects with other stressors as key knowledge gaps. The EFSA Scientific Committee in 2019, in its general guidance on assessment of combined exposure, included a comprehensive set of recommendations on future research and development activities regarding mixture assessment . These range from the development and validation of methods and tools for assessment to the improved access to and sharing of data and cooperation between scientists, EU agencies, member states and international bodies. Box 6 compiles priorities and recommendations on research and development activities from a number of publications, including the ones mentioned above. Box 6. Priorities and recommendations on research and development A number of scientific papers, reports and position papers , , , , , , discuss priorities for future research and development to support and facilitate the assessment and management of chemical mixtures. Some priorities and recommendations put forward in these include: ·Common efforts to generate and make toxicity, exposure and chemicals-monitoring data accessible. This includes data on the overall exposure of the European population. ·Structured approaches for collecting data on simultaneous and sequential exposure to mixtures. ·Research and development regarding modelling approaches to support mixture assessment. ·Increasing the availability of validated and standardised test methods and guidance, including such on how to apply new and innovative methodologies (e.g. in vitro and computer based methods) for mixture assessment. ·Develop methodologies and harmonised guidance on the grouping of chemicals.

·Guidance for analysing uncertainties in risk assessment of combined exposure to multiple chemicals, e.g. associated with the grouping into assessment groups and assumptions concerning similar MoAs/AOPs and application of dose addition ·Knowledge and methods on how to anticipate synergisms and antagonisms between chemicals, predict when such arise and need to be taken into account, and the development of criteria/guidance on this. ·Methodologies to integrate multiple lines of evidence, such as mixture toxicology and epidemiology, into risk assessment. ·Improve the knowledge about effects of mixture exposure on the heath and resilience of ecosystems, vulnerable populations and the importance of sensitive time-windows of exposure. ·Knowledge building about the combination of chemical stressors with other environmental and social stressors. ·Increased collaboration between scientists and regulators on translating science into policy. ·Establish long-term research programs on mixture toxicity on the EU level. 8.INTERNATIONAL ACTIVITIES An overview of international activities relating to mixture assessment, focusing on available guidance documents and ongoing processes under international organisations, as well as on existing regulation and guidance in some countries outside the EU was prepared by JRC in 2014 . The Commission and EU Agencies have participated in several relevant international activities to promote consistent and science-based approaches to the risk assessment of mixtures. Besides the general benefit of the sharing of knowledge, experience and expertise, the joint international activities are important as they can contribute to internationally harmonised approaches to mixtures assessment and management in the long perspective. The Commission contributed to the 2018 OECD consideration document on the risk assessment of combined exposures to chemicals , which describes the essential steps to be considered, independent of legal framework and kind of mixture, scope and assessor (see Section 4). Further, EFSA developed its 2019 guidance on mixture risk assessments based on the WHO/IPCS framework for the assessment of combined exposures to multiple chemicals from 2011, and organised an international consultation (see Section 4). The involvement of the Commission and EU Agencies in the international activities includes the JRC’s co-leading of a working group on Combined Exposures to Multiple Chemicals under the OECD Working Parties on Hazard Assessment and on Exposure Assessment. Further, the JRC together with several EU Agencies, was a member of a WHO informal working group on combined exposure under the WHO Chemical Risk Assessment Network, which was active on the mapping activities, facilitating collaboration, and identifying remaining gaps. The JRC is also coordinating an informal international research group on the testing of mixtures. Further, the Commission and the EU agencies participate in international cooperation aimed at improving the knowledge base on chemical mixtures, toxicity and exposure information. This includes the OECD-led projects on combining different chemicals-related databases (see Section 7), e.g. the OECD eChemPortal and the OECD activities towards a ‘Global Chemicals Knowledge Base’. Box