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Integrating Epidemiology and Experimental Biology to Improve Risk Assessment of Exposure to Mixtures of Endocrine Disruptive Compounds

Periodic Reporting for period 3 - EDC-MixRisk (Integrating Epidemiology and Experimental Biology to Improve Risk Assessment of Exposure to Mixtures of Endocrine Disruptive Compounds)

Reporting period: 2018-05-01 to 2019-04-30

Today we know that the endocrine system is of greatest importance for a healthy development and life for both animals and humans. It is of global concern that the entire human population, foetuses, infants, children and adults, are constantly exposed to low levels of anthropogenic chemicals, some of which are endocrine disrupting chemicals (EDCs) or potential EDCs that interfere with our natural endocrine functions. Exposure to this multitude of chemicals, including EDCs, during windows of susceptibility, even at low doses, is of particular concern as this may program the organism to develop disorders that manifest later in life.

The long reaching goal of EDC-MixRisk was to move forward and meet the societal need for improved decision-making regarding risks that are posed by mixtures of anthropogenic chemicals in general, and EDCs in particular. The project was built on the interaction between advanced expertise in exposure assessment, epidemiology, experimental toxicology and risk assessment.

The overall objectives of the project were:
i) Identification of mixtures of EDCs that are associated with multiple adverse human health outcomes;
ii) Identification of molecular mechanisms and pathways underlying these associations; and
iii) Development of methods for risk assessment of EDC mixtures and interactions with policy makers to increase societal impact.
In the epidemiological module, two sets of reference mixtures were established and created for the experimental testing in cell and animal models. These mixtures addressed specific health domains: metabolism and growth (G), neurodevelopment (N) and sexual development (S). The first set (mixtures 0) was based on exposure data for 20 chemicals, the second set (mixtures 1) on data for 45 chemicals, including phthalate- and PAH metabolites, bisphenols, chlorinated and non-chlorinated pesticides, PCBs, brominated flame retardants and diphenyl phosphate. The mixtures were based on data from the Swedish mother-child pregnancy cohort SELMA including chemical analyses from mother’s urine and serum at pregnancy week 10 and the following health outcomes of their children: birth weight (growth and metabolism), language delay at age 2.5 (neurodevelopment), and anogenital distance (AGD) in boys (sexual development). All of these outcomes are early signs for adversity in the respective domain. Using these data and a novel biostatistical method, we identified so-called bad actors, chemicals that contribute to the association between exposure and adverse health outcome. These chemicals were mixed in ratios corresponding to their mean exposure concentrations to compose a stock solution for the experimental systems. Interestingly, most of the bad actors identified for mixtures 0 were also found in mixtures 1.

In the experimental module, mixtures 0 and 1 were tested in various animal and cell models with relevant read-outs for the respective health domains. These models were used to identify molecular actions of the mixtures that could underlie their adversity. Results obtained in mice, tadpoles, zebrafish, and cell models show that mixtures for all the health domains induce negative effects on the molecular, cellular, and organismal level. In some of the assays, effects were observed even at the lowest concentrations tested, which correspond to the actual levels of the SELMA mothers. Interestingly, the mixtures disrupted common hormone-related pathways in cell and in animal models, which enabled us to link the molecular effects to adverse outcomes such as increased amount of fat tissue, cognitive and behavioural changes, and disruption of sexual organ development. Selected single chemicals were also tested and their effects compared to the mixtures. In most cases, the single compounds did not have an effect at concentrations comparable to the mixtures.

Among the molecular signatures identified in the experimental models were epigenetic changes, i.e. changes in DNA structure that lead to long term changes in gene regulation. Two of these epigenetic changes, one for mixture N and one for mixture S, were selected, analysed in the SELMA samples, and tested for associations with exposure and health outcomes in the children. For the epigenetic pattern identified with mixture N, we found that it partly mediated the association between exposure and cognitive outcomes in the children. This reinforces the experimental finding that this epigenetic change could be a mechanism underlying the association between exposure and health outcome in humans.

The third module focused on improving the regulatory risk assessment of mixtures as well as science-to-policy interaction and dissemination. Three different novel mixture risk assessment methods were established during the project, using EDC-MixRisk and published data. EDC-MixRisk has contributed to increased awareness of EDC-mixture toxicities and actively disseminated project results to the scientific community, policy makers, key stakeholders and the public at large. In order to improve the impact of EU-level mixture efforts and science-to-policy interface, EDC-MixRisk also initiated knowledge exchange and interaction between various EU funded research projects as well as with Commission services and relevant EU agencies.
The EDC-MixRisk approach of identifying EDC mixtures associated with adverse health outcomes in epidemiology, preparing artificial mixtures of the bad actors for toxicological testing, and using the experimental data for mixture risk assessment was a novel approach and one of the major outcomes of the project. This proof-of-concept will enable more systematic integration of epidemiological and experimental evidence into mixture risk assessment strategies.

By applying the novel approach, we could find a higher rate of pregnant women and their children at risk when compared with more traditional models of additivity. This adds to the evidence that cocktail effects are not properly considered in the risk assessment and management of chemicals. Thus, more systematic approaches are needed, both in terms of science and regulations. Furthermore, we have a positive indication that it is possible to identify and test a rather small number of chemical mixtures that are relevant for a large proportion of the population. This is a significant step forward as it is not feasible to test all the possible combinations of environmental chemicals.

The improved testing strategies and risk assessment methodologies developed in the project are important for the regulatory processes to protect public health and to avoid hazardous chemicals, whether they come in mixtures or as single compounds. Thus, the project results will have important implications for the general population globally, for national regulatory agencies and organizations; for chemicals manufacturing industry and down-stream users of these chemicals.
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