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.
