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Periodic Reporting for period 3 - EuroMix (EuroMix)

Reporting period: 2018-05-15 to 2019-05-14

Every day we are exposed to mixtures of chemicals via several exposure routes (e.g. diet, air, cosmetics). To protect human health, risk of exposure to these chemical mixtures needs to be assessed. However, mixture risk assessment (MRA) is in its infancy. The way it should be performed varies between regulatory sectors and geographic regions. Risk assessors have to deal with data gaps, uncertainties and lack of models hampering realistic MRA. Therefore, EuroMix aimed to develop a tiered test strategy and models to perform future MRA with focus on:
1. Reducing uncertainties by generating more refined hazard data of chemical mixtures using cost-effective in-vitro assays;
2. Priority setting for testing chemicals based on in-silico hazards predictions and/or exposure considerations;
3. Developing physiological based-toxicokinetic (PB-TK) models for in-vitro to in-vivo extrapolation (IVIVE) to explore how in-vitro tests and IVIVE can be used as reliable alternatives for animal experiments;
4. Developing hazard and exposure models for MRA and applying these models on the newly generated data.
To increase efficiency in MRA, the second aim of EuroMix was to develop an open-access model and data platform:
• Grouping chemicals into assessment groups based on in-silico predictions, in-vitro and/or in-vivo studies;
• Use of experimental data (in vitro or in vivo) and applying Benchmark Dose Modelling (BMD) to derive relative potency factors for each chemical in a mixture;
• Combined dietary exposure assessment of pesticides, contaminants and/or additives regulated under different regulatory sectors;
• Multiple route (‘aggregated’) exposure assessment;
• Integrating hazard and exposure data into a Margin of Exposure in line with tiered assessment as described in international guidance;
• Comparing risk calculations as required in the regulatory context with data from biomonitoring studies;
• ICT links between the EuroMix model and data platform to international data collections such as foreseen at the European Commission (e.g. IPCHEM) and/or EFSA data warehouse.
As geographic and regulatory differences in MRA exists, EuroMix also aimed for harmonization of mixture risk assessment.
The test strategy consists of the following steps:
• Select an adverse outcome pathway (AOP) for grouping and testing chemicals;
• Align the AOP with in-silico methods, i.e. (quantitative) structure activity relationship and molecular docking, and in-vitro assays;
• Use available literature data, in-silico predictions and/or exposure results for priority setting;
• Test priority chemicals in vitro, model the results to derive chemical potencies and test the appropriateness of the dose addition assumption;
• Develop relevant IVIVE models;
• Compare in-vitro results with in-vivo experiments for verification;
• Perform MRA using the newly generated data.

The test strategy is elaborated for three outcomes: fatty changes in lever, feminisation and cranio-facial malformation. In-silico predictions and Threshold of Toxicological Concern values are available for 1630 substances (10 chemical classes). A probabilistic model for estimating co-exposure of chemicals to prioritise mixtures was programmed and applied for 10 EU countries. These activities have resulted in a list of priority chemicals for further testing.

Relevant in-vitro assays aligning the AOPs have been established and validated. Priority chemicals were tested alone for chemical potencies or as mixtures displaying similar or dissimilar mode of action (MoA). So far, dose-addition seems to be the most common mechanism. In-vivo studies confirmed the in-vitro findings for feminisation and cranio-facial malformation. Results of the in vivo study for liver steatosis were inconclusive and need more investigation. For IVIVE, 9 chemical specific and 1 generic PB-TK models as part of IVIVE were developed.

The web-based EuroMix model and data platform contains relevant models and data for efficient MRA:
• Existing and new developed models for hazard (BMD and PB-TK) and exposure assessment (combined and aggregated);
• Data obtained from in-silico predictions and in-vitro assays;
• Concentration and consumption data.
The EuroMix model and data platform was validated and fits into the European modern ICT infrastructure strategy (e.g. interagency i-cloud).

Case studies using the EuroMix model and data platform were performed. For aggregated exposure, three case studies for multiple pesticides, bisphenols, and pyrethroids were performed. A case study addressing MRA of pesticides, additives and contaminants was performed for 8 EU countries. Examples implementing use of in-silico and in-vitro data in MRA were also performed.

A human biomonitoring study focusing on substances relevant for aggregated exposure was finalised. Biological samples, food consumption data and use of cosmetics were obtained and analysis of samples and data interpretation has been performed. The biomonitoring results were compared with predicted exposure based on residue data and consumption patterns.

Access to the tools was facilitated by physical training and webinars. A practical guidance on how to use the EuroMix tests and models, in line with international developments, was written. Dissemination and harmonization of the approach was achieved by involving key-experts, EFSA, WHO and US-EPA, four harmonization workshops and a WHO expert consultation on how the EuroMix tools can be used in Europe and in a global setting.
EuroMix progress beyond the state of art by:
• Grouping chemicals based on AOP-wise testing;
• Providing a bioassay toolbox for efficient mixture testing;
• Increase efficiency in mixture testing using exposure-wise prioritised chemicals;
• Integrating exposure, kinetic and toxicological models in a single web-based platform;
• Providing practical guidance on 1) regulatory implementation of testing chemical mixtures using the new tests and approaches and 2) use of test results for MRA;
• Discussing MRA overarching regulatory sectors.

Results at the end of the project are:
• Efficient MRA process ensuring adequate protection of public health;
• First tier screening of chemicals for mixture testing based on literature data and in-silico models;
• Guidance on use in-vitro tests for generating hazard data and for understanding mixture effects;
• Harmonized approach to assess health risk of mixtures;
• Harmonized approach to access health risk of multiple route exposure and comparing the results with real life exposure in a biomonitoring study;
• Open-access web-based model and data platform available to stakeholders beyond the project’s lifetime;
• Guidance and training materials on use of the test strategy and refined MRA;
• Four harmonization workshops on acceptance of the test strategy and exposure assessment methodology and an expert consultation by the Codex Alimentarius;
• Reduction in animal tests upon acceptance of the approach.

EuroMix has potential impact on:
• Innovation in the public sector by providing models and data to competent authorities to improve MRA;
• Innovation within the private sector for cost-effective testing of (new) chemicals;
• Improvement of the reliability and efficiency in mixture testing and risk assessment, resulting in improved protection of public health;
• Reduction of animal tests;
• Harmonization of global policies for MRA to ensure global trade without unnecessary barriers.