Regulatory assessment of chemical mixtures: Requirements, current approaches and future perspectives

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Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

Background: A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue® using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene. Results: All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints. Conclusions: We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.JRC.H.5-Rural, water and ecosystem resource

Developing human biomonitoring as a 21st century toolbox within the European exposure science strategy 2020-2030

Human biomonitoring (HBM) is a crucial approach for exposure assessment, as emphasised in the European Commission’s Chemicals Strategy for Sustainability (CSS). HBM can help to improve chemical policies in five major key areas: (1) assessing internal and aggregate exposure in different target populations; 2) assessing exposure to chemicals across life stages; (3) assessing combined exposure to multiple chemicals (mixtures); (4) bridging regulatory silos on aggregate exposure; and (5) enhancing the effectiveness of risk management measures. In this strategy paper we propose a vision and a strategy for the use of HBM in chemical regulations and public health policy in Europe and beyond. We outline six strategic objectives and a roadmap to further strengthen HBM approaches and increase their implementation in the regulatory risk assessment of chemicals to enhance our understanding of exposure and health impacts, enabling timely and targeted policy interventions and risk management. These strategic objectives are: 1) further development of sampling strategies and sample preparation; 2) further development of chemical-analytical HBM methods; 3) improving harmonisation throughout the HBM research life cycle; 4) further development of quality control / quality assurance throughout the HBM research life cycle; 5) obtain sustained funding and reinforcement by legislation; and 6) extend target-specific communication with scientists, policymakers, citizens and other stakeholders. HBM approaches are essential in risk assessment to address scientific, regulatory and societal challenges. HBM requires full and strong support from the scientific and regulatory domain to reach its full potential in public and occupational health assessment and in regulatory decision-making.info:eu-repo/semantics/publishedVersio

Assessing trends and uncertainties in satellite-era ocean chlorophyll using space-time modeling

The presence, magnitude, and even direction of long-term trends in phytoplankton abundance over the past few decades is still debated in the literature, primarily due to differences in the data sets and methodologies used. Recent work has suggested that the satellite chlorophyll record is not yet long enough to distinguish climate change trends from natural variability, despite the high density of coverage in both space and time. Previous work has typically focused on using linear models to determine the presence of trends, where each grid cell is considered independently from its neighbors. However, trends can be more thoroughly evaluated using a spatially resolved approach. Here a Bayesian hierarchical spatio-temporal model is fitted to quantify trends in ocean chlorophyll from September 1997 to December 2013. The approach used in this study explicitly accounts for the dependence between neighboring grid cells, which allows us to estimate trend by ‘borrowing strength’ from the spatial correlation. By way of comparison, a model without spatial correlation is also fitted. This results in a notable loss of accuracy in model fit. Additionally, we find an order of magnitude smaller global trend, and larger uncertainty, when using the spatio-temporal model: -0.023 ± 0.12 % yr-1 as opposed to -0.38 ± 0.045 % yr-1 when the spatial correlation is not taken into account. The improvement in accuracy of trend estimates, and the more complete account of their uncertainty emphasizes the solution that space-time modeling offers for studying global long-term change

Zürich Statement on Future Actions on Per- and Polyfluoroalkyl Substances (PFASs).

Per- and polyfluoroalkyl substances (PFASs) are man-made chemicals that contain at least one perfluoroalkyl moiety, [Formula: see text]. To date, over 4,000 unique PFASs have been used in technical applications and consumer products, and some of them have been detected globally in human and wildlife biomonitoring studies. Because of their extraordinary persistence, human and environmental exposure to PFASs will be a long-term source of concern. Some PFASs such as perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) have been investigated extensively and thus regulated, but for many other PFASs, knowledge about their current uses and hazards is still very limited or missing entirely. To address this problem and prepare an action plan for the assessment and management of PFASs in the coming years, a group of more than 50 international scientists and regulators held a two-day workshop in November, 2017. The group identified both the respective needs of and common goals shared by the scientific and the policy communities, made recommendations for cooperative actions, and outlined how the science-policy interface regarding PFASs can be strengthened using new approaches for assessing and managing highly persistent chemicals such as PFASs. https://doi.org/10.1289/EHP4158

Chemical Mixtures in the EU Population: Composition and Potential Risks

Regulating chemical mixtures is a complex scientific and policy task. The aim of this study was to investigate typical mixtures and their potential risks based on internal exposure levels in the European population. Based on human biomonitoring (HBM) data made available via the HBM4EU project, we derived generic mixtures representative of a median (P50) and a worst-case scenario (P95) for adults and children. We performed a mixture risk assessment based on HBM concentrations, health-based guidance values (HBGVs) as internal thresholds of concern, and the conservative assumption of concentration addition applied across different toxicological endpoints. Maximum cumulative ratios (MCRs) were calculated to characterize the mixture risk. The mixtures comprise 136 biomarkers for adults and 84 for children, although concentration levels could be quantified only for a fraction of these. Due to limited availability of HBGVs, the mixture risk was assessed for a subset of 20 substance-biomarker pairs for adults and 17 for children. The mixture hazard index ranged from 2.8 (P50, children) to 9.2 (P95, adults). Six to seven substances contributed to over 95% of the total risk. MCR values ranged between 2.6 and 5.5, which is in a similar range as in previous studies based on human external exposures assessments. The limited coverage of substances included in the calculations and the application of a hazard index across toxicological endpoints argue for caution in the interpretation of the results. Nonetheless the analyses of MCR and MAFceiling can help inform a possible mixture assessment factor (MAF) applicable to single substance risk assessment to account for exposure to unintentional mixtures