Something from nothing? Ensuring the safety of chemical mixtures

Headlines - Humans and the environment are exposed to a cocktail of chemicals from different sources. - Combined exposure to multiple chemicals can lead to health/ environmental effects even if single substances in the mixture do not exceed safe levels. - The assessment and management of mixtures is only partly covered by current legislation, which focuses on single substances in isolated sectors. - Methodology to address mixture risks is available, yet many knowledge gaps need to be filled. In particular, real co-exposure patterns are mostly unknown. - JRC is performing research on new strategies to assess the combination effects of chemicals.JRC.F.3-Chemicals Safety and Alternative Method

Scientific methodologies for the assessment of combined effects of chemicals - a survey and literature review

Exposure of humans and wildlife to chemicals via food, consumer products, the environment etc. can imply exposure to an infinite number of different combinations of chemicals in mixtures. It is practically impossible to test all these possible mixtures experimentally and it is therefore needed to find smart strategies to assess the potential hazards using new tools that rely less on in vivo testing and incorporate instead alternative experimental and computational tools. In this report the current state of the art for the application of these alternative tools for assessing the hazard of chemical mixtures is briefly reviewed. The focus is hereby on the adverse outcome pathway (AOP) concept, in vitro methods, omics techniques, in silico approaches such as quantitative structure activity relationships (QSARs) and read-across, toxicokinetic and dynamic energy budget (DEB) modelling, and on integrated approaches to testing and assessment (IATA). Furthermore, an expert survey was performed to collect up to date information and experience on the current use of different approaches for assessing human and environmental health risks from exposure to chemical mixtures, with a view to informing the development of a consistent assessment approach. An online survey was performed among experts in the field of combined exposure assessment in the period of January to March 2014, addressing both, human health and environmental risk assessment. Fifty-eight experts from 21 countries, different stakeholder groups and sectors of legislation participated in the survey. The main sectors where most experience is already gained in assessing mixtures are in the area of plant protection products and chemicals. These were also rated highest regarding the priority for performing mixture assessments. Experts have experience with the whole mixture as well as the component-based approaches applying them to both, intentional and unintentional mixtures. Mostly concentration addition (CA) based methods are used for predicting mixture effects. Regarding the use of novel and alternative tools in the risk assessment of mixtures, expert opinions are split between those applying them (often more in a research context) and those that generally think these tools are valuable but their use is currently limited because of lack of guidance, lack of data, or lack of expertise. A general need for clear guidance for combined exposure assessments was identified. Overall, a high potential in applying novel tools and scientific methodologies for the assessment of chemical mixtures can be identified. They allow deriving meaningful information on individual mixture components or whole mixtures, enabling a better understanding of the underlying mechanisms of mixture effects. Their main strengths lie in their integrated use and smart combination to put different aspects regarding the hazard from combined exposure to multiple chemicals into context. In order to benefit from these tools in the hazard assessment of mixtures, more guidance on their use is needed to facilitate a more widespread application.JRC.I.5-Systems Toxicolog

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

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Accelerating progress in the Replacement, Reduction and Refinement of animal testing through better knowledge sharing

In the context of the European Commission's Communication published in response to the European Citizens' Initiative "Stop Vivisection", Action 1 aimed to conduct an assessment of current technologies, information sources and networks from all relevant sectors with potential impact on the advancement of the refinement, reduction and replacement of animals used for scientific purposes (the "Three Rs"). The main objective of this study is to map knowledge sources relevant to the Three Rs, to examine how knowledge is being shared, and to identify possible gaps and opportunities to enhance knowledge sharing. An inventory of knowledge sources (KS) potentially relevant to the Three Rs used in the area of life sciences has been compiled which should be viewed as a snapshot of the current status of knowledge sources available (the supply), and as a starting point for further analysis of knowledge sharing strategies. A public survey was also conducted to complement this study (supply versus demand), and to inform on how to proceed effectively with any knowledge management strategy. The survey has revealed that many involved in this area consider that whilst access to relevant knowledge sources is adequate, there is a need for better coordination of the sources, as well as for the communication of information. Opportunities for face-to-face exchanges are highly valued and there is a firm call for more developments in education and training at professional, university and secondary school levels.JRC.F.3-Chemicals Safety and Alternative Method

Application of the Virtual Cell Based Assay for Simulation of in vitro Chemical fate following Acute Exposure

In order to reliably assess the risk of adverse systemic effects of chemicals by using in vitro methods, there is a need to simulate their absorption, distribution, metabolism, and excretion (ADME) in vivo to determine the target organ bioavailable concentration, and to compare this predicted internal concentration with an effective internal concentration. The effective concentration derived from in vitro toxicity studies should ideally take into account the fate of chemicals in the in vitro test system, since there can be significant differences between the applied nominal concentration and the in vitro bioavailable concentration. Whereas PBK models have been developed to simulate ADME properties in vivo, the Virtual Cell Based Assay (VCBA) has been developed to simulate in vitro fate. In this project, the VCBA model in R code, was applied to better interpret previously obtained in vitro acute toxicity data and study how they can be compared to results from acute toxicity in vivo. For 178 chemicals previously tested in vitro with the 3T3 BALB/c cell line using the Neutral Red Uptake cytotoxicity assay, physicochemical parameters were retrieved and curated. Of these chemicals, 83 were run in the VCBA to simulate a 96-well microplate set up with 5% serum supplementation, and their no effect concentration (NEC) and killing rate (Kr) optimized against the experimental data. Analyses of results of partitioning of the chemicals show a strong relation with their lipophilicity, expressed here as the logarithm of the octanol/water partitioning coefficient, with highly lipophilic chemicals binding mostly to medium lipid. Among the chemicals analysed, only benzene and xylene were modelled to evaporate by more than 10 %, and these were also the chemicals with highest degradation rates during the 48 hours assay. Chemical degradation is dependent not only on the air and water degradation rates but also on the extent of binding of the chemical. Due to the strong binding of some chemicals to medium lipids and proteins we analysed the impact of different serum supplementations (0%, 5% and 10%) on the chemical dissolved concentrations. As expected, for the more lipophilic chemicals, different serum levels result in different dissolved concentrations, with lipid and protein binding reducing chemical loss by evaporation. Still the lack of saturation modelling might mislead the 0 % supplementation since the lipids coming solely from cells exudates are able to sequester chemical to a large extent, eg. after 48 hours, 63% (1.2E-5 M) of dimethyldioctadecylammonium chloride was bound to lipid from the cells. Although highly lipophilic chemicals have a very small bioavailable fraction, cellular uptake rate is also dependent on logKow, which compensates for this lack of bioavailability to some extent. Based on the relevance of lipophilicity on in vitro chemical bioavailability, we have developed an alert system based on logKow, creating four classes of chemicals for the experimental condition with 10% serum supplementation: logKow 5- 10 (A), logKow <5 (B), logKow <2.5 (C), and logKow <2 (D). New chemicals from Classes A and B, which will in the future be tested in vitro, were run first on the VCBA, without considering toxicity (NEC and Kr set to 0). VCBA simulations indicated that these chemicals are more than 50% bound to medium proteins, lipids and plastic. Therefore, for chemicals with logKow falling in these classes, special care should be taken when extrapolating the obtained in vitro toxic concentrations to in vivo relevant doses. A comparison of the VCBA-predicted dissolved concentrations corresponding to nominal IC50 values with the available rat oral LD50 values did not improve the previously obtained correlations. This is probably because other in vivo kinetic processes play an important role but were not considered in this in vitro-in vivo extrapolation. The comparison of the VCBA predicted IC50 dissolved concentrations with the available rat oral LD50 values, did not improve the previously obtained correlations. Nevertheless, other in vivo kinetic processes that are not modelled may play an important role. They should be considered in the in vitro-in vivo extrapolations. A local sensitivity analysis showed the relative low impact of Molar Volume and Molecular Diffusion Volume on the final dissolved concentration, supporting the use of approximated values obtained through the herein created QSARs. The logkow and Henry Law Constant showed, as expected, a high impact in partitioning. Killing rate was shown to also have a relative low impact in the final chemical concentration, indicating that although its optimization is important, finding the Kr that leads to the absolute best correlation between experimental and predicted concentration-viability curves, is not imperative. The VCBA can be applied to virtually any chemical as long as the physicochemical data (for the fate model) and the experimental toxicity data (that include cell growth/death) are available. However, being such a generic model, several assumptions had to be made: i) no distinction of chemical classes (inorganic, polar organic chemicals), ii) no consideration of metabolism, iii) saturation kinetics and iv) external in vitro conditions. The advantages of having a generic model are that the VCBA can fit several experimental set ups and should be used in an exploratory manner, to help refinement of experimental conditions. The herein obtained VCBA results should be double checked experimentally the partition with a set of chemical compounds to better understand to what extent VCBA represents chemicals of different properties. In future developments, it would be important to reduce the uncertainties of the model such as binding-saturation and consider inclusion of other endpoints such as metabolic activity.JRC.F.3-Chemicals Safety and Alternative Method

Novel chemical hazard characterisation approaches

There is a fundamental change in thinking within the regulatory community due to a better understanding of the underlying biology of adverse effects to human health and the environment. The development of alternatives to use laboratory animals has become a priority. In addition, technological progress is impacting greatly on the amount of data available and on the ways to process and analyse it. Topics, such as identification of adverse outcome pathways (AOPs) and modes of action (MoA), together with integrated assessment and testing approaches (IATAs), represent fundamental tools for hazard identification and characterisation of a chemical. Complex endpoints cannot be predicted by a single standalone non-animal test; thus, a major challenge is the complex nature of biological systems. Microphysiological systems (MPS) will enable more complex in vitro human models that better simulate the organ's biology and function by combining different cell types in a specific three-dimensional configuration that simulates functional organs. The process of validation of new approaches needs to be considered in terms of efficiency and length. Regulators might still not have enough confidence to adopt and apply these new approaches: this phase is very challenging and the activities performed by assay developers are not yet addressing the regulatory requirements needs sufficiently. The IATAs provide a framework to consistently evaluate new approach data and could assist in understanding their relevance for specific endpoints. The data need to be reproducible, understandable and statistically sound: indeed, a major issue lies in the interpretation and integration of the results based on subjective assessment, which relies on expert judgement. A well-defined mechanistic characterisation is proposed as a way forward to ensure the relevance of new cell-based test systems

Chemical Safety Assessment Using Read-Across: Assessing the Use of Novel Testing Methods to Strengthen the Evidence Base for Decision Making

Background: Safety assessment for repeated dose toxicity is one of the largest challenges in the process to replace animal testing. This is also one of the proof of concept ambitions of SEURAT-1, the largest ever European Union research initiative on alternative testing, co-funded by the European Commission and Cosmetics Europe. This review is based on the discussion and outcome of a workshop organized on initiative of the SEURAT-1 consortium joined by a group of international experts with complementary knowledge to further develop traditional read-across and include new approach data. Objectives: The aim of the suggested strategy for chemical read-across is to show how a traditional read-across based on structural similarities between source and target substance can be strengthened with additional evidence from new approach data—for example, information from in vitro molecular screening, “-omics” assays and computational models—to reach regulatory acceptance. Methods: We identified four read-across scenarios that cover typical human health assessment situations. For each such decision context, we suggested several chemical groups as examples to prove when read-across between group members is possible, considering both chemical and biological similarities. Conclusions: We agreed to carry out the complete read-across exercise for at least one chemical category per read-across scenario in the context of SEURAT-1, and the results of this exercise will be completed and presented by the end of the research initiative in December 2015

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2013-April 2014)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches since the last report published in April 2013. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and international acceptance of alternative methods and approaches. R&D activities are ongoing for the complex endpoints where the toxicological processes and the mechanistic understanding have not been sufficiently elucidated yet and for which 3Rs solutions are more difficult to find. On the other hand, good progress In the validation and regulatory acceptance is made in areas where non-animal alternative methods have been developed and validated and where the focus lies in an intelligent combination/ integration of the various non-animal approaches.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2015)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches and their dissemination since the last report published in June 2014. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and regulatory/international acceptance of alternative methods and approaches and dissemination activities. R&D activities within large European or International consortia continued in toxicity areas where 3Rs solutions are more difficult to find due to the underlying complexity of the area. On the other hand, toxicity areas where promising non-animal approaches have been developed, their validation and regulatory acceptance/international adoption could be progressed. Particular emphasis was given to the best and most intelligent combination and integration of these different non-animal approaches to ultimately obtain the required information without resorting to animal testing.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2016)

Replacement, Reduction and Refinement of animal testing is anchored in EU legislation. Alternative non-animal approaches facilitate a shift away from animal testing. Cell-based methods and computational technologies are integrated to translate molecular mechanistic understanding of toxicity into safety testing strategies.JRC.F.3-Chemicals Safety and Alternative Method