A retain and refine approach to cumulative risk assessment.

Mixtures of substances to which humans are exposed may lead to cumulative exposure and health effects. To study their effects, it is first necessary to identify a cumulative assessment group (CAG) of substances for risk assessment or hazard testing. Excluding substances from consideration before there is sufficient evidence may underestimate the risk. Conversely, including everything and treating the inevitable uncertainties using conservative assumptions is inefficient and may overestimate the risk, with an unknown level of protection. An efficient, transparent strategy is described to retain a large group, quantifying the uncertainty of group membership and other uncertainties. Iterative refinement of the CAG then focuses on adding information for the substances with high probability of contributing significantly to the risk. Probabilities can be estimated using expert opinion or derived from data on substance properties. An example is presented with 100 pesticides, in which the retain step identified a single substance to target refinement. Using an updated hazard characterisation for this substance reduced the mean exposure estimate from 0.43 to 0.28 µg kg-bw-1 day-1 and reduced the 99.99th percentile exposure from 24.9 to 5.1 µg kg-bw-1 day-1. Other retained substances contributed little to the risk estimates, even after accounting for uncertainty.

The MCRA toolbox of models and data to support chemical mixture risk assessment.

A model and data toolbox is presented to assess risks from combined exposure to multiple chemicals using probabilistic methods. The Monte Carlo Risk Assessment (MCRA) toolbox, also known as the EuroMix toolbox, has more than 40 modules addressing all areas of risk assessment, and includes a data repository with data collected in the EuroMix project. This paper gives an introduction to the toolbox and illustrates its use with examples from the EuroMix project. The toolbox can be used for hazard identification, hazard characterisation, exposure assessment and risk characterisation. Examples for hazard identification are selection of substances relevant for a specific adverse outcome based on adverse outcome pathways and QSAR models. Examples for hazard characterisation are calculation of benchmark doses and relative potency factors with uncertainty from dose response data, and use of kinetic models to perform in vitro to in vivo extrapolation. Examples for exposure assessment are assessing cumulative exposure at external or internal level, where the latter option is needed when dietary and non-dietary routes have to be aggregated. Finally, risk characterisation is illustrated by calculation and display of the margin of exposure for single substances and for the cumulation, including uncertainties derived from exposure and hazard characterisation estimates.

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.