Status of the EU-ToxRisk Project

Dinant Kroese (TNO)

EU-ToxRisk is the flagship research project in new approach methods (NAM)-based toxicity testing in Europe. This six year project is funded with ~30M € through the European Commission’s Horizon 2020 scheme and was launched in 2016. The ultimate aim of the project is to develop human relevant toxicity testing strategies that can be applied in a regulatory or screening context using only NAMs. These approaches will be based on mechanistic understanding guided by adverse outcome pathways (AOPs) that are being developed in the project. The project integrates advancements in cell biology, omics technologies, systems biology and computational modelling to define the complex chains of events that link chemical exposure to toxic outcome. The focus of this project is on two areas of risk assessment: repeated dose systemic toxicity and developmental and reproductive toxicity. The project directly addresses two complementary critical regulatory needs: 1) Pragmatic, solid “read-across” procedures between chemicals as the most important data gap filling method in Europe at present, and 2) ab initio hazard and risk assessment strategies of chemicals with little background information. The EU-ToxRisk work plan is structured along a broad spectrum of case studies, driven by the cosmetics, (agro)-chemical, and pharma industry together with regulators and specialists from academia. Different, tiered test systems are integrated to balance speed, cost and biological complexity. Currently, the first phase of 6 case studies that encompass both read-across and ab initio scenarios is being completed. The next phase case studies will build upon the learnings of the first set of case studies and will be designed to answer important scientific and regulatory questions.

Detection of titanium particles in human liver and spleen and possible health implications
M.B. Heringa1*, R.J.B. Peters2, R.L.A.W. Bleys3, M.K. van der Lee2, P.C. Tromp4, P.C.E. van Kesteren1, J.C.H. van Eijkeren1, A.K. Undas2, A.G. Oomen1, H. Bouwmeester2#

* Corresponding author
1 National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands
2 RIKILT – Wageningen University & Research, Wageningen, The Netherlands
3 Department of Anatomy, University Medical Center Utrecht, Utrecht, The Netherlands
4 TNO Earth, Life and Social Sciences, Utrecht, The Netherlands
# current address: Division of Toxicology, Wageningen University, Wageningen, The Netherlands.

Abstract
Background: Titanium dioxide (TiO2) is produced at high volumes and applied in many consumer and food products. Recent toxicokinetic modelling indicated the potential of TiO2 to accumulate in human liver and spleen upon daily oral exposure, which is not routinely investigated in chronic animal studies. A health risk from nanosized TiO2 particle consumption could not be excluded then.
Results: Here we show the first quantification of both total titanium (Ti) and TiO2 particles in 15 post-mortem human livers and spleens. These low-level analyses were enabled by the use of fully validated (single particle) inductively coupled plasma high resolution mass spectrometry ((sp)ICP-HRMS) detection methods for total Ti and TiO2 particles. The presence of TiO2 in the particles in tissues was confirmed by Scanning Electron Microscopy with energy dispersive X-ray spectrometry.
Conclusions: These results prove that TiO2 particles are present in human liver and spleen, with ≥24% of nanosize (<100 nm). The levels are below the doses regarded as safe in animals, but half are above the dose that is deemed safe for liver damage in humans when taking into account several commonly applied uncertainty factors. With these new and unique human data, we remain with the conclusion that health risks due to oral exposure to TiO2 cannot be excluded.

Keywords: titanium dioxide, tissue level, nanoparticle, risk assessment, sp-ICP-HRMS

The organophosphate insecticides chlorpyrifos and diazinon disturb neuronal network function in a rat primary cortical cultures.
Harm J Heusinkveld1,2, Remco HS Westerink1,

1. Neurotoxicology Research Group, Toxicology and Pharmacology Division, Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands.
2. Department for Innovative Testing Strategies, Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.

Organophosphate (OP) insecticides like chlorpyrifos (CPF) and diazinon (Dz) are widely used in agriculture and household applications. OPs exert their (primary) adverse effect via (irreversible) inhibition of acetylcholine esterase (AChE), leading to neuronal overstimulation and malfunction. Human exposure occurs mainly in occupational settings as well as via the food chain. Recently, disturbance of intracellular calcium homeostasis was identified as a mechanism potentially underlying neurotoxicity of OP insecticides, as OP insecticides can inhibit calcium influx at levels that do not induce inhibition of AChE. Therefore, this study aimed to assess the effects of two widely used OP insecticides on intracellular calcium homeostasis and function of spontaneously active neuronal networks.

Effects of acute exposure to Dz and CPF on parameters of neuronal (network) functionality were assessed in primary rat cortical cultures using single-cell fluorescence imaging of the intracellular calcium homeostasis using Fura-2 AM, while effects on spontaneous neuronal activity were assessed using micro-electrode arrays (MEAs).

Acute exposure to CPF or Dz induces a concentration-dependent inhibition of depolarization-evoked calcium influx, likely via calcium channels block, although with different potency. Similarly, acute exposure to CPF or Dz causes a concentration-dependent inhibition of spontaneous electrical activity. In contrast, chronic MEA experiments demonstrates a concentration-dependent increase of electrical activity with Dz, whereas CPF exposure causes a change of electrical activity only at high (>10µM) concentrations. As these effects occur at concentrations below effect concentrations for AChE inhibition, the combined results indicate that other mechanisms may play a role in the neurotoxicity of OP insecticides.

Funding: ZonMW (#114027001) and the Faculty of Veterinary Medicine (Utrecht University, The Netherlands).