Thijs Moerenhout

In this thesis animal-free methods for next generation risk assessment of organophosphate pesticides were evaluated. These approaches are based on in silico and in vitro methods, and were evaluated for predicting the safety of human exposures, as well as points of departure for acetylcholinesterase (AChE) inhibition by organophosphate pesticides. This was achieved by the development of a generic physiologically based kinetic (PBK) model for organophosphate pesticides, in vitro quantification of AChE inhibition by various organophosphate pesticides, exposure safety evaluation using the dietary comparator ratio (DCR) method, and point of departure prediction for hazard characterization using quantitative in vitro to in vivo extrapolation (QIVIVE) of in vitro AChE inhibition to the in vivo situation. Chapter 1 introduces essential information of organophosphate pesticides on their use, chemistry, kinetics and effects. It is also explained how traditional risk assessment is performed and how animal-free in vitro and in silico methods can be used to perform risk assessment without animal testing. Lastly, the aim of the thesis is presented. In chapter 2, a PBK model was developed to predict blood concentrations of eight organophosphate pesticides and their metabolites in rats and humans. The developed PBK model predicts how organophosphates are absorbed, distributed, metabolized and excreted in/from the body, based on in vitro or in silico derived parameters. The PBK model was validated using literature derived blood concentration-time data from in vivo exposure studies data. When validating the model, blood concentrations of most of the organophosphate pesticides were predicted within 5-fold difference from in vivo values. Those pesticides that were less well predicted were all found to be in the dimethyl-organophosphate pesticide subclass. The developed PBK model was used in chapter 3, to investigate if the DCR method can be reliably used for safety evaluation of organophosphate pesticides. Six organophosphate pesticides were used to evaluate the DCR method for organophosphate pesticides. The DCR method correctly predicted 70 out of 74 exposures to be safe when these were indeed reported to have no adverse effects. Four exposure scenarios were predicted to be dangerous, while in literature these were reported to be safe. The four false positives were all for dimethyl-organophosphates. It was concluded that in the future the DCR method can be used for safety evaluation of exposures to organophosphate pesticides, since it was shown to accurately predict the occurrence of adverse effects after exposure to these pesticides. In chapter 4, it was investigated if the overprediction of the dimethyl-organophosphates fenitro-oxon, methyl-paraoxon and omethoate could be due to a metabolic pathway not incorporating in the PBK model. In vitro metabolism studies showed that methyl-paraoxon and omethoate were metabolized via a glutathione-mediated pathway. Furthermore, rats were found to metabolize methyl-paraoxon four times faster than humans via this pathway. When the glutathione-mediated metabolic pathway was incorporated in the PBK model, blood predictions for methyl-paraoxon and fenitro-oxon improved from over 10-fold difference when glutathione-mediated metabolism was not included, to less than 5- and often even 2-fold differences compared to in vivo rat data. For omethoate, no significant changes in predicted blood concentrations were observed. For humans, predicted maximum concentrations for methyl-parathion and fenitrothion exposures were more than 12-fold lower when glutathione-mediated metabolism was incorporated in the model. The quantitative in vitro to in vivo extrapolation approach was used for point of departure determination of AChE inhibition by organophosphate pesticides in chapter 5. For eight organophosphate pesticides and their active metabolites, concentration-response curves derived from in vitro AChE inhibition assays using blood or pure enzyme. For each pesticide, both whole blood and enzyme concentration-response curves were translated to dose-response curves using the adjusted PBK model developed in chapter 4. Benchmark dose modelling was used on the dose-response curves to predict points of departure, which were then compared to in vivo determined points of departure reported in literature. All predicted points of departure, for both rats and humans, were within 4-fold from in vivo determined points of departure low-observed-adverse-effect levels (LOAELs) and higher than the reported no-observed-adverse-effect levels (NOAELs). For some organophosphate pesticides it was not possible to a point of departure based on whole blood data, possibly due to non-AChE esterases present in whole blood. It was shown that QIVIVE-based point of departure determination can be used for animal-free hazard characterization for organophosphate pesticides when pure enzyme is used for AChE inhibition assays. In chapter 6, the results of each experimental chapter are summarized. Considerations with regard to chronic and mixture exposures are discussed and the association of organophosphate pesticides with neurodegenerative diseases briefly touched upon. For PBK modelling some opportunities are discussed in detail, focusing food effects on oral absorption, lymphatic transport, the required physiological detail that is needed in a PBK model, growth and population modelling and limitations of and solutions for in vitro and in silico derivation of kinetic parameters. Lastly, model validation and adoption of animal-free risk assessment were discussed. The findings in this thesis show how several in vitro and in silico approaches can be used for next generation safety assessment for organophosphate pesticides. Using these approaches for both rats and humans also provided valuable insights into species differences. All these findings support the thought that animals used in toxicity testing do not always represent humans very well. Therefore the human centric data provided by animal-free approaches is essential for chemical risk assessment for humans.