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Pesticide use is essential for control of pests in horticultural crops and adequate production of food supplies for the ever increasing world population as well as for control of vector-borne diseases. Currently, OPs are among the most widely used pesticides in agriculture, and their residues have been found in various foods. Regarding human risk assessment, the points of departure (PODs) to set health based guidance values have been derived from data from animal studies, mostly on OP-induced acetylcholinesterase (AChE) inhibition. The use of such animal data is undesirable due to economical, ethical and scientific limitations. The main aim of this thesis was to develop non-animal approaches for the hazard assessment of OPs to be applied in human risk assessment and application of these novel approach methods (NAMs) to predict the potential of the OPs to inhibit AChE in vivo upon acute exposure, also taking combined exposure into account. Furthermore, this thesis aimed to obtain data on the occurrence of OPs on commonly consumed vegetables in Kenya, as such data, and related estimations of human exposure, are lacking. In the first step, commonly consumed vegetables in Kenya were screened for OPs (and carbamates) and the accompanying exposure and related health risks assessed. A total of 90 samples were analyzed by liquid chromatography/high-resolution tandem mass spectrometry. Residues of acephate, chlorpyrifos (CPF), methamidophos, omethoate and profenofos (PFF) were found in 22% of the samples, at levels ranging from 10 to 1343 μg/kg. The EU MRLs for these OPs was exceeded in 21%, 10%, 8% and 22% of the samples of French beans, kales, spinach, and tomatoes, respectively. CPF levels on spinach were calculated to result in an acute Health Quotient (HQ) of 3.3 and 2.2 for children and adults, respectively, upon high consumption, implying that potential health risks with respect to acute dietary exposure cannot be excluded. Calculations for chronic dietary exposure showed that all chronic HQs were below 1. The Health Index (HI) for the pesticides, i.e. the sum of the chronic HQs, was 0.54 and 0.34 for children and adults, respectively, implying no direct health concern upon chronic average consumption of the vegetables assessed..
In the next step, a combined in vitro-in silico approach to predict AChE inhibition by the OP profenofos (PFF) in rats and humans by applying physiologically based kinetic (PBK) modelling-facilitated quantitative in vitro to in vivo extrapolation (QIVIVE) was developed. A PBK model was developed for both species. PBK model parameter values for PFF conversion to 4-bromo-2-chlorophenol (BCP; detoxification pathway) were derived from in vitro incubations with liver microsomes, liver cytosol, and plasma from rats (catalytic efficiencies of 1.1, 2.8, and 0.19 ml/min/mg protein, respectively) and humans (catalytic efficiencies of 0.17, 0.79, and 0.063 ml/min/mg protein, respectively), whereas other chemical-related PBK model parameter values were derived using in silico calculations. The rat PBK model was evaluated against literature data on urinary excretion of conjugated BCP. Concentration-dependent inhibition of rat and human AChE by PFF was determined in vitro and these data were translated with the PBK models to predicted dose-dependent AChE inhibition in rats and humans in vivo. Comparing predicted dose-dependent AChE inhibition in rats to literature data on PFF-induced AChE inhibition revealed an accurate prediction of in vivo effect levels. Comparison of rat predictions (BMDL10 of predicted dose-response data of 0.45 mg/kg bw) and human predictions (BMDL10 of predicted dose-response data of 0.01 mg/kg bw) suggests that humans are more sensitive than rats, being mainly due to differences in kinetics. Results obtained demonstrate that in vivo AChE inhibition upon acute exposure to PFF was closely predicted in rats, indicating the potential of this NAM in chemical hazard assessment.
In order to consider OP-mixture effects, this study assessed combination effects of PFF and chlorpyrifos’ (CPF) toxic metabolite chlorpyrifos oxon (CPO) on AChE inhibition in vitro, and the effects of CPF and CPO on PFF detoxification by human liver microsomes, cytosol, and plasma. PFF was 199-fold less potent than CPO in AChE inhibition, and combined effects of CPO and PFF followed principles of dose addition. CPF and CPO affected PFF detoxification to BCP in a non-competitive manner at high concentrations that are not expected to be reached in vivo. The PBK model of PFF was extended to include the description of CPF and CPO kinetics. PFF concentrations in the model were expressed as CPO-equivalents allowing prediction of total internal CPO-equivalents upon combined exposure to PFF and CPF. Different exposure scenarios were applied for which PBK model-predicted internal (unbound) CPO-equivalents were compared to CPO concentrations affecting AChE activity in vitro, to assess whether in vivo effects on AChE activity are expected. These results reveals that AChE inhibition upon combined exposure to these OPs follows principles of dose addition, and that no effects of CPF on PFF detoxification are expected.
Finally, this study determined human interindividual differences in PFF detoxification based on the combined in vitro-in silico approach. To that end, PFF conversion to BCP was studied in in vitro incubations with liver S9 fractions of 25 different Caucasian donors and plasma samples of 25 different Caucasian donors. The obtained information on the variation in the in vitro kinetic constants Vmax and Km was applied in the PBK model using a Monte Carlo simulation modelling approach. Analysis of the predicted internal maximum concentrations of the simulated population indicated a 1.4- and 1.5-fold difference between the geometric mean (GM) and the 90th and 99th percentile, respectively, of the simulated population, being smaller than the default uncertainty factor accounting for human variability in toxicokinetics of 3.16. Concentration-response curves for inhibition of human AChE determined in vitro were translated with the PBK model to predicted dose-dependent AChE inhibition in humans in vivo. In line with the differences in Cmax values, the 99th percentile of the simulated population was predicted to be 1.5-fold more sensitive to PFF-induced AChE inhibition than the GM of the simulated population based on the BMDL10 values derived from predicted dose-response data for the virtual population. This predicted BMDL10 of 0.0057 mg/kg bw (99th percentile of population) is comparable to an ARfD reported by EFSA (0.005 mg/kg bw) based on a German evaluation, and the ARfD reported by EPA (0.002 mg/kg bw), but much lower than the ARfD adopted by JMPR (1 mg/kg bw). In conclusion, integrating PBK modelling with Monte Carlo simulations using human in vitro data provides a powerful strategy to quantify human interindividual variation in kinetics, which can be used to refine the hazard characterization of OPs.
Altogether, the present thesis presents a NAM-based hazard assessment of OP-induced AChE inhibition upon single or combined exposure to CPF and PFF. Since a generic bottom-up PBK modelling approach was applied, the approach can be easily extended to include more OPs allowing a mechanism-based hazard and risk assessment of combined OP exposure.
|Qualification||Doctor of Philosophy|
|Award date||7 Jan 2022|
|Place of Publication||Wageningen|
|Publication status||Published - 2022|
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- 1 Finished
Development of combined in vitro-in silico methods for the risk assessment of pesticides and pesticide mixtures as found in Kenya.
1/07/17 → 7/01/22