Consequences of kinetic variability for inter-species, inter-ethnic and inter-individual differences in sensitivity towards organophosphate pesticides quantified by new approach methodologies

The aim of the present thesis was to investigate whether new approach methodologies (NAMs) and especially physiologically based kinetic (PBK) model-based reverse dosimetry using in vitro assays and in silico data, in combination with Monte Carlo simulations or the toxic equivalency factor (TEF) methodology, can be used to derive points of departure (POD) and chemical-specific adjustment factors (CSAFs) for characterizing inter-ethnic, inter-species, and inter-individual differences in toxicokinetics and red blood cell (RBC) acetylcholinesterase (AChE) inhibition by organophosphate (OP) pesticides upon single acute oral exposure.Chapter 1 provided an overview of relevant background information, including an introduction of chlorpyrifos (CPF), diazinon (DZN), and profenofos (PFF) as model compounds, their absorption, distribution, metabolism and excretion (ADME) and toxicity profiles, as well as their reported PODs and health-based guidance values (HBGVs). The aim of the thesis and the NAMs that were applied in the thesis (PBK model-based reverse dosimetry solely, or combined with Monte Carlo simulations or the TEF methodology) were also presented. At the end, the general outline of the thesis was provided.Chapter 2 assessed the inter-ethnic differences in toxicokinetics of CPF and its resulting RBC AChE inhibition between the Chinese and Caucasian populations using PBK model-facilitated reverse dosimetry based on in vitro and in silico data. The results obtained revealed a marked inter-ethnic difference in toxicokinetics of CPF, with a slower CPF bioactivation and faster detoxification of its active metabolite chlorpyrifos oxon (CPO) in the Chinese than in the Caucasian population. This could be explained by the inter-ethnic variation in enzyme profiles and enzyme activities involved in biotransformation of CPF and CPO. The differences resulted in 5- to 6-fold lower predicted POD values of CPF for the Caucasian than the Chinese population, reflecting a lower risk of CPF for the Chinese than the Caucasian at similar levels of exposure. Besides, because of inter-ethnic variation in toxicokinetics of CPF, 2-fold lower cumulative urinary 3,5,6-trichloro-2-pyridinol (TCPy) biomarker levels were observed in Chinese than in Caucasian at similar dose levels, implying the importance of taking possible inter-ethnic differences into account when characterizing CPF exposure for different ethnic groups based on their urinary biomarker levels.Chapter 3 focused on another OP and investigated the inter-species differences between rat and human in PFF-induced RBC AChE inhibition by PBK model-based reverse dosimetry. The obtained results show the predicted POD of human to be 45-fold lower than that of rat, indicating human to be more sensitive than rat to the inhibition from equivalent oral doses of PFF. Such differences appeared mainly due to marked interspecies differences in toxicokinetics of PFF, with rat being more efficient in hepatic and plasma detoxification of PFF than human, while the difference in toxicodynamics appeared limited as reflected by a similar potency of PFF to inhibit rat RBC AChE and human recombinant AChE in vitro. In conclusion, this chapter provided another proof-of-principle that integrating in vitro toxicity data and PBK model-based reverse dosimetry is a promising strategy for non-animal-based safety assessment of OP pesticides. Chapter 4 further studied the applicability of PBK model-based reverse dosimetry to predict the combined inhibitory effect of DZN and its active oxon metabolite diazoxon (DZO) on RBC AChE inhibition by integrating the TEF approach into the model. Use of the TEF coded PBK model enabled calculation of the external exposure dose of DZN that would produce a defined combined free DZN plus DZO concentration expressed in DZO equivalents in human/rat blood, and to translate the observed inhibition effects of DZO in the in vitro concentration-response curve expressed in free in vitro DZO equivalents to the in vivo situation. The obtained results show that DZN contributes substantially to the DZO equivalents, indicating it is of critical importance to take DZN into account in addition to the active metabolite DZO when conducting a risk assessment for DZN. Although no inter-species differences were observed in the in vitro concentration-response curves for rat or human RBC AChE inhibition by DZN or DZO, marked inter-species differences in toxicokinetics were observed, as rat displayed a faster metabolic rate for biotransformation of DZN and DZO than human, resulting in a 6-fold lower POD in human than in rat. In conclusion, this chapter provided a proof-of-principle for derivation of a point of departure (POD) in risk assessment for an OP pesticide for which both the parent OP and its oxon metabolite have the ability to contribute to RBC AChE inhibition.Chapter 5 characterized the inter-individual variation in toxicokinetics of CPF and its resulting RBC AChE inhibition. A CSAF for inter-individual differences in kinetics (HKAF) was quantified on the basis of the PBK model for CPF developed in Caucasian (Chapter 2), in combination with Monte Carlo simulations. To define the metabolic variation in the PBK model, two different approaches were developed and compared, namely a SupersomeTM cytochrome P450 (CYP)-based PBK model approach and a human liver microsome (HLM)-based PBK model approach. The results revealed that bioactivation of CPF exhibits biphasic kinetics due to distinct differences in Km values for CYPs involved in the CPF metabolic pathway, and these kinetic characteristics could be best identified by SupersomeTM CYP. In general, there is a good match between the two approaches, as no substantial differences were found in the predicted HKAF values and the BMDL10 values obtained by the two procedures. The predicted HKAF values for the 99th percentile obtained by the two approaches were slightly higher than the default uncertainty factor (UF) of 3.16, reflecting possible inadequate protection for extremely sensitive adult individuals by the default UF. Altogether, this chapter provided a proof-of-principle for assessing inter-individual variation in toxicokinetics of OP pesticides and its resulting RBC AChE inhibition, and the SupersomeTM CYP-based approach seems to be the preferred option for defining inter-individual differences in CYP-mediated kinetics.Chapter 6 provided an overview of the results and main findings of the present thesis, followed by an in-depth discussion of the results obtained, and the future perspectives. It was concluded that the present thesis generated proofs-of-principle for the use of NAM and especially PBK model-facilitated reverse dosimetry based on in vitro assays and in silico data, in combination with Monte Carlo simulations or the TEF methodology, to derive PODs and CSAF values for defining inter-ethnic, inter-species and inter-individual differences in toxicokinetics and the resulting RBC AChE inhibition following an OP pesticide acute single oral exposure. Altogether, the current work supports the application of such a NAM in regulatory safety decision making on compounds, and contributes to the implementation of the 3Rs (replacement, reduction, and refinement of experimental animal studies), thereby promoting the development of next generation risk assessment.&nbsp