Malathion (Diethyl[(dimethoxyphosphinothioyl)thio] succinate) is an organophosphorus (OP) insecticide. It is a widely used broad-spectrum insecticide to control a variety of insects that attack fruits, vegetables, ornamentals, and shrubs. Malathion products (Fyfanon® EW and Fyfanon® ULV Mosquito) are listed in Word Health Organization (WHO)’s essential vector control products used in public health programs. It is also used in household settings for e.g., to control fleas on pets, and in human personal care products to treat head lice (US EPA, 2024; Malathion-PHS.doc (cdc.gov), accessed in Feb 2024). The insecticidal action of malathion is rendered by malaoxon, an active metabolite, formed by P-450 catalyzed oxidative desulfuration. In target insects, malaoxon inhibits the enzyme acetylcholinesterase (AChE) resulting in the accumulation of acetylcholine (ACh) within synapses leading to over-stimulation of post-synaptic receptors, neurotoxicity, and eventually death (US EPA, 2024). However, in mammals including humans, this oxidative pathway leading to malaoxon constitutes a minor route. The predominant biotransformation accountable for low vertebrate toxicity is detoxification of malathion by esterases via carboxylesterase to form mono- and di-acid metabolites (MMCA and MDCA), which are further converted into phosphoric derivatives (Buratti and Testai, 2005; Chen et al., 2013; Johnson et al., 1952). A schematic of malathion biotransformation in mammals is presented in Fig. 1.

AChE inhibition in red blood cells (RBCs) seems to be the most sensitive toxicology endpoint for various species, life stages, different routes, and duration of exposure to malathion. As such, human health risk assessment of malathion has been based on RBC AChE inhibition (Bouchard et al., 2003; US EPA, 2024). A physiologically based pharmacokinetic-pharmacodynamic (PBPK-PD) model is considered a better approach to derive human points of departure (PODs) for malathion based on benchmark response (BMR) of 10 % AChE inhibition. A toxicokinetic (TK) model was previously published and provide estimates of urinary excretion of malathion metabolites (Bouchard et al., 2003). The malathion PBPK-PD model simulates plasma and red blood cell (RBC) concentrations of malathion and malaoxon and inhibition of AChE by malaoxon. Such PBPK-PD models have been developed for the risk assessments for several OPs including chlorpyrifos, diazinon and dimethoate (Poet et al., 2004; Reiss et al., 2023; Timchalk et al., 2002; US EPA, 2006). As part of development of a similar PBPK-PD model for malathion, data collection was undertaken to measure PK and PD parameters for malathion and its metabolites (Reiss and Loccisano, 2021; US EPA, 2024). The PBPK-PD model also relies on other metabolism and inhibition kinetic data in the literature. The focus of this paper is to describe in vitro approaches utilized to develop key input PK parameters for developing a PBPK-PD model for malathion. Specifically, plasma concentrations were measured in vivo following dosing to rats, and in vitro metabolism rates for malathion were measured in liver microsomes from rat and human sources. The rat in vivo plasma concentrations will be used to validate the PBPK-PK model and the in vitro metabolism rates will be used to estimate clearance rates and to extend the validated rat model to humans. Unlike previous reports, in our study malaoxon kinetics in liver microsomes were determined directly via an LC/MS method. The description of the malathion PBPK-PD model will be reported separately.

Efforts on generating kinetic parameters for development of PBPK-PD models for other OPs have previously been reported, with majority of the studies focused on chlorpyrifos (Ellison et al., 2011; Smith et al., 2011; Timchalk, 2010; Timchalk et al., 2007b). A few studies are available on in vitro metabolism, PK and PBPK/PD assessment of OPs diazinon and methyl parathion (Kramer et al., 2011; Poet et al., 2004; Poet et al., 2003). An evaluation of comparative PK of metabolites of OPs has also been reported in the literature. (Forsberg et al., 2011; Timchalk et al., 2007a). We recently reported in vitro enzymatic kinetics of dimethoate and its oxon metabolite omethoate in rat and human, that were used in the development of PBPK-PD model for dimethoate (Nallani et al., 2023; Reiss et al., 2023).

Studies available in the literature on systematic evaluation of kinetics and metabolism of malathion and its active metabolite malaoxon are limited. Bouchard et al. (2003) proposed a toxicokinetic model to predict the extent of formation of malathion and its metabolites: mono- and dicarboxylic acids (MMCA and MDCA) and phosphoric derivates (dimethyl dithiophosphate, DMDTP, dimethyl thiophosphate (DMTP) and dimethyl phosphate (DMP) in human biological fluids for their potential use as biomarkers of exposure to the insecticide (Bouchard et al., 2003). These metabolites were also detected in human urine following oral dosing with malathion (Aston, 2000). A few studies focused on characterization of distinct types of carboxyl esterases responsible for detoxification of malathion and others characterized different forms of cytochrome P450 (CYP 450) isoforms that lead to bioactivation and toxicity (Buratti et al., 2004; Buratti and Testai, 2005).

In a preliminary feasibility experiment (not presented here), malathion rapidly metabolized to its carboxy metabolite(s) with no detectable levels of the parent, so it was not practical to establish degradation kinetics via the substrate (parent) depletion approach. Therefore, in this study, malathion in vitro clearance was studied in terms of formation kinetics of its metabolites, MMCA and malaoxon. Information on malaoxon formation kinetics was important as this active metabolite is responsible for AChE inhibition. In our study, formation of malaoxon in liver microsomes was facilitated via inhibition of esterase pathway using isomalathion (Buratti and Testai, 2005).