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Proposed metabolic pathway of 2,4-D in dog. In the rat, only 2,4-D was identified. Postulated intermediates are shown in brackets.
Source publication
1. There is a significant species difference in the toxicity of 2,4-dichlorophenoxyacetic acid (2,4-D). The oral no overall adverse effect level (NOAEL) for chronic toxicity of 2,4-D in rat is 5 mg kg(-1) day(-1) and in dog is 1 mg kg(-1) day(-1). The maximum tolerated dose (MTD) in rat is 150 and 75 kg(-1) day(-1) for male and females, respectivel...
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Citations
... Serine is a nonessential amino acid that is the precursor for cysteine, selenocysteine, tryptophan, glycine, and phospholipids, and plays a role in the biosynthesis of purines and pyrimidines (Reitzer, 2009). Serine conjugates with xenobiotics (Steventon and Hutt, 2002;Ravenzwaay et al., 2003;Aloysius et al., 2008) and endogenous serine conjugates with lipids such as N-arachidonoyl-serine (Milman et al., 2006) and N-stearoyl serine (Tan et al., 2010) have been reported in animal species, but the formation of serine conjugate metabolites of xenobiotics in humans seems to be rare. Nevertheless, additional research is needed to better understand the formation of M443 and the relevance of emvododstat metabolites to the efficacy and safety of emvododstat in humans. ...
Emvododstat is a potent inhibitor of dihydroorotate dehydrogenase and is now in clinical development for the treatment of COVID-19 and acute myeloid leukemia. Since the metabolism and pharmacokinetics of emvododstat in humans is time‑dependent, a repeat dose study design using a combination of microtracer radioactivity and high radioactivity doses was employed to evaluate the metabolism and excretion of emvododstat near steady state. Seven healthy male subjects each received 16 mg/0.3 µCi 14C-emvododstat daily oral doses for 6 days followed by a 16 mg/100 µCi high radioactivity oral dose on Day 7. Following the last 16 mg/0.3 µCi 14C‑emvododstat dose on Day 6, total radioactivity in plasma peaked at 6 h post-dose. Following a high radioactivity oral dose (16 mg/100 µCi) of 14C-emvododstat on Day 7, both whole blood and plasma radioactivity peaked at 6 h, rapidly declined from 6 h to 36 h post-dose, and decreased slowly thereafter with measurable radioactivity at 240 h post-dose. The mean cumulative recovery of the administered dose was 6.0% in urine and 19.9% in feces by 240 h post-dose, and the mean extrapolated recovery to infinity was 37.3% in urine and 56.6% in feces. Similar metabolite profiles were observed after repeat daily microtracer radioactivity oral dosing on Day 6 and after a high radioactivity oral dose on Day 7. Emvododstat was the most abundant circulating component, M443 and O-desmethyl emvododstat glucuronide were the major circulating metabolites; M474 was the most abundant metabolite in urine, while O‑desmethyl emvododstat was the most abundant metabolite in feces. Significance Statement This study provides a complete set of the absorption, metabolism and excretion data of emvododstat, a potent inhibitor of dihydroorotate dehydrogenase, at close to steady state in healthy human subjects. Resolution of challenges due to slow metabolism and elimination of a lipophilic compound highlighted in this study can be achieved by repeat daily microtracer radioactivity oral dosing followed by a high radioactivity oral dosing at therapeutically relevant doses.
... 2,4-Dichlorophenoxyacetic acid is also reported to be of low metabolic reactivity in humans. 36 Several features were left after the filtering steps for bifenox, but these had the same retention time as that of the parent pesticide. These features are likely not annotated adducts and in-source fragments rather than metabolites. ...
There is a current need to monitor human exposureto a large number of pesticides and other chemicals of emergingconcern (CECs). This requires screening analysis with highconfidence for these compounds and their metabolites in complexmatrices, which is hampered by the fact that no reference standardsare available for most metabolites. We address this challenge by ahigh-throughput workflow based on incubation of pesticides (orother CECs) with human liver S9, followed by solid-phaseextraction, liquid chromatography−high-resolution mass spectrom-etry (LC−HRMS) analysis, and automated data processing togenerate a database (retention time, precursorm/z, and MS2spectral library) for the annotation in human samples. Themetabolite prioritization consists of statistical comparisons andmass defect andm/zrangefiltering to obtain a subset of probable phase I metabolites, for which molecular formulas and likelymetabolic transformation are retrieved. We tested the workflow on 22 pesticides, for which we could determine 91 metabolitemolecular formulas which are only partly covered by the literature and/or predicted byin silicometabolization. Our workflow allowsfor an efficient generation of metabolite reference information, which can be used directly for annotating LC−HRMS data fromhuman samples. A full structure elucidation of individual metabolites can be limited to those being actually present in humansamples.
... In the present study, the confirmed prothiofos metabolites, other than 3,4,5-trihydroxy-6-(2,4-dichlorophenoxy) oxane-2-carboxylic acid, were also reported to be the metabolites of 2-(2,4-dichlorophenoxy) acetic acid (2,4-D), a phenoxy herbicide. [36][37][38] Although the possibility that the parent compound was 2,4-D was less likely (see Supporting Information for this confirmation), another possibility is that the parent was 2,4-dichlorophenol. The World Health Organization reported the detection of 2,4-dichlorophenol from tap water in the chlorination process. ...
Objectives
The comprehensive detection of environmental chemicals in biospecimens, an indispensable task in exposome research, is advancing. This study aimed to develop an exposomic approach to identify urinary metabolites of organophosphate (OP) pesticides, specifically cadusafos and prothiofos metabolites, as an example chemical group, using an original metabolome dataset generated from animal experiments.
Methods
Urine samples from 73 university students were analyzed using liquid chromatography–high‐resolution mass spectrometry. The metabolome data, including the exact masses, retention time (tR), and tandem mass spectra obtained from the human samples, were compared with the existing reference databases and with our original metabolome dataset for cadusafos and prothiofos, which was produced from mice to whom two doses of these OPs were orally administered.
Results
Using the existing databases, one chromatographic peak was annotated as 2,4‐dichlorophenol, which could be a prothiofos metabolite. Using our original dataset, one peak was annotated as a putative cadusafos metabolite and three peaks as putative prothiofos metabolites. Of these, all three peaks suggestive of prothiofos metabolites, 2,4‐dichlorophenol, 3,4,5‐trihydroxy‐6‐(2,4‐dichlorophenoxy) oxane‐2‐carboxylic acid, and (2,4‐dichlorophenyl) hydrogen sulfate were confirmed as authentic compounds by comparing their peak data with both the original dataset and peak data of the standard reagents. The putative cadusafos metabolite was identified as a level C compound (metabolite candidate with limited plausibility).
Conclusions
Our developed method successfully identified prothiofos metabolites that are usually not a target of biomonitoring studies. Our approach is extensively applicable to various environmental contaminants beyond OP pesticides.
... The exact form of conjugated 2,4-D was not identified in the urine of children. b-glucuronidase deconjugated 2,4-D in this study may not represent all the conjugated forms of 2,4-D in urine, as glycine and taurine conjugates of 2,4-D may exist (Griffin et al., 1997;van Ravenzwaay et al., 2003). Further studies are needed to identify different forms of 2,4-D conjugates in humans exposed to environmentally relevant doses. ...
Herbicide 2,4-Dichlorophenoxyacetic acid (2,4-D) and its analogues are widely used in agriculture. Although the occurrence of 2,4-D in urine has been widely reported in North America, it has scarcely been investigated in China, especially in young children. In addition, both in vivo and in vitro studies have shown that high-level 2,4-D exposure is associated with oxidative stress, but their association in a general sensitive population has rarely been studied. In this study, 2,4-D and its analogues were measured in 417 urine samples collected from 139 children aged 0–7 during the non-peak season of pesticide application in Wuhan, central China, and Shenzhen, south China. Each of them provided three samples in three consecutive days. An oxidative stress biomarker, 8-hydroxy-2-deoxyguanosine (8-OHdG), was also measured. The geometric mean (GM) of unconjugated urinary 2,4-D concentration was 0.10 μg/L (corrected by urinary specific gravity, SG-corrected). After β-glucuronidase hydrolysis, the GM of SG-corrected urinary 2,4-D was 0.15 μg/L, and the detection frequency was 100%. Moderate inter-day reproducibility was found within individuals, with an intraclass correlation coefficient of 0.68 for SG-corrected urinary deconjugated 2,4-D. The GM of estimated daily intake of 2,4-D was 6.05 ng/kg-bw/day. A significant positive correlation was found between urinary 2,4-D and 8-OHdG, whereas no association was found after SG-correction. This is the first study to characterize the occurrence of urinary 2,4-D, its inter-day reliability, and its association with urinary 8-OHdG in young children from China.
... human versus rat model) offers insight regarding temporal and concentration constraints of detecting 2,4-D in various biological matrices. Extensive pharmacokinetic (Timchalk, 2004;Sauerhoff et al., 1977) and metabolomic (Van Ravenzwaay et al., 2003) assessments have been conducted, and PBPK models for 2,4-D (Kim et al., 1994;Durkin et al., 2004) have been developed, but an update of current models that incorporate salivary clearance will allow for quantitative interpretation of non-invasive salivary biomonitoring. These types of quantitative models could also promote development and application of field deployable 2,4-D sensors (Wang et al., 2017) and allow for rapid exposure assessment, particularly among those exposed occupationally. ...
The objective of this study was to evaluate the potential for non-invasive biomonitoring of 2,4-dichlorophenoxyacetic acid (2,4-D) in saliva. Using an in vitro rat salivary gland epithelial cell (SGEC) system, a collection of experiments investigating chemical protein binding, temporal and directional transport, as well as competitive transport with para-aminohippuric acid (PAH), a substrate for renal organic anion transporters, was conducted to identify cellular transport parameters required to computationally model salivary transport of 2,4-D. Additionally, a physiological protein gradient was implemented to mimic physiologically relevant concentrations of protein in rat plasma and saliva, and under these conditions the transfer of 2,4-D was markedly slower, driven by increased protein binding (i.e. reduced free 2,4-D species available to cross salivary barrier). The rate of transfer was directly proportional to the amount of unbound 2,4-D and demonstrated no indication of active transport. An in vivo assessment of 2,4-D exposure in rats revealed a non-linear protein binding in plasma, indicating saturated protein binding and increased levels of unbound 2,4-D species at higher doses. A strong correlation between 2,4-D concentrations in saliva and unbound 2,4-D in plasma was observed (Pearson correlation coefficient = 0.95). Saliva:plasma 2,4-D ratios measured in vivo (0.0079) were consistent within the linear protein binding range and expected 2,4-D levels from occupational exposures, but significantly different than ratios measured in vitro (physiological conditions) (0.034), possibly due to 2,4-D concentrations in saliva not being at equilibrium with 2,4-D concentrations in blood, as well as physiological features absent in in vitro settings (e.g. blood flow). We demonstrated that 2,4-D is consistently transported into saliva using both in vitro and in vivo models, making 2,4-D a potential candidate for human non-invasive salivary biomonitoring. Further work is needed to understand whether current sensor limits of detection are sufficient to measure occupationally relevant exposures.
... Some HPLC methods have been established for the determination of 2,4-D residues in vegetables, fruits [10] and environmental samples [11,12], but only a few studies have been carried out for biological samples [13,14]. There is only limited data with respect to 2,4-D pharmacokinetic properties [15]. According to Chinese Standard Bureau (GB) 15670 "Toxicological Test Methods of Pesticides for Registration", rat species share many of the pharmacokinetics properties of 2,4-D with humans. ...
2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlorophenoxy herbicide used worldwide. We describe a high-performance liquid chromatography (HPLC) method with UV detection for the determination of 2,4-D in female and male rat serum. This allows to observe the change of serum 2,4-D concentration in rats with time and its pharmacokinetics characteristics with a simple, rapid, optimized and validated method. The serum samples are pretreated and introduced into the HPLC system. The analytes are separated in a XDB-C18 column with a mobile phase of acetonitrile (solvent A) and 0.02 M ammonium acetate (containing 0.1% formic acid) (solvent B) using a gradient elution at a flow rate of 1.0 mL/min. The wavelength for UV detection was set at 230 nm. Calibration curve for 2,4-D was constructed over a range of 0.1–400 mg/L. The method was successfully applied to study the pharmacokinetics of 2,4-D in rats in this study. After oral administration of 300 mg/kg and 60 mg/kg 2,4-D, the mean Cmax values were 601.9 and 218.4 mg/L, the AUC0→∞ values were 23,722 and 4,127 mg×h/L and the clearance (Cl) were 1.10 and 0.02 L/(h×kg), respectively. The developed method was found to be specific, precise, reproducible and rapid.
... The WoE reflects an assessment of whether results might signal a specific endocrine pathway interaction, the relative weight or rank placed on that parameter for specifically and sensitively flagging a potential interaction, and whether a finding (if any) was made only at a systemically toxic or otherwise excessive dose, as discussed above. The WoE tables developed for each pathway provide a visual representation that assists in identifying patterns of findings within or across studies that may indicate a potential endocrine pathway interaction; the Impact of toxicokinetic data for 2,4-D on study design, data interpretation and risk assessment Extensive research has been done to characterize 2,4-D TK. 2,4-D clearly exhibits species-, dose-and sex-dependent nonlinear TK in animal test species (Gorzinski et al. 1987;Van Ravenswaay et al. 2003;Timchalk 2004;Saghir et al. 2006;. The non-linear TK is directly and primarily attributable to high-dose saturation of a renal anion transporter, OAT-1, that is responsible for rapid renal clearance of 2,4-D (Hasegawa et al. 2003;Saghir et al. 2013). ...
... In addition, high-dose administration of 2,4-D to mice and rabbits results in increased distribution to and/or retention in brain (Kim et al. 1988). The overall TK data suggest that potential central nervous system (CNS) effects are secondary to two sequential and mechanistically related dose-disproportionate events resulting in increases in 2,4-D brain concentrations: (1) initial saturation of OAT-1 renal clearance leading to dose-disproportionate elevation in plasma 2,4-D plasma concentration allowing for increased organ distribution of non-plasma-protein bound 2,4-D (Timchalk 2004;van Ravenswaay et al. 2003); and (2) followed by augmented nonlinear increases in brain concentration associated with highdose specific saturation of OAT-1 clearance from brain (Kim et al. 1988). Although the quantitative contribution of each of these saturation events to altered distribution of 2,4-D within the brain is unknown, such alterations to and within an endocrine-modulatory organ such as brain have the potential to initiate high-dose specific secondary modes of action. ...
... "Knowledge of the dose and species-dependent pharmacokinetic behavior of 2,4-D significantly enhances the understanding of the relevance of toxicity findings of 2,4-D in rodents, and particularly in dogs, to predicting potential human health risks. Once absorbed, 2,4-D is rapidly and completely excreted in urine by both rats and humans, but not dogs (Van Ravenswaay et al. 2003;Timchalk, 2004). In rodents and humans, renal excretion of 2,4-D is facilitated by a saturable organic anion active transporter located in the renal tubules (Timchalk, 2004). ...
A comprehensive weight-of-the-evidence evaluation of 2,4-dichlorophenoxyacetic acid (2,4-D) was conducted for potential interactions with the estrogen, androgen and thyroid pathways and with steroidogenesis. This assessment was based on an extensive database of high quality in vitro, in vivo ecotoxicological and in vivo mammalian toxicological studies. Epidemiological studies were also considered. Toxicokinetic data provided the basis for determining rational cutoffs above which exposures were considered irrelevant to humans based on exceeding thresholds for saturation of renal clearance (TSRC); extensive human exposure and biomonitoring data support that these boundaries far exceed human exposures and provide ample margins of exposure. 2,4-D showed no evidence of interacting with the estrogen or androgen pathways. 2,4-D interacts with the thyroid axis in rats through displacement of thyroxine from plasma binding sites only at high doses exceeding the TSRC in mammals. 2,4-D effects on steroidogenesis parameters are likely related to high-dose specific systemic toxicity at doses exceeding the TSRC and are not likely to be endocrine mediated. No studies, including high quality studies in the published literature, predict significant endocrine-related toxicity or functional decrements in any species at environmentally relevant concentrations, or, in mammals, at doses below the TSRC that are relevant for human hazard and risk assessment. Overall, there is no basis for concern regarding potential interactions of 2,4-D with endocrine pathways or axes (estrogen, androgen, steroidogenesis or thyroid), and thus 2,4-D is unlikely to pose a threat from endocrine disruption to wildlife or humans under conditions of real-world exposures.
... The ether-soluble wild radish metabolite could be an amino acid conjugate, but these are usually resistant to acid hydrolysis (Chkanikov et al., 1976), which was not the case in this study. An in vitro glutathione S-transferase assay using wild radish leaf extract with [ 14 C]2,4-D as a substrate gave no reaction products (data not shown), suggesting that glutathione is not conjugated to 2,4-D in wild radish as is potentially the case in potato (Peixoto et al., 2008) and dog (van Ravenzwaay et al., 2003). Characterization of the 2,4-D metabolites produced by wild radish, initiated in this study but requiring more work, is necessary so that any future populations evolving metabolic 2,4-D resistance and displaying 'new' metabolites can be rapidly identified. ...
Resistance to auxinic herbicides is increasing in a range of dicotyledonous weed species, but in most cases the biochemical
mechanism of resistance is unknown. Using 14C-labelled herbicide, the mechanism of resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) in two wild radish (Raphanus raphanistrum L.) populations was identified as an inability to translocate 2,4-D out of the treated leaf. Although 2,4-D was metabolized
in wild radish, and in a different manner to the well-characterized crop species wheat and bean, there was no difference in
metabolism between the susceptible and resistant populations. Reduced translocation of 2,4-D in the latter was also not due
to sequestration of the herbicide, or to reduced uptake by the leaf epidermis or mesophyll cells. Application of auxin efflux
or ABCB transporter inhibitors to 2,4-D-susceptible plants caused a mimicking of the reduced-translocation resistance phenotype,
suggesting that 2,4-D resistance in the populations under investigation could be due to an alteration in the activity of a
plasma membrane ABCB-type auxin transporter responsible for facilitating long-distance transport of 2,4-D.
... Subchronic and Chronic Toxicity (Including Carcinogenicity). Once absorbed, 2,4-D is rapidly and completely excreted in urine (Timchalk 2004;Van Ravenzwaay et al. 2003). Therefore, it does not accumulate in animal tissues. ...
Since its discovery and initial commercialization in the 1940s, 2,4-D has been an important tool for weed control in a wide variety of crop and noncrop uses. Work studying its chemistry, physiology, mode of action, toxicology, environmental behavior, and efficacy has not only helped elucidate the characteristics of 2,4-D but also provided basic methods that have been used to investigate the properties of hundreds of herbicides that followed it. Much of the information published by researchers over 60 yr ago is still pertinent to understanding the performance of 2,4-D today. Further, new studies continue to be published, especially regarding the mechanisms of 2,4-D action at the molecular level. New uses for 2,4-D, sometimes enabled by biotechnology, continue to be developed. This review strives to provide an overall understanding of 2,4-D activity in plants, plant sensitivity to 2,4-D, toxicological impacts, and current and future uses.
... There was some initial concern over a nonstatistically significant increase in male rat astrocytomas at 45 mg/kg/d in the earlier rat study. However, a subsequent study conducted with doses of 75 and 150 mg/kg/d [42], and the nonlinear toxicokinetics of 2,4-D because of saturation of renal clearance [3,44,45] indicate that this was a spurious finding bearing no relationship to treatment [43]. ...
Despite evidence from experimental studies indicating that the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D), is not carcinogenic, several epidemiology studies have evaluated links between 2,4-D and cancer. Some suggest that 2,4-D is associated with non-Hodgkin's lymphoma (NHL), gastric cancer, and prostate cancer, but results have been inconsistent. We conducted meta-analyses to evaluate the weight of epidemiology evidence for these cancers.
We identified articles from PubMed, Scopus, and TOXLINE databases and reference lists of review articles. We evaluated study quality and calculated summary risk estimates using random effects models. We conducted subgroup and sensitivity analyses when possible.
We identified nine NHL, three gastric cancer, and two prostate cancer studies for inclusion in our meta-analyses. We found that 2,4-D was not associated with NHL (relative risk [RR] = 0.97, 95% confidence interval [CI] = 0.77-1.22, I(2) = 28.8%, Pheterogeneity = .19), and this result was generally robust to subgroup and sensitivity analyses. 2,4-D was not associated with gastric (RR = 1.14, 95% CI = 0.62-2.10, I(2) = 54.9%, Pheterogeneity = .11) or prostate cancer (RR = 1.32, 95% CI = 0.37-4.69, I(2) 87.0%, Pheterogeneity = .01).
The epidemiology evidence does not support an association between 2,4-D and NHL, gastric cancer, or prostate cancer risk.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
