Life-Stage-, Sex-, and Dose-Dependent Dietary Toxicokinetics and Relationship to Toxicity of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Rats: Implications for Toxicity Test Dose Selection, Design, and Interpretation

Abstract

Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating, <2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥1200 ppm (63mg/kg/day) for P1 males and between 200 and 400 ppm (14–27mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21–35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies.

An F1-Extended One-Generation Reproductive Toxicity Study in Crl:CD(SD) Rats With 2,4-Dichlorophenoxyacetic Acid

“…One advantage of this study design is that it promotes the use of TK data to set a kinetically derived maximum dose (KMD) compared with use of the traditional maximum tolerated dose (MTD) approach to define study dose selection (Saghir et al ., 2012, 2013). An MTD-based dose selection strategy based primarily on body weight and/or other evidence of toxicity would likely have resulted in selection of mid and high study doses higher than the threshold dose for onset of nonlinear TK in rats (Saghir et al , 2013).…”

“…In a dietary probe study conducted to characterize TK across life stages and support TK-derived dose selection for the 2,4-D EOGRTS (Saghir et al , 2013), P1 females exhibited nonlinear TK at ≤ 200 ppm (approximately 14mg/kg/day), whereas P1 male nonlinearity was evident at ≥ 800 ppm (approximately 41mg/kg/day). Nonlinear TK was seen in postnatal day (PND) 35 pups at 400 ppm (females; lowest dose tested in the probe study) and 800 ppm (males).…”

Weight-of-the-evidence evaluation of 2,4-D potential for interactions with the estrogen, androgen and thyroid pathways and steroidogenesis

“…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). Non-linear TK, in which metabolism or excretion pathways available at lower blood concentrations are partly or fully saturated with increasing dose, may be a clear confounder both in appropriately designing toxicological studies and evaluating results for hazard and human risk assessment.…”

“…The single exception to the guidelines for these assays was that it was considered appropriate to limit the high concentration in the first four EDSP in vitro assays to 100 lM, rather than the guideline-recommended 1 mM, because the 100 lM concentration was equivalent to serum concentrations at or slightly above the TSRC (slightly above the inflection point for non-linear TK) in rats dosed with 2,4-D in the diet in the EOGRT study (Marty et al 2010;Marty et al 2013;Saghir et al 2013). As noted previously, responses seen only in the non-linear TK range are not regarded as relevant to human risk assessment.…”

High-dose exposure to synthetic chemicals, hormones, or homeostatic substances in experimental animals or humans can induce artefactual pathology

“…PK dose modelers have posited that doses exceeding saturation levels are not biologically relevant and can lead to overestimates of risk in hazard and risk assessments. [11][12][13][14][15][16] Another proposed approach for dose selection involves the employment of "safety factors." This strategy first estimates the highest concentration of a chemical in the environment to which the general public is potentially exposed, or to the highest occupational exposure.…”