Bioaccumulation Screening of Neutral Hydrophobic Organic Chemicals in Air-Breathing Organisms Using In Vitro Rat Liver S9 Biotransformation Assays

Abstract

To advance methods for bioaccumulation assessment of organic substances in air‐breathing organisms, the present study developed an in vitro approach for screening neutral hydrophobic organic substances for their bioaccumulation potential in air‐breathing organisms consisting of (1) depletion assays for chemicals in rat liver S9 subcellular fractions, (2) in vitro–in vivo extrapolation, and (3) whole‐organism bioaccumulation modeling to assess the biomagnification potential of neutral organic substances in the rat. Testing of the in vitro method on 14 test chemicals of potentially biomagnifying substances showed that the bioassays could be conducted with a high level of reproducibility and that in vitro–derived elimination rate constants were in good agreement with in vivo–determined elimination rate constants in the rat. Exploring the potential of the in vitro approach for screening organic chemicals for bioaccumulation in air‐breathing organisms indicated that chemical substances that exhibit a depletion rate constant in the S9 in vitro bioassay ≥0.3 h−1 are not expected to biomagnify in rats independent of their octanol–water partitioning coefficient (KOW) or octanol–air partitioning coefficient (KOA). The high level of reproducibility achieved in the test, combined with the good agreement between in vitro–derived and in vivo–determined depuration rates, suggests that the in vitro approach in combination with a KOA‐ and KOW‐based screening approach has good potential for screening chemicals in commerce for their bioaccumulation potential in air‐breathing organisms in a cost‐effective and expedient manner, especially if the bioassay can be automated. Environ Toxicol Chem 2022;41:2565–2579. © 2022 SETAC

Methods for assessing the bioaccumulation of hydrocarbons and related substances in terrestrial organisms: A critical review

“…The major uptake and elimination processes of chemicals in mammals that are often included in the model are respiratory and dietary uptake, respiratory elimination, fecal elimination, urinary elimination, biliary elimination, biotransformation, lactation, and growth dilution: $$$\frac{{{dC}}_{\mathrm{organism}}}{{dt}}=({k}_{\mathrm{respiratory}\unicode{x02007}\mathrm{uptake}}{\times C}_{{air}})+({k}_{\mathrm{dietary}\unicode{x02007}\mathrm{uptake}}\times {C}_{\mathrm{diet}})-({k}_{\mathrm{respiratory}\unicode{x02007}\mathrm{elimination}}+{k}_{\mathrm{fecal}\unicode{x02007}\mathrm{elimination}}+{k}_{\mathrm{urinary}\unicode{x02007}\mathrm{elimination}}+{k}_{\mathrm{biliary}\unicode{x02007}\mathrm{elimination}}+{k}_{\mathrm{met}}+{k}_{\mathrm{lactation}}+{k}_{\mathrm{growth}})\times {C}_{\mathrm{organism}},$$$where C organism , C air , and C diet are the concentrations of the chemical in the organism (g/kg organism), air (g L air −1 ), and diet (g kg food −1 ), respectively; k respiratory uptake and k dietary uptake are the clearance rates (L‐air kg‐organism −1 day –1 and kg‐food kg‐organism −1 day −1 ) describing the kinetics of chemical uptake from the air and the diet, respectively; and k respiratory elimination , k fecal elimination , k urinary elimination , k biliary elimination , k met , k lactation , and k growth are the rate constants (day –1 ) describing the first‐order kinetics of chemical elimination via respiratory elimination, fecal elimination, urinary elimination, biliary elimination, biotransformation, lactation, and growth dilution, respectively. Models for deriving k respiratory uptake , k dietary uptake , k respiratory elimination , k fecal elimination , k urinary elimination , k biliary elimination , and k lactation are described in Lee et al (2022). The steady‐state biomagnification factor (BMF C ) of the parent substance as a result of dietary uptake is …”

“…While these guidelines are developed for bioaccumulation assessments in fish, they provide useful information for conducting in vitro assays in mammalian hepatocytes and liver S9. Guidelines for conducting liver S9 in vitro assays in rats have been developed by Lee et al (2022) and involve a two‐tiered approach, where preliminary experiments are carried out to optimize experimental conditions for a final experiment that determines the depletion rate constant that is used for estimating the in vivo biotransformation rate. The preliminary experiments aim to confirm (i) the enzymatic activity of the hepatocytes or liver S9 considering both phase I and phase II enzymes through enzyme assays; (ii) method detection limits to ensure that experimental concentrations allow depletion to be measured; (iii) the effect of the concentrations of the parent substance on the enzymatic reaction rate constant; and (iv) the appropriate spacing of sampling times throughout the duration of the incubation period to minimize measurement error of the depletion rate constant.…”

“…Testing of the in vitro bioaccumulation assessment approach in rats has been conducted by Lee et al (2022) (Figure 3). This involved conducting in vitro biotransformation assays using rat liver S9, IVIVE as described above, and rat bioaccumulation modeling to derive depuration rate constants of the test chemicals in the rat, which were then compared to depuration rate constants derived from in vivo studies involving rats dosed to the same test chemicals.…”

Identification of Mid-Polar and Polar AhR Agonists in Cetaceans from Korean Coastal Waters: Application of Effect-Directed Analysis with Full-Scan Screening

“…2 This is because compounds with greater K OA values are less volatile and tend to partition into lipid-rich substances instead of being released into the atmosphere. 3 A recent report identified several polar aryl hydrocarbon receptor (AhR) agonists, such as canrenone, mepanipyrim, medroxyprogesterone, rutaecarpine, genistein, tretinoin, etofenprox, [10]-gingerol, and eupatilin, in livers of black-tailed gulls from South Korea. 4 These rater polar compounds demonstrated significant biomagnification potential in the marine food webs.…”

“…In the marine-mammalian food web, which includes air-breathing organisms, chemicals with lesser log K OW values (2.0–5.0) could be biomagnified due to greater log K OA values (>5.0) . This is because compounds with greater K OA values are less volatile and tend to partition into lipid-rich substances instead of being released into the atmosphere . A recent report identified several polar aryl hydrocarbon receptor (AhR) agonists, such as canrenone, mepanipyrim, medroxyprogesterone, rutaecarpine, genistein, tretinoin, etofenprox, [10]-gingerol, and eupatilin, in livers of black-tailed gulls from South Korea .…”