Interspecies scaling of toxicity reference values in human health versus ecological risk assessments: A critical review.

Risk assessments that focus on anthropogenic chemicals in environmental media-whether considering human health or ecological effects-often rely on toxicity data from experimentally studied species to estimate safe exposures for species that lack similar data. Current default extrapolation approaches used in both human health risk assessments and ecological risk assessments (ERAs) account for differences in body weight between the test organisms and the species of interest, but the two default approaches differ in important ways. Human health risk assessments currently employ a default based on body weight raised to the three-quarters power. Ecological risk assessments for wildlife (i.e., mammals and birds) are typically based directly on body weight, as measured in the test organism and receptor species. This review describes differences in the experimental data underlying these default practices and discusses the many factors that affect interspecies variability in chemical exposures. The interplay of these different factors can lead to substantial departures from default expectations. Alternative methodologies for conducting more accurate interspecies extrapolations in ERAs for wildlife are discussed, including tissue-based toxicity reference values, physiologically based toxicokinetic and/or toxicodynamic modeling, chemical read-across, and a system of categorical defaults based on route of exposure and toxic mode of action. Integr Environ Assess Manag 2024;20:749-764. © 2023 SETAC.

PBPK modeling to evaluate maximum tolerated doses: A case study with 3-chloroallyl alcohol.

Introduction: A physiologically based pharmacokinetic (PBPK) model for 3-chloroallyl alcohol (3-CAA) was developed and used to evaluate the design of assays for the in vivo genotoxicity of 3-CAA. Methods: Model development was supported by read across from a published PBPK model for ethanol. Read across was motivated by the expectation that 3-CAA, which like ethanol is a primary alcohol, is metabolized largely by hepatic alcohol dehydrogenases. The PBPK model was used to evaluate how two metrics of tissue dosimetry, maximum blood concentration (Cmax; mg/L) and area under the curve (AUC; mg-hr/L) vary with dose of 3-CAA and with dose route (oral gavage, drinking water). Results: The model predicted that oral gavage results in a 6-fold higher Cmax than the same dose administered in drinking water, but in similar AUCs. Predicted Cmax provided the best correlation with severe toxicity (e.g., lethality) from 3-CAA, consistent with the production of a reactive metabolite. Therefore, drinking water administration can achieve higher sustained concentration without severe toxicity in vivo. Discussion: This evaluation is significant because cytotoxicity is a potential confounder of mutagenicity testing. The PBPK model can be used to ensure that studies meet OECD and USEPA test guidelines and that the highest dose used is not associated with severe toxicity. In addition, PBPK modeling provides assurance of target tissue (e.g., bone marrow) exposure even in the absence of laboratory data, by defining the relationship between applied dose and target tissue dose based on accepted principles of pharmacokinetics, relevant physiology and biochemistry of the dosed animals, and chemical-specific information.

Relative contributions of endogenous and exogenous formaldehyde to formation of deoxyguanosine monoadducts and DNA-protein crosslink adducts of DNA in rat nasal mucosa.

Understanding the dose-response for formaldehyde-induced nasal cancer in rats is complicated by (1) the uneven distribution of inhaled formaldehyde across the interior surface of the nasal cavity and, (2) the presence of endogenous formaldehyde (endoF) in the nasal mucosa. In this work, we used computational fluid dynamics (CFD) modeling to predict flux of inhaled (exogenous) formaldehyde (exogF) from air into tissue at the specific locations where DNA adducts were measured. Experimental work has identified DNA-protein crosslink (DPX) adducts due to exogF and deoxyguanosine (DG) adducts due to both exogF and endoF. These adducts can be considered biomarkers of exposure for effects of endoF and exogF on DNA that may be part of the mechanism of tumor formation. We describe a computational model linking CFD-predicted flux of formaldehyde from air into tissue, and the intracellular production of endoF, with the formation of DPX and DG adducts. We assumed that, like exogF, endoF can produce DPX. The model accurately reproduces exogDPX, exogDG, and endoDG data after inhalation from 0.7 to 15 ppm. The dose-dependent concentrations of exogDPX and exogDG are predicted to exceed the concentrations of their endogenous counterparts at about 2 and 6 ppm exogF, respectively. At all concentrations examined, the concentrations of endoDPX and exogDPX were predicted to be at least 10-fold higher than that of their DG counterparts. The modeled dose-dependent concentrations of these adducts are suitable to be used together with data on the dose-dependence of cell proliferation to conduct quantitative modeling of formaldehyde-induced rat nasal carcinogenicity.

Incorporation of rapid association/dissociation processes in tissues into the monkey and human physiologically based pharmacokinetic models for manganese.

In earlier physiologically based pharmacokinetic (PBPK) models for manganese (Mn), the kinetics of transport of Mn into and out of tissues were primarily driven by slow rates of association and dissociation of Mn with tissue binding sites. However, Mn is known to show rapidly reversible binding in tissues. An updated Mn model for primates, following similar work with rats, was developed that included rapid association/dissociation processes with tissue Mn-binding sites, accumulation of free Mn in tissues after saturation of these Mn-binding sites and rapid rates of entry into tissues. This alternative structure successfully described Mn kinetics in tissues in monkeys exposed to Mn via various routes including oral, inhalation, and intraperitoneal, subcutaneous, or intravenous injection and whole-body kinetics and tissue levels in humans. An important contribution of this effort is showing that the extension of the rate constants for binding and cellular uptake established in the monkey were also able to describe kinetic data from humans. With a consistent model structure for monkeys and humans, there is less need to rely on cadaver data and whole-body tracer studies alone to calibrate a human model. The increased biological relevance of the Mn model structure and parameters provides greater confidence in applying the Mn PBPK models to risk assessment. This model is also well-suited to explicitly incorporate emerging information on the role of transporters in tissue disposition, intestinal uptake, and hepatobiliary excretion of Mn.

Considerations for Improving Metabolism Predictions for In Vitro to In Vivo Extrapolation.

High-throughput (HT) in vitro to in vivo extrapolation (IVIVE) is an integral component in new approach method (NAM)-based risk assessment paradigms, for rapidly translating in vitro toxicity assay results into the context of in vivo exposure. When coupled with rapid exposure predictions, HT-IVIVE supports the use of HT in vitro assays for risk-based chemical prioritization. However, the reliability of prioritization based on HT bioactivity data and HT-IVIVE can be limited as the domain of applicability of current HT-IVIVE is generally restricted to intrinsic clearance measured primarily in pharmaceutical compounds. Further, current approaches only consider parent chemical toxicity. These limitations occur because current state-of-the-art HT prediction tools for clearance and metabolite kinetics do not provide reliable data to support HT-IVIVE. This paper discusses current challenges in implementation of IVIVE for prioritization and risk assessment and recommends a path forward for addressing the most pressing needs and expanding the utility of IVIVE.

Read-across and new approach methodologies applied in a 10-step framework for cosmetics safety assessment - A case study with parabens.

Parabens are esters of para-hydroxybenzoic acid that have been used as preservatives in many types of products for decades including agrochemicals, pharmaceuticals, food and cosmetics. This illustrative case study with propylparaben (PP) demonstrates a 10-step read-across (RAX) framework in practice. It aims at establishing a proof-of-concept for the value added by new approach methodologies (NAMs) in read-across (RAX) for use in a next-generation risk assessment (NGRA) in order to assess consumer safety after exposure to PP-containing cosmetics. In addition to structural and physico-chemical properties, in silico information, toxicogenomics, in vitro toxicodynamic, toxicokinetic data from PBK models, and bioactivity data are used to provide evidence of the chemical and biological similarity of PP and analogues and to establish potency trends for observed effects in vitro. The chemical category under consideration is short (C1-C4) linear chain n-alkyl parabens: methylparaben, ethylparaben, propylparaben and butylparaben. The goal of this case study is to illustrate how a practical framework for RAX can be used to fill a hypothetical data gap for reproductive toxicity of the target chemical PP.

An in vitro approach to determine the human relevance of anti-spermatogenic effects of 4-methylmorpholine 4-oxide, monohydrate (NMMO) in rat reproductive toxicity studies.

Reduced sperm counts have been observed in male rats in an extended one generation reproductive toxicity study (EOGRTS, OECD 443) following repeated administration of 300 mg/kg/day N-Methylmorpholine N-oxide (NMMO). However, no adverse effects on reproductive organs have been reported in studies conducted with NMMO, and the mode of action (MOA) for the effects of NMMO on spermatogenesis is unknown, which complicates the interpretation of these data for human risk assessment. Here, a New Approach Method (NAM) strategy was used to evaluate NMMO MOA and compare interspecies susceptibility for anti-spermatogenic effects using organotypic in vitro assays combined with in vitro metabolism and in vitro to in vivo extrapolation (IVIVE) biokinetic modeling to compare predicted oral equivalent doses (OEDs) in human and rat. Dose-response data were collected in isolated germ cells and in an ex vivo seminiferous tubule model that recapitulates the interaction between the somatic environment and differentiating germ cells to account for potential direct and indirect effects on germ cells. With regard to direct spermatogenic effects, the human isolated germ cell model showed no toxicity at doses ≤300 µM (OED ≤ 86 mg/kg/day). With regard to indirect effects, the rat ex vivo model demonstrated dose-dependent decreases in secondary spermatocyte populations at OEDs ≥89 mg/kg/day, and reduced expression of RNAs specific to several stages of spermatogenesis (spermatogonia, pachytene spermatocytes, round spermatids) at OED = 267 mg/kg/day, consistent with in vivo observations. In contrast, the monkey ex vivo model did not show dose-dependent decreases in these same RNAs, and often demonstrated increased trends instead. These studies demonstrate clear quantitative and qualitative differences in the rat and primate response to NMMO. Furthermore, effects observed in the rat in vitro culture were not observed in the monkey at concentrations equivalent to in vivo doses of up to 1376 mg/kg/day, which is higher than the in vivo dose limit in the EOGRT study, indicating that the isolated findings on spermatogenesis in the rat studies are not likely to be relevant to humans.

Time-dependent genomic response in primary human uroepithelial cells exposed to arsenite for up to 60 days.

Evidence from both in vivo and in vitro studies suggests that gene expression changes from long-term exposure to arsenite evolve markedly over time, including reversals in the direction of expression change in key regulatory genes. In this study, human uroepithelial cells from the ureter segments of 4 kidney-donors were continuously treated in culture with arsenite at concentrations of 0.1 or 1 µM for 60 days. Gene expression at 10, 20, 30, 40, and 60 days was determined using Affymetrix human genome microarrays and signal pathway analysis was performed using GeneGo Metacore. Arsenic treated cells continued to proliferate for the full 60-day period, whereas untreated cells ceased proliferating after approximately 30 days. A peak in the number of gene changes in the treated cells compared to untreated controls was observed between 30 and 40 days of exposure, with substantially fewer changes at 10 and 60 days, suggesting remodeling of the cells over time. Consistent with this possibility, the direction of expression change for a number of key genes was reversed between 20 and 30 days, including CFOS and MDM2. While the progression of gene changes was different for each subject, a common pattern was observed in arsenic treated cells over time, with early upregulation of oxidative stress responses (HMOX1, NQ01, TXN, TXNRD1) and down-regulation of immune/inflammatory responses (IKKα). At around 30 days, there was a transition to increased inflammatory and proliferative signaling (AKT, CFOS), evidence of epithelial-to-mesenchymal transition (EMT), and alterations in DNA damage responses (MDM2, ATM). A common element in the changing response of cells to arsenite over time appears to involve up-regulation of MDM2 by inflammatory signaling (through AP-1 and NF-κB), leading to inhibition of P53 function.

Why is elevation of serum cholesterol associated with exposure to perfluoroalkyl substances (PFAS) in humans? A workshop report on potential mechanisms.

Serum concentrations of cholesterol are positively correlated with exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) in humans. The associated change in cholesterol is small across a broad range of exposure to PFOA and PFOS. Animal studies generally have not indicated a mechanism that would account for the association in humans. The extent to which the relationship is causal is an open question. Nonetheless, the association is of particular importance because increased serum cholesterol has been considered as an endpoint to derive a point of departure in at least one recent risk assessment. To gain insight into potential mechanisms for the association, both causal and non-causal, an expert workshop was held Oct 31 and Nov 1, 2019 to discuss relevant data and propose new studies. In this report, we summarize the relevant background data, the discussion among the attendees, and their recommendations for further research.

Using quantitative modeling tools to assess pharmacokinetic bias in epidemiological studies showing associations between biomarkers and health outcomes at low exposures.

Biomarkers of exposure can be measured at lower and lower levels due to advances in analytical chemistry. Using these sensitive methods, some epidemiology studies report associations between biomarkers and health outcomes at biomarker levels much below those associated with effects in animal studies. While some of these low exposure associations may arise from increased sensitivity of humans compared with animals or from species-specific responses, toxicology studies with drugs, commodity chemicals and consumer products have not generally indicated significantly greater sensitivity of humans compared with test animals for most health outcomes. In some cases, these associations may be indicative of pharmacokinetic (PK) bias, i.e., a situation where a confounding factor or the health outcome itself alters pharmacokinetic processes affecting biomarker levels. Quantitative assessment of PK bias combines PK modeling and statistical methods describing outcomes across large numbers of individuals in simulated populations. Here, we first provide background on the types of PK models that can be used for assessing biomarker levels in human population and then outline a process for considering PK bias in studies intended to assess associations between biomarkers and health outcomes at low levels of exposure. After providing this background, we work through published examples where these PK methods have been applied with several chemicals/chemical classes - polychlorinated biphenyls (PCBs), perfluoroalkyl substances (PFAS), polybrominated biphenyl ethers (PBDE) and phthalates - to assess the possibility of PK bias. Studies of the health effects of low levels of exposure will be improved by developing some confidence that PK bias did not play significant roles in the observed associations.

New framework for a non-animal approach adequately assures the safety of cosmetic ingredients - A case study on caffeine.

This case study on the model substance caffeine demonstrates the viability of a 10-step read-across (RAX) framework in practice. New approach methodologies (NAM), including RAX and physiologically-based kinetic (PBK) modelling were used to assess the consumer safety of caffeine. Appropriate animal systemic toxicity data were used from the most relevant RAX analogue while assuming that no suitable animal toxicity data were available for caffeine. Based on structural similarities, three primary metabolites of the target chemical caffeine (theophylline, theobromine and paraxanthine) were selected as its most relevant analogues, to estimate a point of departure in order to support a next generation risk assessment (NGRA). On the basis of the pivotal mode of action (MOA) of caffeine and other methylxanthines, theophylline appeared to be the most potent and suitable analogue. A worst-case aggregate exposure assessment determined consumer exposure to caffeine from different sources, such as cosmetics and food/drinks. Using a PBK model to estimate human blood concentrations following exposure to caffeine, an acceptable Margin of Internal Exposure (MOIE) of 27-fold was derived on the basis of a RAX using theophylline animal data, which suggests that the NGRA approach for caffeine is sufficiently conservative to protect human health.

Paraquat pharmacokinetics in primates and extrapolation to humans.

By extending our Paraquat (PQ) work to include primates we have implemented a modelling and simulation strategy that has enabled PQ pharmacokinetic data to be integrated into a single physiologically based pharmacokinetic (PBPK) model that enables more confident extrapolation to humans. Because available data suggested there might be differences in PQ kinetics between primates and non-primates, a radiolabelled study was conducted to characterize pharmacokinetics and excretion in Cynomolgus monkeys. Following single intravenous doses of 0.01 or 0.1 mg paraquat dichloride/kg bw, plasma PQ concentration-time profiles were dose-proportional. Excretion up to 48 h (predominantly urinary) was 82.9%, with ca. 10% remaining unexcreted. In vitro blood binding was similar across Cynomolgus monkeys, humans and rat. Our PBPK model for the rat, mouse and dog, employing a single set of PQ-specific parameters, was scaled to Cynomolgus monkeys and well represented the measured plasma concentration-time profiles over 14 days. Addition of a cartilage compartment to the model better captured the percent remaining in the monkeys at 48 h, whilst having negligible effect on model predictions for the other species. The PBPK model performed well for all four species, demonstrating there is little difference in PQ kinetics between non-primates and primates enabling a more confident extrapolation to humans. Scaling of the PBPK model to humans, with addition of a human-specific dermal submodel based on in vitro human dermal absorption data, provides a valuable tool that could be employed in defining internal dosimetry to complement human health risk assessments.

Integration of paraquat pharmacokinetic data across species using PBPK modelling.

Paraquat dichloride (PQ) is a non-selective herbicide which has been the subject of numerous toxicology studies over more than 50 years. This paper describes the development of a physiologically-based pharmacokinetic (PBPK) model of PQ kinetics for the rat, mouse and dog, firstly to aid the interpretation of studies in which no kinetic measurements were made, and secondly to enable the future extension of the model to humans. Existing pharmacokinetic data were used to develop a model for the rat and mouse. Simulations with this preliminary model were then used to identify key data gaps and to design a new blood binding study to reduce uncertainty in critical aspects of the model. The new data provided evidence to support the model structure, and its predictive performance was then assessed against dog and rat datasets not used in model development. The PQ-specific model parameters are the same for all three species, with only the physiological parameters varying between species. This consistency across species provides a strong basis for extrapolation to other species, as demonstrated here for the dog. The model enables a wide range of PQ data to be linked together to provide a broad understanding of PQ pharmacokinetics in rodents and the dog, showing that the key aspects of PQ kinetics in these species are understood and adequately encapsulated within the model.

Development of a rat and human PBPK model for bromate and estimation of human equivalent concentrations in drinking water.

A physiologically based pharmacokinetic (PBPK) model was developed to described uptake, disposition and clearance of bromate in the rat using published experimental data in rat. The rodent bromate model was extrapolated to human using species-specific physiological parameters and standard interspecies scaling of rate constants. The bromate model is kinetically linear (i.e. AUC and Cmax) across the range of drinking water concentrations used in the cancer bioassays (15 to 500 ppm). This is likely the result of the poor oral bioavailability of bromate due to high reduction rates in the intestinal tract. The bromate PBPK model was used to assess the human equivalent drinking water concentration (HEC) consistent with average plasma concentrations in the rodent bioassays. At drinking water concentrations <500 mg/L, the predicted HEC was two to three fold lower than the bioassay concentration and was dependent on the reported drinking water intake reported in the bioassay.

Kinetics of metabolism of deltamethrin and cis- and trans-permethrin in vitro. Studies using rat and human liver microsomes, isolated rat hepatocytes and rat liver cytosol.

The kinetics of metabolism of deltamethrin (DLM) and cis- and trans-permethrin (CPM and TPM) was studied in male Sprague-Dawley rat and human liver microsomes. DLM metabolism kinetics was also studied in isolated rat hepatocytes, liver microsomes and cytosol. Apparent intrinsic clearance (CLint) values for the metabolism of DLM, CPM and TPM by cytochrome P450 (CYP) and carboxylesterase (CES) enzymes in rat and human liver microsomes decreased with increasing microsomal protein concentration. However, when apparent CLint values were corrected for nonspecific binding to allow calculation of unbound (i.e., corrected) CLint values, the unbound values did not vary greatly with microsomal protein concentration. Unbound CLint values for metabolism of 0.05-1 µM DLM in rat liver microsomes (CYP and CES enzymes) and cytosol (CES enzymes) were not significantly different from rates of DLM metabolism in isolated rat hepatocytes. This study demonstrates that the nonspecific binding of these highly lipophilic compounds needs to be taken into account in order to obtain accurate estimates of rates of in vitro metabolism of these pyrethroids. While DLM is rapidly metabolised in vitro, the hepatocyte membrane does not appear to represent a barrier to the absorption and hence subsequent hepatic metabolism of this pyrethroid.

Development of a physiologically based pharmacokinetic model of diisononyl phthalate (DiNP) in pregnant rat and human.

A physiologically based pharmacokinetic (PBPK) model for di-isononyl phthalate (DiNP) was developed by adapting the existing models for di(2-ethylhexyl) phthalate (DEHP) and di-butylphthalate (DBP). Both pregnant rat and human time-course plasma and urine data were used to address the hydrolysis of DiNP in intestinal tract, plasma, and liver as well as hepatic oxidative metabolism and conjugation of the monoester and primary oxidative metabolites. Data in both rats and humans were available to inform the uptake and disposition of mono-isononyl phthalate (MiNP) as well as the three primary oxidative metabolites including hydroxy (7-OH)-, oxo (7-OXO)-, and carboxy (7-COX)-monoisononyl phthalate in plasma and urine. The DiNP model was reliable over a wide range of exposure levels in the pregnant rat as well as the two low exposure levels in humans including capturing the nonlinear behavior in the pregnant rat after repeated 750 mg/kg/day dosing. The presented DiNP PBPK model in pregnant rat and human, based upon an extensive kinetic dataset in both species, may provide a basis for assessing human equivalent exposures based upon either rodent or in vitro points of departure.

Differential lymphatic versus portal vein uptake of the synthetic pyrethroids deltamethrin and cis-permethrin in rats.

The objective of this study was to obtain data on pathways of absorption of the synthetic pyrethroids deltamethrin (DLM) and cis-permethrin (CPM) following oral administration to rats. Adult male Sprague-Dawley rats with cannulated mesenteric lymph ducts and hepatic portal veins were given single doses of either 5 mg/kg DLM or 60 mg/kg CPM via the duodenum and lymph and portal blood samples collected for up to 300 min. The pyrethroid dosing vehicles (5 mL/kg body weight) were either corn oil or glycerol formal. Levels of DLM and CPM in lymph and portal blood samples were determined by high-performance liquid chromatography-mass spectrometry-mass spectrometry. Over the time period studied, levels of both DLM and CPM following administration in either corn oil or glycerol formal were greater in lymph than in portal blood. Lymphatic uptake of both DLM and CPM was enhanced following dosing in glycerol formal than in corn oil. The results of this study suggest that after oral administration to rats, these two pyrethroids are predominantly absorbed via the lymphatic system rather than via portal blood. The data obtained in this study thus support a recently developed physiologically-based pharmacokinetic (PBPK) model to evaluate age-related differences in pyrethroid pharmacokinetics in the rat, where it was assumed that absorption of pyrethroids was predominantly via lymphatic uptake.

Physiologically Based Pharmacokinetic Modeling in Risk Assessment: Case Study With Pyrethroids.

The assessment of potentially sensitive populations is an important application of risk assessment. To address the concern for age-related sensitivity to pyrethroid insecticides, life-stage physiologically based pharmacokinetic (PBPK) modeling supported by in vitro to in vivo extrapolation was conducted to predict age-dependent changes in target tissue exposure to 8 pyrethroids. The purpose of this age-dependent dosimetry was to calculate a Data-derived Extrapolation Factor (DDEF) to address age-related pharmacokinetic differences for pyrethroids in humans. We developed a generic human PBPK model for pyrethroids based on our previously published rat model that was developed with in vivo rat data. The results demonstrated that the age-related differences in internal exposure to pyrethroids in the brain are largely determined by the differences in metabolic capacity and in physiology for pyrethroids between children and adults. The most important conclusion from our research is that, given an identical external exposure, the internal (target tissue) concentration is equal or lower in children than in adults in response to the same level of exposure to a pyrethroid. Our results show that, based on the use of the life-stage PBPK models with 8 pyrethroids, DDEF values are essentially close to 1, resulting in a DDEF for age-related pharmacokinetic differences of 1. For risk assessment purposes, this indicates that no additional adjustment factor is necessary to account for age-related pharmacokinetic differences for these pyrethroids.

Pharmacokinetic bias analysis of an association between clinical thyroid disease and two perfluoroalkyl substances.

Exposure to perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) has been associated with the occurrence of thyroid disease in some epidemiologic studies. We hypothesized that in a specific epidemiologic study based on the National Health and Nutrition Examination Survey, the association of clinical thyroid disease with serum concentration of PFOA and PFOS was due to reverse causality. Thyroid hormone affects glomerular filtration, which in turn affects excretion of PFOA and PFOS. We evaluated this by linking a model of thyroid disease status over the lifetime to a physiologically based pharmacokinetic model of PFOA and PFOS. Using Monte Carlo methods, we simulated the target study population and analyzed the data using multivariable logistic regression. The target and simulated populations were similar with respect to age, estimated glomerular filtration rate, serum concentrations of PFOA and PFOS, and prevalence of clinical thyroid disease. The analysis showed little or no evidence of bias from the hypothesized mechanism. The largest bias was for the fourth quartile of PFOA in females, with an odds ratio of 0.93 (95% CI, 0.90, 0.97). The reported odds ratio of clinical thyroid disease for this group was 1.63 (1.07, 2.47), and if it were corrected for the bias would have been 1.74 (1.14, 2.65). Our results suggest that little of the reported association in the target study was due to reverse causality.