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Interpretation of untargeted metabolomics data from both in vivo and physiologically relevant in vitro model systems continues to be a significant challenge for toxicology research. Potency-based modeling of toxicological responses has served as a pillar of interpretive context and translation of testing data. In this study, we leverage the resolving power of concentration-response modeling through benchmark concentration (BMC) analysis to interpret untargeted metabolomics data from differentiated cultures of HepaRG cells exposed to a panel of reference compounds and integrate data in a potency-aligned framework with matched transcriptomic data. For this work, we characterized biological responses to classical human liver injury compounds and comparator compounds, known to not cause liver injury in humans, at 10 exposure concentrations in spent culture media by untargeted liquid chromatography-mass spectrometry analysis. The analyte features observed (with limited metabolites identified) were analyzed using BMC modeling to derive compound-induced points of departure. The results revealed liver injury compounds produced concentration-related increases in metabolomic response compared to those rarely associated with liver injury (ie, sucrose, potassium chloride). Moreover, the distributions of altered metabolomic features were largely comparable with those observed using high throughput transcriptomics, which were further extended to investigate the potential for in vitro observed biological responses to be observed in humans with exposures at therapeutic doses. These results demonstrate the utility of BMC modeling of untargeted metabolomics data as a sensitive and quantitative indicator of human liver injury potential.

A 5-day in vivo rat model was evaluated as an approach to estimate chemical exposures that may pose minimal risk by comparing benchmark dose (BMD) values for transcriptional changes in the liver and kidney to BMD values for toxicological endpoints from traditional toxicity studies. Eighteen chemicals, most having been tested by the National Toxicology Program in 2-year bioassays, were evaluated. Some of these chemicals are potent hepatotoxicants (eg, DE71, PFOA, and furan) in rodents, some exhibit toxicity but have minimal hepatic effects (eg, acrylamide and α,β-thujone), and some exhibit little overt toxicity (eg, ginseng and milk thistle extract) based on traditional toxicological evaluations. Male Sprague Dawley rats were exposed once daily for 5 consecutive days by oral gavage to 8–10 dose levels for each chemical. Liver and kidney were collected 24 h after the final exposure and total RNA was assayed using high-throughput transcriptomics (HTT) with the rat S1500+ platform. HTT data were analyzed using BMD Express 2 to determine transcriptional gene set BMD values. BMDS was used to determine BMD values for histopathological effects from chronic or subchronic toxicity studies. For many of the chemicals, the lowest transcriptional BMDs from the 5-day assays were within a factor of 5 of the lowest histopathological BMDs from the toxicity studies. These data suggest that using HTT in a 5-day in vivo rat model provides reasonable estimates of BMD values for traditional apical endpoints. This approach may be useful to prioritize chemicals for further testing while providing actionable data in a timely and cost-effective manner.

Ulcerative dermatitis in laboratory mice remains an ongoing clinical problem and animal welfare issue. Many products have been used to treat dermatitis in mice, with varying success. Recently, the topical administration of healing clays, such as bentonite and green clays, has been explored as a viable, natural treatment. We found high concentrations of arsenic and lead in experimental samples of therapeutic clay. Given the known toxic effects of these environmental heavy metals, we sought to determine whether the topical administration of a clay product containing bioavailable arsenic and lead exerted a biologic effect in mice that potentially could introduce unwanted research variability. Two cohorts of 20 singly housed, shaved, dermatitis free, adult male CD1 mice were dosed daily for 2 wk by topical application of saline or green clay paste. Samples of liver, kidney and whole blood were collected and analyzed for total arsenic and lead concentrations. Hepatic and renal concentrations of arsenic were not different between treated and control mice in either cohort; however, hepatic and renal concentrations of lead were elevated in clay treated mice compared to controls in both cohorts. In addition, in both cohorts, the activity of δ-aminolevulinate acid dehydratase, an enzyme involved with heme biosynthesis and a marker of lead toxicity, did not differ significantly between the clay-treated mice and controls. We have demonstrated that these clay products contain high concentrations of arsenic and lead and that topical application can result in the accumulation of lead in the liver and kidneys; however, these concentrations did not result in measurable biologic effects. These products should be used with caution, especially in studies of lead toxicity, heme biosynthesis, and renal α2 microglobulin function.

Botanical dietary supplements are complex mixtures with numerous potential sources of variation along the supply chain from raw plant material to the market. Approaches for determining sufficient similarity (ie, complex mixture read-across) may be required to extrapolate efficacy or safety data from a tested sample to other products containing the botanical ingredient(s) of interest. In this work, screening-level approaches for generating both chemical and biological-response profiles were used to evaluate the similarity of black cohosh (Actaea racemosa) and Echinacea purpurea samples to well-characterized National Toxicology Program (NTP) test articles. Data from nontargeted chemical analyses and gene expression of toxicologically important hepatic receptor pathways (aryl hydrocarbon receptor [AhR], constitutive androstane receptor [CAR], pregnane X receptor [PXR], farnesoid X receptor [FXR], and peroxisome proliferator-activated receptor alpha [PPARα]) in primary human hepatocyte cultures were used to determine similarity through hierarchical clustering. Although there were differences in chemical profiles across black cohosh samples, these differences were not reflected in the biological-response profiles. These findings highlight the complexity of biological-response dynamics that may not be reflected in chemical composition profiles. Thus, biological-response data could be used as the primary basis for determining similarity among black cohosh samples. Samples of E. purpurea displayed better correlation in similarity across chemical and biological-response measures. The general approaches described herein can be applied to complex mixtures with unidentified active constituents to determine when data from a tested mixture (eg, NTP test article) can be used for hazard identification of sufficiently similar mixtures, with the knowledge of toxicological targets informing assay selection when possible.

The Reference Dose (RfD) and Reference Concentration (RfC) are human health reference values (RfVs) representing exposure concentrations at or below which there is presumed to be little risk of adverse effects in the general human population. The 2009 National Research Council report Science and Decisions recommended redefining RfVs as “a risk-specific dose (for example, the dose associated with a 1 in 100,000 risk of a particular end point).” Distributions representing variability in human response to environmental contaminant exposures are critical for deriving risk-specific doses. Existing distributions estimating the extent of human toxicokinetic and toxicodynamic variability are based largely on controlled human exposure studies of pharmaceuticals. New data and methods have been developed that are designed to improve estimation of the quantitative variability in human response to environmental chemical exposures. Categories of research with potential to provide new database useful for developing updated human variability distributions include controlled human experiments, human epidemiology, animal models of genetic variability, in vitro estimates of toxicodynamic variability, and in vitro-based models of toxicokinetic variability. In vitro approaches, with further development including studies of different cell types and endpoints, and approaches to incorporate non-genetic sources of variability, appear to provide the greatest opportunity for substantial near-term advances.

Graphical abstract, Highlights • In rats, the half-life of perfluoroalkyl sulfonic acids decreased with shorter chain lengths. • Sex differences in kinetics were found for PFBS and PFHxS but not PFOS. • Perfluoroalkyl sulfonic acids were highly present in the liver but not the brain., Perfluorinated alkyl substances (PFAS) are persistent contaminants that have been detected in the environment and in humans. With the PFAS chemical class, there are perfluorinated alkyl acids, many of which have been associated with certain toxicities. Because toxicity testing cannot feasibly be conducted for each individual PFAS, the National Toxicology Program (NTP) designed studies to compare toxicities across different subclasses of PFAS and across PFAS of different chain lengths to better understand the structure-toxicity relationship. Pharmacokinetic studies were conducted in parallel to these toxicity studies to facilitate comparisons across PFAS and to provide context for human relevance. Here, the toxicokinetic parameters of perfluorobutane sulfonate (PFBS), perfluorohexane-1-sulphonic acid (PFHxS), or perfluorooctane sulfonate (PFOS) after a single intravenous or gavage administration in male and female Hsd:Sprague-Dawley rats are reported. Concentrations of these PFAS were measured in the liver, kidney, and brain. Plasma half-life increased with longer chain length after gavage administration: PFBS- males averaged 3.3 h, females 1.3 h; PFHxS- males averaged 16.3 days, females 2.1 days; PFOS- males and females averaged ˜ 20 days. There were dose-dependent changes in clearance and systemic exposure for all administered chemicals and the direction of change was different in PFOS compared to the others. Liver:plasma ratios of PFOS were the highest followed by PFHxS and PFBS, while brain:plasma ratios were low in all three sulfonates. Sex differences in plasma half-life and tissue distribution were observed for PFBS and PFHxS, but not PFOS. These data provide a direct comparison of the kinetics of three different perfluoroalkyl sulfonic acids and allow for the contextualization of toxicity data in rats for human risk assessment of this chemical class.

The brominated flame retardant, hexabromocyclododecane (HBCD), is added—but not bound—to consumer products and is eventually found in the environment and human tissues. Commercial-grade HBCD mixtures contain three major stereoisomers, alpha (α), beta (β), and gamma (γ), that are typically at a ratio of 12%:6%:82%, respectively. Although HBCD is widely used, the toxicological effects from its exposure in humans are not clearly understood. Using a physiologically based pharmacokinetic (PBPK) model could help improve our understanding of the toxicity of HBCD. The aim of this work was to develop a PBPK model, consisting of five permeability limited compartments (i.e., brain, liver, adipose tissue, blood, and rest of the body), to evaluate the pharmacokinetics of γ-HBCD in C57BL/6 mice. Physiological parameters related to body size, organ weights, and blood flow were taken from the literature. All partition coefficients were calculated based on the log K(ow). The elimination in urine and feces was optimized to reflect the percent dose eliminated, as published in the literature. Compared with data from the literature for brain, liver, blood, and adipose tissue, the model simulations accurately described the mouse data set within 1.5-fold of the data points. Also, two examples showing the utility of the PBPK model supplement the information regarding the internal dose that caused the health effects observed during these studies. Although this version of the PBPK model expressly describes γ-HBCD, more efforts are needed to clarify and improve the model to discriminate between the α, β, and γ stereoisomers.

Graphical abstract, Highlights • 8:2 fluorotelomer is rapidly distributed and eliminated in rats. • Perfluorooctanoic acid and 7:3-fluorotelomer acid are detectable metabolites. • Sex differences in kinetics were seen only in perfluorooctanoic acid., Fluorotelomer alcohols (FTOHs) are used in the production of persistent per- and polyfluorinated alkyl substances (PFAS). Rodents and humans metabolize FTOHs to perfluoralkyl carboxylic acids which have several associated toxicities. Thus, understanding the toxicokinetics of these FTOHs and their metabolites will be useful for interpreting their toxicity for humans. Here, male and female Hsd:Sprague-Dawley SD rats were administered a single dose of 8:2-FTOH via gavage (males: 12, 24, 48 mg/kg; females: 40, 80, 160 mg/kg) or IV (males: 12 mg/kg; females: 40 mg/kg). Toxicokinetics of 8:2-FTOH and two primary metabolites, perfluorooctanoic acid (PFOA) and 7:3-fluorotelomer acid (7:3-FTA) were determined in plasma. Concentrations (total) of these chemicals were determined in the liver, kidney, and brain. There was rapid absorption and distribution of 8:2-FTOH after gavage administration in male rats. The plasma elimination half-life ranged from 1.1 to 1.7 hours. Kinetic parameters of 8:2-FTOH in females were similar to that in males. Bioavailability of 8:2-FTOH ranged from 22 to 41% for both sexes with no dose-dependent trends. 8:2-FTOH metabolites, PFOA and 7:3-FTA were detected in plasma following administration of the parent FTOH. Consistent with existing literature, the plasma half-life of PFOA was longer in males than in females (198–353 hours and 4.47–6.9 hours, respectively). The plasma half-life of 7:3-FTA was around 2–3 days in both sexes. 8:2-FTOH and 7:3-FTA were detected in all tissues; PFOA was found in the liver and kidney but not the brain. Detectable concentrations of metabolites persisted longer than the parent FTOH. These data demonstrate that in rats given a single gavage dose, 8:2-FTOH is rapidly absorbed, metabolized to form PFOA and 7:3-FTA, distributed to tissues, and eliminated faster than its metabolites. Sex differences were observed in the tissue distribution and elimination of PFOA, but not 8:2-FTOH and 7:3-FTA.

Botanical dietary supplements are complex mixtures containing one or more botanical ingredient(s), each containing numerous constituents potentially responsible for its purported biological activity. Absorption, distribution, metabolism, and excretion (ADME) data are critical to understand the safety of botanical dietary supplements, including their potential for pharmacokinetic botanical-drug or botanical-botanical interactions. However, ADME data for botanical dietary supplements are rarely available and frequently inadequate to characterize their fate in vivo. Based on an assessment of the current status of botanical dietary supplements ADME research, the following key areas are identified that require robust data for human safety assessment: 1) phytochemical characterization including contaminant analysis and botanical authentication; 2) in vitro and/or in vivo data for identifying potential botanical-botanical or botanical-drug interactions and active/marker constituents; 3) robust ADME study design to include systemic exposure data on active/marker constituents using traditional or novel analytical chemistry and statistical approaches such as poly-pharmacokinetics; and 4) investigation of human relevance. A case study with Ginkgo biloba extract is used to highlight the challenges and proposed approaches in using ADME data for human safety assessment of botanical dietary supplements.

Background An increasing number of reports suggest early life exposures result in adverse effects in offspring who were never directly exposed; this phenomenon is termed “transgenerational inheritance.” Given concern for public health implications for potential effects of exposures transmitted to subsequent generations, it is critical to determine how widespread and robust this phenomenon is and to identify the range of exposures and possible outcomes. Objectives This scoping report examines the evidence for transgenerational inheritance associated with exposure to a wide range of stressors in humans and animals to identify areas of consistency, uncertainty, data gaps, and to evaluate general risk of bias issues for the transgenerational study design. Methods A protocol was developed to collect and categorize the literature into a systematic evidence map for transgenerational inheritance by health effects, exposures, and evidence streams following the Office of Health Assessment and Translation (OHAT) approach for conducting literature-based health assessments. Results A PubMed search yielded 63,758 unique records from which 257 relevant studies were identified and categorized into a systematic evidence map by evidence streams (46 human and 211 animal), broad health effect categories, and exposures. Data extracted from the individual studies are available in the Health Assessment Workspace Collaborative (HAWC) program. There are relatively few bodies of evidence where multiple studies evaluated the same exposure and the same or similar outcomes. Studies evaluated for risk of bias generally had multiple issues in design or conduct. Conclusions The evidence mapping illustrated that risk of bias, few studies, and heterogeneity in exposures and endpoints examined present serious limitations to available bodies of evidence for assessing transgenerational effects. Targeted research is suggested to addressed inconsistencies and risk of bias issues identified, and thereby establish more robust bodies of evidence to critically assess transgenerational effects - particularly by adding data on exposure-outcome pairs where there is some evidence (i.e., reproductive, metabolic, and neurological effects).

The effects of body fat mass on the elimination of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in mice. When male C57BL/6J mice are fed a high-fat, simple carbohydrate diet (HFD) for 13 weeks, they develop an obese phenotype. In contrast, A/J mice fed an HFD do not become obese. After 13 weeks on a normal diet (ND) or HFD, male C57BL/6J and A/J mice received a single dose by gavage of 0.1 or 5.0 µg of 2,3,7,8-tetrachloro[1,6-3H] dibenzo-p-dioxin per kg body weight. Using classical pharmacokinetics, the blood elimination half-life of TCDD was approximately 10 and 2 times longer in the C57BL/6J on the HFD compared with the mice on the ND at 0.1 and 5.0 μg/kg doses, respectively. The diet did not increase the blood half-life of TCDD in the A/J mice, which did not get obese. Using a physiologically based pharmacokinetic model for TCDD that incorporated experimentally derived percent body fat mass and tissue partition coefficients, as well as data on hepatic sequestration, did not provide accurate predictions to the data and could not explain the increase in half-life of TCDD in the HFD groups. This work demonstrates that obesity influences the half-life of TCDD, but other undetermined factors are involved in its elimination because the increase in body fat mass, decreases in cytochrome P4501A2, and altered partition coefficients could not completely explain the prolonged half-life.

Graphical abstract, Highlights • Pentabrominated diphenyl ether (PBDE) mixture was a multispecies carcinogen causing liver tumors in male and female rats and mice. • Hras or Ctnnb1 mutations characterized the PBDE-induced liver tumors. • PBDE-induced liver tumors increased with increasing PBDE exposure., Pentabrominated diphenyl ether (PBDE) flame retardants have been phased out in Europe and in the United States, but these lipid soluble chemicals persist in the environment and are found human and animal tissues. PBDEs have limited genotoxic activity. However, in a 2-year cancer study of a PBDE mixture (DE-71) (0, 3, 15, or 50 mg/kg (rats); 0, 3, 30, or 100 mg/kg (mice)) there were treatment-related liver tumors in male and female Wistar Han rats [Crl:WI(Han) after in utero/postnatal/adult exposure, and in male and female B6C3F1 mice, after adult exposure. In addition, there was evidence for a treatment-related carcinogenic effect in the thyroid and pituitary gland tumor in male rats, and in the uterus (stromal polyps/stromal sarcomas) in female rats. The treatment-related liver tumors in female rats were unrelated to the AhR genotype status, and occurred in animals with wild, mutant, or heterozygous Ah receptor. The liver tumors in rats and mice had treatment-related Hras and Ctnnb mutations, respectively. The PBDE carcinogenic activity could be related to oxidative damage, disruption of hormone homeostasis, and molecular and epigenetic changes in target tissue. Further work is needed to compare the PBDE toxic effects in rodents and humans.

Effective prediction of human responses to chemical and drug exposure is of critical importance in environmental toxicology research and drug development. While significant progress has been made to address this challenge using invitro liver models, these approaches often fail due to inadequate tissue model functionality. Herein, we describe the development, optimization, and characterization of a novel three-dimensional (3D) spheroid model using differentiated HepaRG cells that achieve and maintain physiologically relevant levels of xenobiotic metabolism (CYP1A2, CYP2B6, and CYP3A4/5). This invitro model maintains a stable phenotype over multiple weeks in both 96- and 384-well formats, supports highly reproducible tissue-like architectures and models pharmacologically- and environmentally important hepatic receptor pathways (ie AhR, CAR, and PXR) analogous to primary human hepatocyte cultures. HepaRG spheroid cultures use 50-100× fewer cells than conventional two dimensional cultures, and enable the identification of metabolically activated toxicants. Spheroid size, time in culture and culture media composition were important factors affecting basal levels of xenobiotic metabolism and liver enzyme inducibility with activators of hepatic receptors AhR, CAR and PXR. Repeated exposure studies showed higher sensitivity than traditional 2D cultures in identifying compounds that cause liver injury and metabolism-dependent toxicity. This platform combines the well-documented impact of 3D culture configuration for improved tissue functionality and longevity with the requisite throughput and repeatability needed for year-over-year toxicology screening.

Safe drinking water at the point of use (tapwater, TW) is a public-health priority. TW exposures and potential human-health concerns of 540 organics and 35 inorganics were assessed in 45 Chicago-area United States (US) homes in 2017. No US Environmental Protection Agency (EPA) enforceable Maximum Contaminant Level(s) (MCL) were exceeded in any residential or water treatment plant (WTP) pre-distribution TW sample. Ninety percent (90%) of organic analytes were not detected in treated TW, emphasizing the high quality of the Lake Michigan drinking-water source and the efficacy of the drinking-water treatment and monitoring. Sixteen (16) organics were detected in >25% of TW samples, with about 50 detected at least once. Low-level TW exposures to unregulated disinfection byproducts (DBP) of emerging concern, per/polyfluoroalkyl substances (PFAS), and three pesticides were ubiquitous. Common exceedances of non-enforceable EPA MCL Goal(s) (MCLG) of zero for arsenic [As], lead [Pb], uranium [U]), bromodichloromethane, and tribromomethane suggest potential human-health concerns and emphasize the continuing need for improved understanding of cumulative effects of low-concentration mixtures on vulnerable sub-populations. Because DBP dominated TW organics, residential-TW concentrations are potentially predictable with expanded pre-distribution DBP monitoring. However, several TW chemicals, notably Pb and several infrequently detected organic compounds, were not readily explained by pre-distribution samples, illustrating the need for continued broad inorganic/organic TW characterization to support consumer assessment of acceptable risk and point-of-use treatment options.

Introduction: Recent nationwide surveys found that natural products, including botanical dietary supplements, are used by ∼18% of adults. In many cases, there is a paucity of toxicological data available for these substances to allow for confident evaluations of product safety. The National Toxicology Program (NTP) has received numerous nominations from the public and federal agencies to study the toxicological effects of botanical dietary supplements. The NTP sought to evaluate the utility of in vitro quantitative high-throughput screening (qHTS) assays for toxicological assessment of botanical and dietary supplements. Materials and Methods: In brief, concentration–response assessments of 90 test substances, including 13 distinct botanical species, and individual purported active constituents were evaluated using a subset of the Tox21 qHTS testing panel. The screen included 20 different endpoints that covered a broad range of biologically relevant signaling pathways to detect test article effects upon endocrine activity, nuclear receptor signaling, stress response signaling, genotoxicity, and cell death signaling. Results and Discussion: Botanical dietary supplement extracts induced measurable and diverse activity. Elevated biological activity profiles were observed following treatments with individual chemical constituents relative to their associated botanical extract. The overall distribution of activity was comparable to activities exhibited by compounds present in the Tox21 10K chemical library. Conclusion: Botanical supplements did not exhibit minimal or idiosyncratic activities that would preclude the use of qHTS platforms as a feasible method to screen this class of compounds. However, there are still many considerations and further development required when attempting to use in vitro qHTS methods to characterize the safety profile of botanical/dietary supplements.

Prediction of human response to chemical exposures is a major challenge in both pharmaceutical and toxicological research. Transcriptomics has been a powerful tool to explore chemical-biological interactions, however, limited throughput, high-costs, and complexity of transcriptomic interpretations have yielded numerous studies lacking sufficient experimental context for predictive application. To address these challenges, we have utilized a novel high-throughput transcriptomics (HTT) platform, TempO-Seq, to apply the interpretive power of concentration-response modeling with exposures to 24 reference compounds in both differentiated and non-differentiated human HepaRG cell cultures. Our goals were to (1) explore transcriptomic characteristics distinguishing liver injury compounds, (2) assess impacts of differentiation state of HepaRG cells on baseline and compound-induced responses (eg, metabolically-activated), and (3) identify and resolve reference biological-response pathways through benchmark concentration (BMC) modeling. Study data revealed the predictive utility of this approach to identify human liver injury compounds by their respective BMCs in relation to human internal exposure plasma concentrations, and effectively distinguished drug analogs with varied associations of human liver injury (eg, withdrawn therapeutics trovafloxacin and troglitazone). Impacts of cellular differentiation state (proliferated vs differentiated) were revealed on baseline drug metabolizing enzyme expression, hepatic receptor signaling, and responsiveness to metabolically-activated toxicants (eg, cyclophosphamide, benzo(a)pyrene, and aflatoxin B1). Finally, concentration-response modeling enabled efficient identification and resolution of plausibly-relevant biological-response pathways through their respective pathway-level BMCs. Taken together, these findings revealed HTT paired with differentiated in vitro liver models as an effective tool to model, explore, and interpret toxicological and pharmacological interactions.

The Tox21 Program has investigated thousands of chemicals with high-throughput screening assays using cell-based assays to link thousands of chemicals to individual molecular targets/pathways. However, these systems have been widely criticized for their suspected lack of ‘metabolic competence’ to bioactivate or detoxify chemical exposures. In this study, 9 cell line backgrounds used in Tox21 assays (i.e., HepG2, HEK293, Hela, HCT116, ME180, CHO-K1, GH3.TRE-Luc, C3H10T1/2 and MCF7) were evaluated via metabolite formation rates, along with metabolic clearance and metabolite profiling for HepG2, HEK293, and MCF-7aroERE, in comparison to pooled donor (50) suspensions of primary human hepatocytes (PHHs). Using prototype clinical drug substrates for CYP1A2, CYP2B6, and CYP3A4/5, extremely low-to-undetectable CYP450 metabolism was observed (24 h), and consistent with their purported ‘lack’ of metabolic competence. However, for Phase II metabolizing enzymes and metabolic clearance, surprisingly proficient metabolism was observed for bisphenol AF, bisphenol S, and 7-hydroxycoumarin. Here, comparatively low glucuronidation relative to sulfation was observed in contrast to equivalent levels in PHHs. Overall, while a lack of CYP450 metabolism was confirmed in this benchmarking effort, Tox21 cell lines were not ‘incompetent’ for xenobiotic metabolism, and displayed surprisingly high proficiency for sulfation that rivaled PHHs. These findings have implications for the interpretation of Tox21 assay data, and establish a framework for evaluating of ‘metabolic competence’ with in vitro models.

Virginia cedarwood oil is widely used as a fragrance material in household and personal products and as a naturally derived pesticide alternative. Due to conflicting literature on dermal exposures in animals and humans, concern for safe levels of human exposure remains. The present study evaluated the toxicity of cedarwood oil applied dermally to F344/N rats and B6C3F1/N mice for 13 weeks. Groups of 10 male and female rats and mice received no treatment (untreated control) or were administered cedarwood oil in 95% aqueous ethanol dermally at concentrations ranging from 0% (vehicle control), 6.25%, 12.5%, 25%, 50%, and 100% (undiluted). Rats and mice developed extensive skin lesions at the site of application. Benchmark dose modeling (BMD) was performed for the significantly increased skin lesions observed in the rat, to provide perspective for risk assessment applications. Benchmark dose modeling levels (BMDL) of 0.65 to 2.1% and 1.2 to 4.4% (equivalent to 13 to 42 mg/kg and 24 to 48 mg/kg, respectively) cedarwood oil were calculated for the most sensitive endpoint of epidermal hyperplasia in female rats and chronic active inflammation in male rats, respectively. These BMDL levels coincide with reported use levels in cosmetics and pesticides, raising the concern for human exposure.

On July 10, 1976, an explosion at a chemical plant in Seveso, Italy, released up to 30 kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)—the most potent dioxin congener. Twenty years later, the Seveso Women's Health Study (SWHS) initiated a follow-up assessment of a cohort of female Seveso residents. Researchers collected serial blood, measured for TCDD levels, and recorded information about the women's medical history after the explosion. The study's aims were to: 1) modify the human PBPK model for TCDD (Emond et al. 2004; Emond et al. 2005; NCEA-USEPA, 2010) to include repetitive gestation and lactation; 2) simulate TCDD blood concentrations during different life stages including pregnancy and lactation, under different exposure scenarios; and 3) use this PBPK model to compare the influence of gestation and lactation on elimination of TCDD. After optimization of the model, it was assessed using data from the SWHS cohort. The 23 women in Subcohort A, were 4–39 years old and in Subcohort B, the 18 women were 3–17 years old when the explosion occurred. The model accurately predicted the blood concentrations during the 20 years post-exposure, including periods of pregnancy and lactation. The model was also used to analyze the contribution of gestation and lactation to the mother's elimination of TCDD. The results suggest that gestation and lactation do not significantly impact TCDD blood elimination. Future efforts will focus on using additional data to evaluate the PBPK model and improving the mathematical descriptions of lactation and multiple gestations. Keywords: PBPK, Pharmacokinetics, Dioxin, TCDD, Developmental

CAsE-PE cells are an arsenic-transformed, human prostate epithelial line containing oncogenic mutations in KRAS compared to immortalized, normal KRAS parent cells, RWPE-1. We previously reported increased copy number of mutated KRAS in CAsE-PE cells, suggesting gene amplification. Here, KRAS flanking genomic and transcriptomic regions were sequenced in CAsE-PE cells for insight into KRAS amplification. Comparison of DNA-Seq and RNA-Seq showed increased reads from background aligning to all KRAS exons in CAsE-PE cells, while a uniform DNA-Seq read distribution occurred in RWPE-1 cells with normal transcript expression. We searched for KRAS fusions in DNA and RNA sequencing data finding a portion of reads aligning to KRAS and viral sequence. After generation of cDNA from total RNA, short and long KRAS probes were generated to hybridize cDNA and KRAS enriched fragments were PacBio sequenced. More KRAS reads were captured from CAsE-PE cDNA versus RWPE-1 by each probe set. Only CAsE-PE cDNA showed KRAS viral fusion transcripts, primarily mapping to LTR and endogenous retrovirus sequences on either 5'- or 3'-ends of KRAS. Most KRAS viral fusion transcripts contained 4 to 6 exons but some PacBio sequences were in unusual orientations, suggesting viral insertions within the gene body. Additionally, conditioned media was extracted for potential retroviral particles. RNA-Seq of culture media isolates identified KRAS retroviral fusion transcripts in CAsE-PE media only. Truncated KRAS transcripts suggested multiple retroviral integration sites occurred within the KRAS gene producing KRAS retroviral fusions of various lengths. Findings suggest activation of endogenous retroviruses in arsenic carcinogenesis should be explored.

Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5μM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q

Summary: Per- and polyfluoroalkyl substances (PFASs) are a group of fluorinated substances of interest to researchers, regulators, and the public due to their widespread presence in the environment. A few PFASs have comparatively extensive amounts of human epidemiological, exposure, and experimental animal toxicity data (e.g., perfluorooctanoic acid), whereas little toxicity and exposure information exists for much of the broader set of PFASs. Given that traditional approaches to generate toxicity information are resource intensive, new approach methods, including in vitro high-throughput toxicity (HTT) testing, are being employed to inform PFAS hazard characterization and further (in vivo) testing. The U.S. Environmental Protection Agency (EPA) and the National Toxicology Program (NTP) are collaborating to develop a risk-based approach for conducting PFAS toxicity testing to facilitate PFAS human health assessments. This article describes the construction of a PFAS screening library and the process by which a targeted subset of 75 PFASs were selected. Multiple factors were considered, including interest to the U.S. EPA, compounds within targeted categories, structural diversity, exposure considerations, procurability and testability, and availability of existing toxicity data. Generating targeted HTT data for PFASs represents a new frontier for informing priority setting. https://doi.org/10.1289/EHP4555

Background: To effectively incorporate in vitro data into regulatory use, confidence must be established in the quantitative extrapolation of in vitro activity to relevant end points in animals or humans. Objective: Our goal was to evaluate and optimize in vitro to in vivo extrapolation (IVIVE) approaches using in vitro estrogen receptor (ER) activity to predict estrogenic effects measured in rodent uterotrophic studies. Methods: We evaluated three pharmacokinetic (PK) models with varying complexities to extrapolate in vitro to in vivo dosimetry for a group of 29 ER agonists, using data from validated in vitro [U.S. Environmental Protection Agency (U.S. EPA) ToxCast™ ER model] and in vivo (uterotrophic) methods. In vitro activity values were adjusted using mass-balance equations to estimate intracellular exposure via an enrichment factor (EF), and steady-state model calculations were adjusted using fraction of unbound chemical in the plasma (fu) to approximate bioavailability. Accuracy of each model-adjustment combination was assessed by comparing model predictions with lowest effect levels (LELs) from guideline uterotrophic studies. Results: We found little difference in model predictive performance based on complexity or route-specific modifications. Simple adjustments, applied to account for in vitro intracellular exposure (EF) or chemical bioavailability (fu), resulted in significant improvements in the predictive performance of all models. Conclusion: Computational IVIVE approaches accurately estimate chemical exposure levels that elicit positive responses in the rodent uterotrophic bioassay. The simplest model had the best overall performance for predicting both oral (PPK_EF) and injection (PPK_fu) LELs from guideline uterotrophic studies, is freely available, and can be parameterized entirely using freely available in silico tools. https://doi.org/10.1289/EHP1655

Background: To effectively incorporate in vitro data into regulatory use, confidence must be established in the quantitative extrapolation of in vitro activity to relevant end points in animals or ...

Background: A central challenge in toxicity testing is the large number of chemicals in commerce that lack toxicological assessment. In response, the Tox21 program is re-focusing toxicity testing from animal studies to less expensive and higher throughput in vitro methods using target/pathway-specific, mechanism-driven assays. Objectives: Our objective was to use an in-depth mechanistic study approach to prioritize and characterize the chemicals affecting mitochondrial function. Methods: We used a tiered testing approach to prioritize for more extensive testing 622 compounds identified from a primary, quantitative high-throughput screen of 8,300 unique small molecules, including drugs and industrial chemicals, as potential mitochondrial toxicants by their ability to significantly decrease the mitochondrial membrane potential (MMP). Based on results from secondary MMP assays in HepG2 cells and rat hepatocytes, 34 compounds were selected for testing in tertiary assays that included formation of reactive oxygen species (ROS), upregulation of p53 and nuclear erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE), mitochondrial oxygen consumption, cellular Parkin translocation, and larval development and ATP status in the nematode Caenorhabditis elegans. Results: A group of known mitochondrial complex inhibitors (e.g., rotenone) and uncouplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and uncouplers, were detected. From this study, we identified four not well-characterized potential mitochondrial toxicants (lasalocid, picoxystrobin, pinacyanol, and triclocarban) that merit additional in vivo characterization. Conclusions: The tier-based approach for identifying and mechanistically characterizing mitochondrial toxicants can potentially reduce animal use in toxicological testing. https://doi.org/10.1289/EHP2589

Effective prediction of human responses to chemical and drug exposure is of critical importance in environmental toxicology research and drug development. While significant progress has been made to address this challenge using invitro liver models, these approaches often fail due to inadequate tissue model functionality. Herein, we describe the development, optimization, and characterization of a novel three-dimensional (3D) spheroid model using differentiated HepaRG cells that achieve and maintain physiologically relevant levels of xenobiotic metabolism (CYP1A2, CYP2B6, and CYP3A4/5). This invitro model maintains a stable phenotype over multiple weeks in both 96- and 384-well formats, supports highly reproducible tissue-like architectures and models pharmacologically- and environmentally important hepatic receptor pathways (ie AhR, CAR, and PXR) analogous to primary human hepatocyte cultures. HepaRG spheroid cultures use 50–100× fewer cells than conventional two dimensional cultures, and enable the identification of metabolically activated toxicants. Spheroid size, time in culture and culture media composition were important factors affecting basal levels of xenobiotic metabolism and liver enzyme inducibility with activators of hepatic receptors AhR, CAR and PXR. Repeated exposure studies showed higher sensitivity than traditional 2D cultures in identifying compounds that cause liver injury and metabolism-dependent toxicity. This platform combines the well-documented impact of 3D culture configuration for improved tissue functionality and longevity with the requisite throughput and repeatability needed for year-over-year toxicology screening.

In vitro-in vivo extrapolation (IVIVE) analyses translating high-throughput screening (HTS) data to human relevance have been limited. This study represents the first report applying IVIVE approaches and exposure comparisons using the entirety of the Tox21 federal collaboration chemical screening data, incorporating assay response efficacy and quality of concentration-response fits, and providing quantitative anchoring to first address the likelihood of human in vivo interactions with Tox21 compounds. This likelihood was assessed using a maximum blood concentration to in vitro response ratio approach (Cmax/AC50), analogous to decision-making methods for clinical drug-drug interactions. Fraction unbound in plasma (fup) and intrinsic hepatic clearance (CLint) parameters were estimated in silico and incorporated in a 3-compartment toxicokinetic (TK) model to first predict Cmax for in vivo corroboration using therapeutic scenarios. Toward lower exposure scenarios, 36 compounds of 3,925 with curated activity in the HTS data using high quality dose-response model fits and ≥40% efficacy gave ‘possible’ human in vivo interaction likelihoods lower than median human exposures predicted in EPA’s ExpoCast program. A publicly available web application has been designed to provide all Tox21/ToxCast dose likelihood predictions. Overall, this approach provides an intuitive framework to relate in vitro toxicology data rapidly and quantitatively to exposures using either in vitro or in silico derived TK parameters, and can be thought of as an important step towards estimating plausible biological interactions in a high throughput risk assessment framework.

The use of brominated flame retardants and incineration of bromine-containing materials has lead to an increase in polybrominated dibenzo-p-dioxins and dibenzofurans (PBDD/Fs) in the environment. Measurable amounts of PBDD/Fs have been detected in soil, seafood, and human breast milk and serum. Studies indicate that the relative potencies of some PBDD/Fs based on enzyme induction are equivalent to those of some polychlorinated dibenzo-p-dioxins and dibenzofurans. To assess the humoral immunity relative potencies of PBDD/Fs and compare them to their chlorinated analogs, female B6C3F1/N mice received a single oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrabromodibenzofuran (TBDF), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentabromodibenzofuran (1PeBDF), 1,2,3,7,8-pentachlorodibenzofuran (1PeCDF), 2,3,4,7,8-pentabromodibenzofuran (4PeBDF), 2,3,4,7,8-pentachlorodibenzofuran (4PeCDF), 2,3-dibromo-7,8-dichlorodibenzo-p-dioxin (DBDCDD), or 2,3,7-tribromodibenzo-p-dioxin (TriBDD). Inhibition of the immunoglobulin M (IgM) antibody forming cell response was measured 4 days following immunization with sheep red blood cells. The data were fit to a Hill model to estimate the ED50 for inhibition. Expression of xenobiotic metabolizing enzyme (XME) and thyroxine transport protein (Ttr) genes in liver was measured by PCR to assess aryl hydrocarbon-mediated responses. TCDD, TBDF, TCDF, 1PeBDF, 4PeBDF, 4PeCDF, and DBDCDD suppressed the IgM antibody response and Ttr gene expression, and upregulated phase I XME genes. 1PeCDF suppressed the IgM antibody response but only upregulated phase I XME genes; TriBDD had no effect on antibody response. The rank order of potency (ED50) for these chemicals was TCDD>TBDF>4PeBDF>TCDF/4PeCDF/1PeBDF>1PeCDF. Whereas TCDD was the most potent compound tested, the brominated analogs were more potent than their chlorinated analogs, suggesting that these compounds should be considered in toxic equivalency factor evaluation and risk assessment.

1. The liver metabolizes thyroxine (T(4)) through two major pathways: deiodination and conjugation. Following exposure to xenobiotics, T(4) conjugation increases through the induction of hepatic uridine diphosphate glucuronosyltransferase (UGT) in rodents; however, it is uncertain to what degree different species employ deiodination and conjugation in the metabolism of T(4). The objective of this study was to compare the metabolism of T4 in untreated and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153)-treated primary sandwich-cultured hepatocytes from rat (SCRH) and human (SCHH). 2. Basal metabolite concentrations were 13 times higher in the medium of SCRH compared to SCHH. Metabolite distribution in the medium of SCRH versus SCHH was as follows: T(4)G, (91.6 versus 5.3%); T4S, (3.6 versus 4.4%) and T(3) + rT(3), (4.9 versus 90.3%). PCB 153 induced T(4)G in the medium of SCRH and SCHH; however, T(4)S and T(3) + rT(3) were changed but to a much lesser degree. 3. The results indicate that baseline T(4) glucuronidation is greater in SCRH compared to SCHH. These data also suggest that glucuronidation may be a more important pathway for T(4) metabolism in rats and deiodination may be a favored pathway in humans; however, with PCB 153 treatment these data support glucuronidation as a primary route of T(4) metabolism in both rat and humans.

In this study we assessed the relative toxicity and potency of the chlorinated naphthalenes 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Chemicals were administered in corn oil:acetone (99:1) by gavage to female Harlan Sprague–Dawley rats at dosages of 0 (vehicle), 500, 1500, 5000, 50,000 and 500,000 ng/kg (PCN 66 and PCN 67) and 1, 3, 10, 100, and 300 ng/kg (TCDD) for 2 weeks. Histopathologic changes were observed in the thymus, liver and lung of TCDD treated animals and in the liver and thymus of PCN treated animals. Significant increases in CYP1A1 and CYP1A2 associated enzyme activity were observed in all animals exposed to TCDD, PCN 66 and PCN 67. Dose response modeling of CYP1A1, CYP1A2 and thymic atrophy gave ranges of estimated relative potencies, as compared to TCDD, of 0.0015–0.0072, for PCN 66 and 0.00029–0.00067 for PCN 67. Given that PCN 66 and PCN 67 exposure resulted in biochemical and histopathologic changes similar to that seen with TCDD, this suggests that they should be included in the WHO toxic equivalency factor (TEF) scheme, although the estimated relative potencies indicate that these hexachlorinated naphthalenes should not contribute greatly to the overall human body burden of dioxin-like activity.

Aging adults are a growing segment of the U.S. population and are likely to exhibit increased susceptibility to many environmental toxicants. However, there is little information on the susceptibility of the aged to toxicants. The toxicity of toluene has been well characterized in young adult rodents but there is little information in the aged. Three approaches were used: (1) pharmacokinetic (PK), (2) cardiac biomarkers, and (3) whole-animal physiology to assess whether aging increases susceptibility to toluene in the Brown Norway (BN) rat. Three life stages, young adult, middle aged, and aged (4, 12, and 24 mo, respectively), were administered toluene orally at doses of 0, 0.3, 0.65, or 1 g/kg and subjected to the following: terminated at 45 min or 4 h post dosing, and blood and brain toluene concentration were measured; terminated at 4 h post dosing, and biomarkers of cardiac function were measured; or monitor heart rate (HR), core temperature (Tc), and motor activity (MA) by radiotelemetry before and after dosing. Brain toluene concentration was significantly elevated in aged rats at 4 h after dosing with either 0.3 or 1 g/kg. Blood toluene concentrations were unaffected by age. There were various interactions between aging and toluene-induced effects on cardiac biomarkers. Most notably, toluene exposure led to reductions in mRNA markers for oxidative stress in aged but not younger animals. Toluene also produced a reduction in cardiac endothelin-1 in aged rats. Higher doses of toluene led to tachycardia, hypothermia, and a transient elevation in MA. Aged rats were less sensitive to the tachycardic effects of toluene but showed a prolonged hypothermic response. Elevated brain levels of toluene in aged rats may be attributed to their suppressed cardiovascular and respiratory responses. The expression of several cardiac biochemical markers of toluene exposure in the aged may also reflect differential susceptibility to this toxicant.

Triclosan (5-chloro-2-(2,4-dichlorophenoxy)-phenol) is a chlorinated phenolic antibacterial compound found in consumer products. In vitro human pregnane X receptor activation, hepatic phase I enzyme induction, and decreased in vivo total thyroxine (T4) suggest adverse effects on thyroid hormone homeostasis. Current research tested the hypothesis that triclosan decreases circulating T4 via upregulation of hepatic catabolism and transport. Weanling female Long-Evans rats received triclosan (0–1000 mg/kg/day) by gavage for 4 days. Whole blood and liver were collected 24 h later. Total serum T4, triiodothyronine (T3), and thyroid-stimulating hormone (TSH) were measured by radioimmunoassay. Hepatic microsomal assays measured ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase (PROD), and uridine diphosphate glucuronyltransferase enzyme activities. The messenger RNA (mRNA) expression of cytochrome P450s 1a1, 2b1/2, and 3a1/23; UGTs 1a1, 1a6, and 2b5; sulfotransferases 1c1 and 1b1; and hepatic transporters Oatp1a1, Oatp1a4, Mrp2, and Mdr1b was measured by quantitative reverse transcriptase PCR. Total T4 decreased dose responsively, down to 43% of control at 1000 mg/kg/day. Total T3 was decreased to 89 and 75% of control at 300 and 1000 mg/kg/day. TSH did not change. Triclosan dose dependently increased PROD activity up to 900% of control at 1000 mg/kg/day. T4 glucuronidation increased nearly twofold at 1000 mg/kg/day. Cyp2b1/2 and Cyp3a1/23 mRNA expression levels were induced twofold and fourfold at 300 mg/kg/day. Ugt1a1 and Sult1c1 mRNA expression levels increased 2.2-fold and 2.6-fold at 300 mg/kg/day. Transporter mRNA expression levels were unchanged. These data denote important key events in the mode of action for triclosan-induced hypothyroxinemia in rats and suggest that this effect may be partially due to upregulation of hepatic catabolism but not due to mRNA expression changes in the tested hepatic transporters.

Hypothyroxinemia in rats has been well documented as a result of exposure to polychlorinated biphenyls (PCBs). Hypothetical mechanisms include induction of hepatic catabolic enzymes and cellular hormone transporters, and/or interference with plasma transport proteins. We hypothesized that if thyroxine displacement from transport proteins by PCBs occurs in vivo, it would result in increased free thyroxine (FT4). This study investigates the effects of a single oral dose of 2,2’,4,4’,5,5'-hexachlorobiphenyl (PCB 153 at 60 mg/kg) or 3,3’,4,4’,5,5'-hexachlorobiphenyl (PCB 169 at 1 mg/kg) on rats at 28 or 76 days of age. Total thyroxine (TT4) and FT4 were measured at 0.5, 1, 2, 4, 8, 24, or 48 hours post -dosing. Microsomal ethoxy- and pentoxy-resorufin-O-deethylase (EROD and PROD) activity and uridine diphosphoglucuronosyl transferase (UGT) activity were determined. No significant increase in TT4 or FT4 concentrations was seen at any time point. PCB 153 significantly decreased TT4 and FT4 in young and adult rats, with young rats showing a time-by-treatment interaction from 2 to 48 hours post -dosing in serum FT4. With PCB 169 exposure, young rats showed a decrease in FT4 only, whereas adult rats showed decreases in TT4 only. Hepatic EROD and PROD activities were both dramatically increased following PCB 169 and 153, respectively. Uridine diphosphoglucuronosyl transferase activity was increased only after PCB 169 exposure. These data demonstrate that neither PCB 153 nor PCB169 increased FT4, which supports the conclusion that these PCBs do not displace thyroxine from serum TTR, or if it does occur, there is no subsequent increase in serum FT4 in vivo.

Thyroid hormone (TH) disrupting compounds interfere with both thyroidal and extrathyroidal mechanisms to decrease circulating thyroxine (T4). This research tested the hypothesis that serum T4 concentrations of rodents exposed to a mixture of both TH synthesis inhibitors (pesticides) and stimulators of T4 clearance in the liver (polyhalogenated aromatic hydrocarbons, PHAHs) could be best predicted by an integrated addition model. Female Long-Evans rats, 23 days of age, were dosed with dilutions of a mixture of 18 PHAHs (2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin like PCBs) and a mixture of 3 pesticides (thiram, pronamide, and mancozeb) for four consecutive days. Serum was collected 24 hours after the last exposure and T4 concentrations were measured by radioimmunoassay. Animals exposed to the highest dose of the mixture experienced a 45% decrease in serum T4. Three additivity model predictions (dose addition, effect addition, and integrated addition) were generated based on single chemical data, and the results were compared. Effect addition overestimated the effect produced by the combination of all 21 chemicals. The results of the dose- and integrated-addition models were similar, and both provided better predictions than the effect-addition model. These results support the use of dose- and integrated additivity models in predicting the effects of complex mixtures.

The toxic equivalency factor (TEF) approach was employed to compare immunotoxic potency of mixtures containing polychlorinated dibenzo- p -dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls relative to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), using the antibody response to sheep erythrocytes (SRBC). Mixture-1 (MIX-1) contained TCDD, 1,2,3,7,8-pentachlorodibenzo- p -dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,6,7,8,9-octachlorodibenzofuran (OCDF). Mixture-2 (MIX-2) contained MIX-1 and the following PCBs, 3,3′,4,4′-tetrachlorobiphenyl (IUPAC No. 77), 3,3′,4,4′,5-pentachlorobiphenyl (126), 3,3′,4,4′,5,5 N -hexachlorobiphenyl (169), 2,3,3′,4,4′-pentachlorobiphenyl (105), 2,3′,4,4′,5-pentachlorobiphenyl (118), and 2,3,3′,4,4′,5-hexachlorobiphenyl (156). The mixture compositions were based on relative chemical concentrations in food and human tissues. TCDD equivalents (TEQ) of the mixture were estimated using relative potency factors from hepatic enzyme induction in mice [DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Dose–response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I .CYP1A1 and CYP1A2 enzyme activity in liver, lung and skin. Toxicol. Appl. Pharmacol. 130, 197–208; DeVito, M.J., Menache, G., Diliberto, J.J., Ross, D.G., Birnbaum L.S., 2000. Dose–response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 167, 157–172] Female mice received 0, 1.5, 15, 150 or 450 ng TCDD/kg/day or approximately 0, 1.5, 15, 150 or 450 ng TEQ/kg/day of MIX-1 or MIX-2 by gavage 5 days per week for 13 weeks. Mice were immunized 3 days after the last exposure and 4 days later, body, spleen, thymus, and liver weights were measured, and antibody response to SRBCs was observed. Exposure to TCDD, MIX-1, and MIX-2 suppressed the antibody response in a dose-dependent manner. Two-way ANOVA indicated no differences in the response between TCDD and the mixtures for body weight, spleen/body weight and decreased antibody responses. The results support the use of the TEF methodology and suggest that immune suppression by dioxin-like chemicals may be of concern at or near background human exposures.

1. Pyrethroids are neurotoxic and parent pyrethroid appears to be toxic entity. This study evaluated the oral disposition and bioavailability of bifenthrin in the adult male Long-Evans rat. 2. In the disposition study, rats were administered bifenthrin (0.3 or 3 mg/kg) by oral gavage and serially sacrificed (0.25 h to 21 days). Blood, liver, brain and adipose tissue were removed. In the bioavailability study, blood was collected serially from jugular vein cannulated rats (0.25 to 24 h) following oral (0.3 or 3 mg/kg) or intravenous (0.3 mg/kg) administration of bifenthrin. Tissues were extracted and analyzed for bifenthrin by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). 3. Bifenthrin concentration in blood and liver peaked 1-2-h postoral administration and were approximately 90 ng/ml (or g) and 1000 ng/ml (or g) for both tissues at 0.3 and 3 mg/kg, respectively. Bifenthrin was rapidly cleared from both blood and liver. Brain concentrations peaked at 4-6 h and were lower than in blood at both doses (12 and 143 ng/g). Bifenthrin in adipose tissue peaked at the collected time points of 8 (157 ng/g) and 24 (1145 ng/g) h for the 0.3 and 3 mg/kg doses, respectively and was retained 21 days postoral administration. Following intravenous administration, the blood bifenthrin concentration decreased bi-exponentially, with a distribution half-life of 0.2 h and an elimination half-life of 8 h. Bifenthrin bioavailability was approximately 30%. These disposition and kinetic bifenthrin data may decrease uncertainties in the risk assessment for this pyrethroid insecticide.

The metabolism of (alphaS)-cyano-3-phenoxybenzyl (1R, 3R)-cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane carboxylate (deltamethrin) and (alphaS)-cyano-3-phenoxybenzyl 2-(4-chlorophenyl)-3-methylbutyrate (esfenvalerate) by rat and human liver microsomes differs with respect to the biotransformation pathway (oxidation versus hydrolysis) responsible for their clearance. This study aims to further explore the species differences in the metabolism of these chemicals. Using a parent depletion approach, rat and human cytochromes P450 (P450s) were screened for their ability to eliminate deltamethrin or esfenvalerate during in vitro incubations. Rat P450 isoforms CYP1A1, CYP2C6, CYP2C11, and CYP3A2 and human P450 isoforms CYP2C8, CYP2C19, and CYP3A5 were capable of metabolizing either pyrethroid. Human CYP2C9 metabolized esfenvalerate but not deltamethrin. Rat and human P450s that metabolize esfenvalerate and deltamethrin do so with similar kinetics. In addition to the liver, a potential site of metabolic elimination of pyrethroids is the blood via serum carboxylesterase (CE) hydrolysis. The serum of rats, but not humans, contains significant quantities of CE. Deltamethrin and esfenvalerate were metabolized effectively by rat serum and a purified rat serum CE. In contrast, neither pyrethroid was metabolized by human serum or purified human serum esterases (acetylcholinesterase and butyrylcholinesterase). These studies suggest that the difference in rates of oxidative metabolism of pyrethroids by rat and human hepatic microsomes is dependent on the expression levels of individual P450 isoforms rather than their specific activity. Furthermore, these studies show that the metabolic elimination of deltamethrin and esfenvalerate in blood may be important to their disposition in rats but not in humans.

When an interaction has been detected among the chemicals in a mixture, it may be of interest to predict the interaction threshold. A method is presented for estimation of an interaction threshold along a mixture ray which allows differences in the shapes of the dose-response curves of the individual components (e.g., mixtures of full and partial agonists with differing response maxima). A point estimate and confidence interval for the interaction threshold may be estimated. The methods are illustrated with data from a study of a mixture of 18 polyhalogenated aromatic hydrocarbons (PHAHs) in rats exposed by oral gavage for four consecutive days. Serum total thyroxine (T4) was the response variable. Previous analysis of these data demonstrated a dose-dependent interaction among the 18 chemicals in the mixture, with additivity suggested in the lower portion of the dose-response curve and synergy (greater than additive response) in the higher portion of the dose-response curve. The present work builds on this analysis by construction of an interaction threshold model along the mixture ray. This interaction threshold model has two components: an implicit additivity region and an explicit region that describes the departure from additivity; the interaction threshold is the boundary between the two regions. Estimation of the interaction threshold within the observed experimental region suggested evidence of additivity in the low dose region. Total doses of the mixture that exceed the upper limit of the confidence interval on the interaction threshold were associated with a greater-than-additive interaction.

Breast cancer is the most common cancer and the second-leading cause of cancer mortality in women in the United States. A recent 2-year National Toxicology Program carcinogenicity study showed an increased incidence of proliferative mammary lesions (hyperplasia, fibroadenoma, adenocarcinoma) in F344/NTac rats exposed to bromodichloroacetic acid (BDCA), a disinfection by-product in finished drinking water with widespread human exposure. We hypothesized that the increase in mammary tumors observed in BDCA-exposed F344/NTac rats may be due to underlying molecular changes relevant for human breast cancer. The objective of the study was to compare (1) gene and protein expression and (2) mutation spectra of relevant human breast cancer genes between normal untreated mammary gland and mammary tumors from control and BDCA-exposed animals to identify molecular changes relevant for human cancer. Histologically, adenocarcinomas from control and BDCA-exposed animals were morphologically very similar, were estrogen/progesterone receptor positive, and displayed a mixed luminal/basal phenotype. Gene expression analysis showed a positive trend in the number of genes associated with human breast cancer, with proportionally more genes represented in the BDCA-treated tumor group. Additionally, a 5-gene signature representing possible Tgfβ pathway activation in BDCA-treated adenocarcinomas was observed, suggesting that this pathway may be involved in the increased incidence of mammary tumors in BDCA-exposed animals.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly lipophilic chemical that distributes into adipose tissue, especially at low doses. However, at high doses TCDD sequesters in liver because it induces cytochrome P450 1A2 (CYP1A2) that binds TCDD. A physiologically based pharmacokinetic (PBPK) model was developed that included an inducible elimination rate of TCDD in the Sprague-Dawley rat. Objectives of this work were to characterize the influence of induction of CYP1A2 and adipose tissue mass fraction on the terminal elimination half-life (t1/2) of TCDD using this PBPK model. When the model assumes a fixed elimination of TCDD, t1/2 increases with dose, due to hepatic sequestration. Because experimental data indicate that the t1/2 of TCDD decreases with dose, the model was modified to include an inducible elimination rate. The PBPK model was then used to compare the t1/2 after an increase of adipose tissue mass fraction from 6.9 to 70%. The model suggests that at low exposures, increasing adipose tissue mass increases the terminal t1/2. However, at higher exposures, as CYP1A2 is induced, the relationship between adipose tissue mass and t1/2 reaches a plateau. This demonstrates that an inducible elimination rate is needed in a PBPK model in order to describe the pharmacokinetics of TCDD. At low exposures these models are more sensitive to parameters related to partitioning into adipose tissue.

Endocrine disruption from environmental contaminants has been linked to a broad spectrum of adverse outcomes. One concern about endocrine-disrupting xenobiotics is the potential for additive or synergistic (i.e., greater-than-additive) effects of mixtures. A short-term dosing model to examine the effects of environmental mixtures on thyroid homeostasis has been developed. Prototypic thyroid-disrupting chemicals (TDCs) such as dioxins, polychlorinated biphenyls (PCBs), and poly-brominated diphenyl ethers have been shown to alter thyroid hormone homeostasis in this model primarily by up-regulating hepatic catabolism of thyroid hormones via at least two mechanisms. Our present effort tested the hypothesis that a mixture of TDCs will affect serum total thyroxine (T4) concentrations in a dose-additive manner. Young female Long-Evans rats were dosed via gavage with 18 different polyyhalogenated aromatic hydrocarbons [2 dioxins, 4 dibenzofurans, and 12 PCBs, including dioxin-like and non-dioxin-like PCBs] for 4 consecutive days. Serum total T4 was measured via radioimmunoassay in samples collected 24 hr after the last dose. Extensive dose–response functions (based on seven to nine doses per chemical) were determined for individual chemicals. A mixture was custom synthesized with the ratio of chemicals based on environmental concentrations. Serial dilutions of this mixture ranged from approximately background levels to 100-fold greater than background human daily intakes. Six serial dilutions of the mixture were tested in the same 4-day assay. Doses of individual chemicals that were associated with a 30% TH decrease from control (ED30), as well as predicted mixture outcomes were calculated using a flexible single-chemical-required method applicable to chemicals with differing dose thresholds and maximum-effect asymptotes. The single-chemical data were modeled without and with the mixture data to determine, respectively, the expected mixture response (the additivity model) and the experimentally observed mixture response (the empirical model). A likelihood-ratio test revealed statistically significant departure from dose additivity. There was no deviation from additivity at the lowest doses of the mixture, but there was a greater-than-additive effect at the three highest mixtures doses. At high doses the additivity model underpredicted the empirical effects by 2- to 3-fold. These are the first results to suggest dose-dependent additivity and synergism in TDCs that may act via different mechanisms in a complex mixture. The results imply that cumulative risk approaches be considered when assessing the risk of exposure to chemical mixtures that contain TDCs.

Emissions of endocrine disrupting chemicals (EDCs) from combustion sources are poorly characterized due to the large number of compounds present in the emissions, the complexity of the analytical separations required, and the uncertainty regarding identification of chemicals with endocrine effects. In this work, multidimensional gas chromatographic-mass spectrometry (MDGC-MS) was used to characterize emissions from both controlled (diesel engine) and uncontrolled (open burning of domestic waste) combustion sources. The results of this study suggest that, by using MDGC-MS, one can resolve a much greater percentage of the chromatogram and identify about 84% of these resolved compounds. This increase in resolution helped to identify and quantify various classes of polycyclic aromatic hydrocarbons (PAHs) in the combustion emissions that had not been identified previously. Significant emissions (when compared to industrial sources) of known EDCs, dioctyl phthalate (over ∼2,500,000 kg year−1) and bisphenol A (over ∼75,000 kg year−1) were estimated from uncontrolled domestic waste burning. Emissions of several suspected EDCs (oxygenated PAHs) were observed in both diesel soot and the uncontrolled domestic waste burn samples. The emission rates of known and suspected EDCs estimated in this study suggest that combustion emissions need to be characterized for EDCs to further assess its importance as a source of EDC exposure.