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Heritage agrochemicals like myclobutanil, oxyfluorfen, and pronamide, are extensively used in agriculture, with well-established studies on their animal toxicity. Yet, human toxicity assessment relies on conventional human risk assessment approaches including the utilization of animal-based ADME (Absorption, Distribution, Metabolism, and Excretion) data. In recent years, Physiologically Based Pharmacokinetic (PBPK) modelling approaches have played an increasing role in human risk assessment of many chemicals including agrochemicals. This study addresses the absence of PBPK-type data for myclobutanil, oxyfluorfen, and pronamide by generating in vitro data for key input PBPK parameters (Caco-2 permeability, rat plasma binding, rat blood to plasma ratio, and rat liver microsomal half-life), followed by generation of PBPK models for these three chemicals via the GastroPlus TM software. Incorporating these experimental input parameters into PBPK models, the prediction accuracy of plasma AUC (area under curve) was significantly improved. Validation against rat oral administration data demonstrated substantial enhancement. Steady-state plasma concentrations (Css) of pronamide aligned well with published data using measured PBPK parameters. Following validation, parent-based tissue concentrations for these agrochemicals were predicted in humans and rats after single or 30-day repeat exposure of 10 mg/kg/day. These predicted concentrations contribute valuable information for future human toxicity risk assessments of these agrochemicals.

The in vivo working group (WG) considered three topics: acceptable maximum doses for negative erythrocyte micronucleus (MN) tests, validation status of MN assays in non-hematopoietic tissues, and nuisance factors in the comet assay. The WG reached agreement on many issues, including: negative erythrocyte MN studies should be acceptable if dosing is conducted to Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 474 recommendations and if sufficient bone marrow exposure is demonstrated; consensus on the evidence required to demonstrate "sufficient" exposure was not reached. The liver MN test using six-week-old rats is sufficiently validated to develop an OECD TG, but the impact of animal age warrants additional study. Ki-67 is a reliable marker for cellular proliferation in hepatocytes. The gastrointestinal tract MN test is useful for detecting poorly absorbed or rapidly degraded aneugens, and for genotoxic metabolites formed in the colon. Although current validation data are insufficient to support the development of an OECD TG, the methodologies are sufficient to consider as an appendix to OECD TG474. Comparison of comet assay results to laboratory historical control data (HCD) should not be used in data evaluation, unless the HCD distribution is demonstrated to be stable and the predominant source of HCD variation is due to animal, not study, factors. No universally acceptable negative control limit for any tissue was identified. Methodological differences in comet studies can result in variable data interpretations; more data are required before best practice recommendations can be made. Hedgehogs alone are unreliable indicators of cytotoxicity and additional investigations into cytotoxicity markers are required., (© 2023 Environmental Mutagenesis and Genomics Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

1,4-Dioxane is an environmental contaminant that has been shown to cause cancer in rodents after chronic high dose exposures. We reviewed and integrated information from recently published studies to update our understanding of the cancer mode of action of 1,4-dioxane. Tumor development in rodents from exposure to high doses of 1,4-dioxane is preceded by pre-neoplastic events including increased hepatic genomic signaling activity related to mitogenesis, elevation of Cyp2E1 activity and oxidative stress leading to genotoxicity and cytotoxicity. These events are followed by regenerative repair and proliferation and eventual development of tumors. Importantly, these events occur at doses that exceed the metabolic clearance of absorbed 1,4-dioxane in rats and mice resulting in elevated systemic levels of parent 1,4-dioxane. Consistent with previous reviews, we found no evidence of direct mutagenicity from exposure to 1,4-dioxane. We also found no evidence of CAR/PXR, AhR or PPARα activation resulting from exposure to 1,4-dioxane. This integrated assessment supports a cancer mode of action that is dependent on exceeding the metabolic clearance of absorbed 1,4-dioxane, direct mitogenesis, elevation of Cyp2E1 activity and oxidative stress leading to genotoxicity and cytotoxicity followed by sustained proliferation driven by regenerative repair and progression of heritable lesions to tumor development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mark Lafranconi reports financial support and article publishing charges were provided by American Chemistry Council., (Copyright © 2023 The Dow Chemical Company, The Authors. Published by Elsevier Inc. All rights reserved.)

A physiologically based pharmacokinetic (PBPK) model for the important chemical phenoxyethanol (PhE) and its metabolite phenoxyacetic acid (PhAA) was built via GastroPlus TM software (version 9.0) using currently available analytically measured plasma and urinary time-courses of both PhE and its metabolite PhAA. This model was validated and used to predict tissue and urine concentrations of PhE and its metabolite PhAA in rats and humans after oral and dermal exposures. The prediction results showed that most predicted tissue concentrations of PhE or PhAA were lower than the experimental tissue concentrations based on total radioactivity. The predicted cumulative excretion of PhAA in both rats and humans fits very well with most experimental data. With this GastroPlus TM -based model, the margins of exposure (MOE) of PhE and PhAA were also calculated as 194 and 73.7, respectively. The predicted MOE of PhE is two-fold higher than the previous PBPK model built using total radioactivity-based tissue time courses, and the predicted MOE of PhAA was comparable to the previous PBPK model. These data indicate that for chemicals like PhE, GastroPlus TM can integrate multiple data sets into PBPK models to predict PK parameters for parent and metabolites in both rats and humans following intravenous, dermal, or oral exposures.

Nitrapyrin, a nitrification inhibitor, produces liver tumors in B6C3F1 mice. In a 2-year oncogenicity study, increased incidence of mice with hepatocellular tumors was observed following exposure to 125 (females only) or 250 mg/kg/day (males and females) nitrapyrin in the diet. Previous data was generated in male mice to support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor (NR) activation, increased hepatocellular proliferation, and subsequent hepatocellular foci and tumor formation. Uncertainty as to the relevance of this MoA for females remained given the increased sensitivity to tumor formation in female mice. A targeted MoA study was conducted to evaluate CAR activation and hepatic responses in female mice treated with the female carcinogenic dose of nitrapyrin for 4 days. Nitrapyrin induced a treatment-related increase in hepatocellular hypertrophy and hepatocellular proliferation. Nitrapyrin also induced a dose-related increase in the Cyp2b10 /CAR-associated transcript and liver weights. Nitrapyrin-induced liver weights and Cyp2b10 gene expression for both males and females were compared to data generated from three other established CAR activators; methyl isobutyl ketone, phenobarbital, and sulfoxaflor. The response observed in female mice following exposure to nitrapyrin was within range of the degree of change observed in mice following exposure to tumorigenic doses of other CAR activators. Consistent with the liver MoA in male mice, these data support a CAR-mediated mode of action for nitrapyrin-induced liver tumors in female mice, with the understanding that a focused approach minimizing animal use can bridge male and female datasets when sex-specific carcinogenic differences are observed., Competing Interests: Authors LM, KJ, RR, XW, JL, and ML were employed by the company Corteva Agriscience and The Dow Chemical Company. The funders had no involvement with study design or interpretation., (Copyright © 2021 Murphy, LeBaron, Johnson, Rasoulpour, Wang and LaRocca.)

The in vivo comet assay can evaluate the genotoxic potential of a chemical in theoretically any tissue that can be processed to a single cell suspension. This flexibility enables evaluation of point-of-contact tissues using a relevant route of test material administration; however, assessing cytotoxicity is essential for the interpretation of comet results. Histopathological evaluation is routinely utilized to assess cytotoxicity, but temporal- and cell-specific considerations may compromise applicability to the comet assay. In the present study, 1,1'-methylenebis(4-isocyanatobenzene) (4,4'-MDI) was administered to rats for 6 h by nose-only inhalation, and the comet assay was conducted to evaluate genotoxicity in the site-of-contact tissue (bronchoalveolar lavage cells) and distal tissues (liver and glandular stomach). Given the reactive nature of MDI, cellular and molecular metrics at the site-of-contact- including inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress- were used to set appropriate exposure concentrations, in addition to the standard systemic measures of toxicity. In the range-finding study, a concentration of 4 mg/m 3 was considered the maximum noninflammatory concentration; hence target concentrations of 2, 5, and 11 mg/m 3 were selected for the comet study. In the lung lavage, MDI exposure substantially increased total protein and β-glucuronidase, along with cellular apoptosis. Although MDI did not increase the comet assay response (% tail DNA) in any of the tissues examined, the positive control (ethyl methanesulfonate, EMS) significantly increased % tail DNA in all tissues. In total, these data indicate that appropriate cellular and molecular measurements may facilitate dose selection to discern cellular status in the comet assay., (© 2021 The Dow Chemical Company. Environmental and Molecular Mutagenesis published by Wiley Periodicals LLC on behalf of Environmental Mutagen Society.)

The regulatory community is transitioning to the use of nonanimal methods for dermal sensitization assessments; however, some in vitro assays have limitations in their domain of applicability depending on the properties of chemicals being tested. This study explored the utility of epidermal sensitization assay (EpiSensA) to evaluate the sensitization potential of complex and/or "difficult to test" chemicals. Assay performance was evaluated by testing a set of 20 test chemicals including 10 methacrylate esters, 5 silicone-based compounds, 3 crop protection formulations, and 2 surfactant mixtures; each had prior in vivo data plus some in silico and in vitro data. Using the weight of evidence (WoE) assessments by REACH Lead Registrants, 14 of these chemicals were sensitizers and, six were nonsensitizers based on in vivo studies (local lymph node assay [LLNA] and/or guinea pig studies). The EpiSensA correctly predicted 16/20 materials with three test materials as false positive and one silane as false negative. This silane, classified as weak sensitizer via LLNA, also gave a "false negative" result in the KeratinoSens™ assay. Overall, consistent with prior evaluations, the EpiSensA demonstrated an accuracy level of 80% relative to available in vivo WoE assessments. In addition, potency classification based on the concentration showing positive marker gene expression of EpiSensA was performed. The EpiSensA correctly predicted the potency for all seven sensitizing methacrylates classified as weak potency via LLNA (EC3 ≥ 10%). In summary, EpiSensA could identify dermal sensitization potential of these test substances and mixtures, and continues to show promise as an in vitro alternative method for dermal sensitization., (© 2020 John Wiley & Sons, Ltd.)

1,2-dichloroethane (DCE or EDC) is a chlorinated hydrocarbon used as a chemical intermediate, including in the synthesis of polyvinyl chloride. Although DCE has induced tumors in both rats and mice, the overall weight-of-evidence suggests a lack of in vivo mutagenicity. The present study was conducted to explore a potential mode of action further for tumor formation in rat mammary tissue. Fischer 344 rats were exposed to target concentrations of 0 or 200 ppm of DCE vapors (6 hours/day, 7 days/week) for at least 28 days; 200 ppm represents a concentration of ~20% higher than that reported to induce mammary tumors. Endpoints examined included DNA damage (via Comet assay), glutathione (reduced, oxidized and conjugated), tissue DNA adducts, cell proliferation and serum prolactin levels. Exposure to DCE did not alter serum prolactin levels with consistent estrous stage, did not cause cell proliferation in mammary epithelial cells, nor result in histopathological alterations in the mammary gland. DNA adducts were identified, including the N 7 -guanylethyl glutathione adduct, with higher adduct levels measured in liver (nontumorigenic target) compared with mammary tissue isolated from the same rats; no known mutagenic adducts were identified. DCE did not increase the Comet assay response in mammary epithelial cells, whereas DNA damage in the positive control (N-nitroso-N-methylurea) was significantly increased. Although the result of this study did not identify a specific mode of action for DCE-induced mammary tumors in rats, the lack of any exposure-related genotoxic responses further contributes to the weight-of-evidence suggesting that DCE is a nongenotoxic carcinogen., (© 2020 John Wiley & Sons, Ltd.)

We previously demonstrated that the Connectivity Map (CMap) (Lamb et al., 2006) concept can be successfully applied to a predictive toxicology paradigm to generate meaningful MoA-based connections between chemicals (De Abrew et al., 2016). Here we expand both the chemical and biological (cell lines) domain for the method and demonstrate two applications, both in the area of read across. In the first application we demonstrate CMap's utility as a tool for testing biological relevance of source chemicals (analogs) during a chemistry led read across exercise. In the second application we demonstrate how CMap can be used to identify functionally relevant source chemicals (analogs) for a structure of interest (SOI)/target chemical with minimal knowledge of chemical structure. Finally, we highlight four factors: promiscuity of chemical, dose, cell line and timepoint as having significant impact on the output. We discuss the biological relevance of these four factors and incorporate them into a work flow., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Developmental neurotoxicity (DNT) is an important endpoint for the safety assessment of chemicals. However, the current in vivo animal model for DNT assessment is resource-intensive and may not fully capture all mechanisms that may be relevant to DNT in humans. As a result, there is a growing need for more reliable, time- and cost-efficient approaches for DNT evaluation. Toward this end, many stem/progenitor cell-based in vitro models and alternative organism-based models are becoming available with the potential for high throughput screening of DNT. Meanwhile, with advances in the knowledgebase of DNT molecular mechanisms and the identification of DNT-related adverse outcome pathways (AOP) there is potential to develop a mechanism-based integrated testing strategy for DNT assessment. This review summarizes the state of science regarding currently available human stem/progenitor cell-based in vitro models and alternative organism-based models that could be used for DNT testing. In addition, the current knowledge regarding DNT AOPs is reviewed to identify common key events that could serve as critical endpoints to assess multiple AOPs that underlie DNT. Following the identification of common key events, a streamlined strategy is proposed using alternative models to assess the DNT potential of chemicals as an early screening approach for chemicals in development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A database of 91 chemicals with published data from both transgenic rodent mutation (TGR) and rodent comet assays has been compiled. The objective was to compare the sensitivity of the two assays for detecting genotoxicity. Critical aspects of study design and results were tabulated for each dataset. There were fewer datasets from rats than mice, particularly for the TGR assay, and therefore, results from both species were combined for further analysis. TGR and comet responses were compared in liver and bone marrow (the most commonly studied tissues), and in stomach and colon evaluated either separately or in combination with other GI tract segments. Overall positive, negative, or equivocal test results were assessed for each chemical across the tissues examined in the TGR and comet assays using two approaches: 1) overall calls based on weight of evidence (WoE) and expert judgement, and 2) curation of the data based on a priori acceptability criteria prior to deriving final tissue specific calls. Since the database contains a high prevalence of positive results, overall agreement between the assays was determined using statistics adjusted for prevalence (using AC1 and PABAK). These coefficients showed fair or moderate to good agreement for liver and the GI tract (predominantly stomach and colon data) using WoE, reduced agreement for stomach and colon evaluated separately using data curation, and poor or no agreement for bone marrow using both the WoE and data curation approaches. Confidence in these results is higher for liver than for the other tissues, for which there were less data. Our analysis finds that comet and TGR generally identify the same compounds (mainly potent mutagens) as genotoxic in liver, stomach and colon, but not in bone marrow. However, the current database content precluded drawing assay concordance conclusions for weak mutagens and non-DNA reactive chemicals., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

The Pig-a assay is an emerging and promising in vivo method to determine mutagenic potential of chemicals. Since its development in 2008, remarkable progress has been made in harmonizing and characterizing the test procedures, primarily using known mutagenic chemicals. The purpose of the present study was to evaluate specificity of the Pig-a assay using two nongenotoxic and well-characterized rodent liver carcinogens, phenobarbital and clofibrate, in male F344/DuCrl rats. Daily oral administration of phenobarbital or clofibrate at established hepatotoxic doses for 28 days resulted in substantial hepatic alterations, however, did not increase the frequency of Pig-a mutation markers (RET CD59- and RBC CD59- ) compared to vehicle control or pre-exposure (Day -5) mutant frequencies. These results are consistent with the existing literature on the nonmutagenic mode of action (MoA) of phenobarbital and clofibrate liver tumors. The present study contributes to the limited, but expanding evidence on the specificity of the Pig-a assay and further for the investigations of carcinogenic MoAs, i.e., mutagenic or nonmutagenic potential of chemicals. Environ. Mol. Mutagen. 60:42-46, 2019. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Methods for investigating the Mode of Action (MoA) for rodent liver tumors via constitutive androstane receptor (CAR) activation are outlined here, based on current scientific knowledge about CAR and feedback from regulatory agencies globally. The key events (i.e., CAR activation, altered gene expression, cell proliferation, altered foci and increased adenomas/carcinomas) can be demonstrated by measuring a combination of key events and associative events that are markers for the key events. For crop protection products, a primary dataset typically should include a short-term study in the species/strain that showed the tumor response at dose levels that bracket the tumorigenic and non-tumorigenic dose levels. The dataset may vary depending on the species and the test compound. As examples, Case Studies with nitrapyrin (in mice) and metofluthrin (in rats) are described. Based on qualitative differences between the species, the key events leading to tumors in mice or rats by this MoA are not operative in humans. In the future, newer approaches such as a CAR biomarker signature approach and/or in vitro CAR3 reporter assays for mouse, rat and human CAR may eventually be used to demonstrate a CAR MoA is operative, without the need for extensive additional studies in laboratory animals., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Nitrapyrin, a nitrification inhibitor, produces liver tumors in mice at high doses. Several experiments were performed to investigate molecular, cellular, and apical endpoints to define the key events leading to the tumor formation. These data support a mode-of-action (MoA) characterized by constitutive androstane receptor (CAR) nuclear receptor activation, increased hepatocellular proliferation leading to hepatocellular foci and tumor formation. Specifically, nitrapyrin induced a dose-related increase in the Cyp2b10/CAR-associated transcript and protein. Interestingly, the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD) was not enhanced due to nitrapyrin-mediated suicide inhibition of PROD activity. Nitrapyrin exposure elicited a clear dose-responsive increase in hepatocellular proliferation in wild-type mice, but not in CAR knock-out mice, informing that CAR activation is an obligatory key event in this test material-induced hepatocarcinogenesis. Furthermore, nitrapyrin exposure induced a clear, concentration-responsive increase in cell proliferation in mouse, but not human, hepatocytes in vitro . Evaluation of the data from repeat dose and MoA studies by the Bradford Hill criteria and a Human Relevance Framework (HRF) suggested that nitrapyrin-induced mouse liver tumors are not relevant to human health risk assessment because of qualitative differences between these two species.

The Pig-a assay, a recently developed in vivo somatic gene mutation assay, is based on the identification of mutant erythrocytes that have an altered repertoire of glycosylphosphatidylinositol (GPI)-anchored cell surface markers. We hypothesized that the erythrocyte Pig-a assay concept could be applied to rat cauda epididymal spermatozoa (sperm) for germ cell mutagenicity evaluation. We used GPI-anchored CD59 as the Pig-a mutation marker and examined the frequency of CD59-negative sperm using flow cytometry. A reconstruction experiment that spiked un-labeled sperm (mutant-mimic) into labeled sperm at specific ratios yielded good agreement between the detected and expected frequencies of mutant-mimic sperm, demonstrating the analytical ability for CD59-negative sperm detection. Furthermore, this methodology was assessed in F344/DuCrl rats administered N-ethyl-N-nitrosourea (ENU), a prototypical mutagen, or clofibrate, a lipid-lowering drug. Rats treated with 1, 10, or 20 mg/kg body weight/day (mkd) ENU via daily oral garage for five consecutive days showed a dose-dependent increase in the frequency of CD59-negative sperm on study day 63 (i.e., 58 days after the last ENU dose). This ENU dosing regimen also increased the frequency of CD59-negative erythrocytes. In rats treated with 300 mkd clofibrate via daily oral garage for consecutive 28 days, no treatment-related changes were detected in the frequency of CD59-negative sperm on study day 85 (i.e., 57 days after the last dose) or in the frequency of CD59-negative erythrocytes on study day 29. In conclusion, these data suggest that the epidiymal sperm Pig-a assay in rats is a promising method for evaluating germ cell mutagenicity. Environ. Mol. Mutagen. 58:485-493, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Mutagenicity is an important toxicological endpoint that requires thorough evaluation during the industrial chemical registration process. Regulatory requirements for mutagenicity assessment in registration of industrial chemicals vary in geographic regions (and in some cases by intended application). Here we compile the mutagenicity testing requirements for registration of industrial chemicals from representative geographic regions (in alphabetical order), that is Australia, Brazil, Canada, China, European Union (EU), India, Japan, South Korea, Taiwan, and United States (US). We further discuss the challenges that industry is facing to meet global regulations, for example, different testing requirements among geographic regions, different strategies in follow-up tests to in vitro positive findings, no-observed-adverse-effect-levels in genetic toxicity testing, and human relevance of mutagenicity. Environ. Mol. Mutagen. 58:345-353, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

For several decades, regulatory testing schemes for genetic damage have been standardized where the tests being utilized examined mutations and structural and numerical chromosomal damage. This has served the genetic toxicity community well when most of the substances being tested were amenable to such assays. The outcome from this testing is usually a dichotomous (yes/no) evaluation of test results, and in many instances, the information is only used to determine whether a substance has carcinogenic potential or not. Over the same time period, mechanisms and modes of action (MOAs) that elucidate a wider range of genomic damage involved in many adverse health outcomes have been recognized. In addition, a paradigm shift in applied genetic toxicology is moving the field toward a more quantitative dose-response analysis and point-of-departure (PoD) determination with a focus on risks to exposed humans. This is directing emphasis on genomic damage that is likely to induce changes associated with a variety of adverse health outcomes. This paradigm shift is moving the testing emphasis for genetic damage from a hazard identification only evaluation to a more comprehensive risk assessment approach that provides more insightful information for decision makers regarding the potential risk of genetic damage to exposed humans. To enable this broader context for examining genetic damage, a next generation testing strategy needs to take into account a broader, more flexible approach to testing, and ultimately modeling, of genomic damage as it relates to human exposure. This is consistent with the larger risk assessment context being used in regulatory decision making. As presented here, this flexible approach for examining genomic damage focuses on testing for relevant genomic effects that can be, as best as possible, associated with an adverse health effect. The most desired linkage for risk to humans would be changes in loci associated with human diseases, whether in somatic or germ cells. The outline of a flexible approach and associated considerations are presented in a series of nine steps, some of which can occur in parallel, which was developed through a collaborative effort by leading genetic toxicologists from academia, government, and industry through the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Genetic Toxicology Technical Committee (GTTC). The ultimate goal is to provide quantitative data to model the potential risk levels of substances, which induce genomic damage contributing to human adverse health outcomes. Any good risk assessment begins with asking the appropriate risk management questions in a planning and scoping effort. This step sets up the problem to be addressed (e.g., broadly, does genomic damage need to be addressed, and if so, how to proceed). The next two steps assemble what is known about the problem by building a knowledge base about the substance of concern and developing a rational biological argument for why testing for genomic damage is needed or not. By focusing on the risk management problem and potential genomic damage of concern, the next step of assay(s) selection takes place. The work-up of the problem during the earlier steps provides the insight to which assays would most likely produce the most meaningful data. This discussion does not detail the wide range of genomic damage tests available, but points to types of testing systems that can be very useful. Once the assays are performed and analyzed, the relevant data sets are selected for modeling potential risk. From this point on, the data are evaluated and modeled as they are for any other toxicology endpoint. Any observed genomic damage/effects (or genetic event(s)) can be modeled via a dose-response analysis and determination of an estimated PoD. When a quantitative risk analysis is needed for decision making, a parallel exposure assessment effort is performed (exposure assessment is not detailed here as this is not the focus of this discussion; guidelines for this assessment exist elsewhere). Then the PoD for genomic damage is used with the exposure information to develop risk estimations (e.g., using reference dose (RfD), margin of exposure (MOE) approaches) in a risk characterization and presented to risk managers for informing decision making. This approach is applicable now for incorporating genomic damage results into the decision-making process for assessing potential adverse outcomes in chemically exposed humans and is consistent with the ILSI HESI Risk Assessment in the 21st Century (RISK21) roadmap. This applies to any substance to which humans are exposed, including pharmaceuticals, agricultural products, food additives, and other chemicals. It is time for regulatory bodies to incorporate the broader knowledge and insights provided by genomic damage results into the assessments of risk to more fully understand the potential of adverse outcomes in chemically exposed humans, thus improving the assessment of risk due to genomic damage. The historical use of genomic damage data as a yes/no gateway for possible cancer risk has been too narrowly focused in risk assessment. The recent advances in assaying for and understanding genomic damage, including eventually epigenetic alterations, obviously add a greater wealth of information for determining potential risk to humans. Regulatory bodies need to embrace this paradigm shift from hazard identification to quantitative analysis and to incorporate the wider range of genomic damage in their assessments of risk to humans. The quantitative analyses and methodologies discussed here can be readily applied to genomic damage testing results now. Indeed, with the passage of the recent update to the Toxic Substances Control Act (TSCA) in the US, the new generation testing strategy for genomic damage described here provides a regulatory agency (here the US Environmental Protection Agency (EPA), but suitable for others) a golden opportunity to reexamine the way it addresses risk-based genomic damage testing (including hazard identification and exposure). Environ. Mol. Mutagen. 58:264-283, 2017. © 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc., (© 2016 The Authors. Environmental and Molecular Mutagenesis Published by Wiley Periodicals, Inc.)

In a National Toxicology Program (NTP) chronic inhalation study with methyl isobutyl ketone (MIBK), increases in hepatocellular adenomas and hepatocellular adenomas and carcinomas (combined) were observed in male and female B6C3F 1 mice at 1800 ppm. A DNA reactive Mode-of-Action (MOA) for this liver tumor response is not supported by the evidence as MIBK and its major metabolites lack genotoxicity in both in vitro and in vivo studies. Constitutive androstane receptor (CAR) nuclear receptor-mediated activation has been hypothesized as the MOA for MIBK-induced mouse liver tumorigenesis. To further investigate the MOA for MIBK-induced murine liver tumors, male and female B6C3F1, C57BL/6, and CAR/PXR Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/day, 5 days/week for a total of 10 days. On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1-3 h following the final exposure. B6C3F 1 and C57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F 1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, and no increase was observed in MIBK exposed CAR/PXR KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (CAR-associated) transcript and a slight increase in Cyp3a11(PXR-associated) transcript in B6C3F 1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR-associated) or Cyp4a10 (PPAR-α-associated) transcripts. CAR/PXR KO mice exposed to 1800 ppm MIBK showed no evidence of activation of AhR, CAR, PXR or PPAR-α nuclear receptors via their associated transcripts. MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the CAR and PXR nuclear receptors and resultant hepatocellular proliferation leading to rodent liver tumors., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The nature of the dose-response relationship for various in vivo endpoints of exposure and effect were investigated using the alkylating agents, methyl methanesulfonate (MMS) and methylnitrosourea (MNU). Six male F344 rats/group were dosed orally with 0, 0.5, 1, 5, 25 or 50mg/kg bw/day (mkd) of MMS, or 0, 0.01, 0.1, 1, 5, 10, 25 or 50 mkd of MNU, for 4 consecutive days and sacrificed 24h after the last dose. The dose-responses for multiple biomarkers of exposure and genotoxic effect were investigated. In MMS-treated rats, the hemoglobin adduct level, a systemic exposure biomarker, increased linearly with dose (r (2) = 0.9990, P < 0.05), indicating the systemic availability of MMS; however, the N7MeG DNA adduct, a target exposure biomarker, exhibited a non-linear dose-response in blood and liver tissues. Blood reticulocyte micronuclei (MN), a genotoxic effect biomarker, exhibited a clear no-observed-genotoxic-effect-level (NOGEL) of 5 mkd as a point of departure (PoD) for MMS. Two separate dose-response models, the Lutz and Lutz model and the stepwise approach using PROC REG both supported a bilinear/threshold dose-response for MN induction. Liver gene expression, a mechanistic endpoint, also exhibited a bilinear dose-response. Similarly, in MNU-treated rats, hepatic DNA adducts, gene expression changes and MN all exhibited clear PoDs, with a NOGEL of 1 mkd for MN induction, although dose-response modeling of the MNU-induced MN data showed a better statistical fit for a linear dose-response. In summary, these results provide in vivo data that support the existence of clear non-linear dose-responses for a number of biologically significant events along the pathway for genotoxicity induced by DNA-reactive agents., (© The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glutathione (GSH), glutathione disulfide (GSSG) and 2-hydroxyethylated glutathione (HESG) are important biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) or ethylene in vivo. A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of GSH, GSSG and HESG in mouse lung tissues after inhalation exposure to EO. The lower limit of quantitation for all these biomarkers was 0.002 µg/mL. The linearity of the calibration curves for all analytes was >0.998. The intra-day assay precision relative standard deviation (RSD) values for quality control samples for all analytes were ≤12.8% with accuracy values ranging from 87.2 to 113%. The inter-day assay precision (RSD) values for all analytes were ≤13.1% with accuracy values ranging from 86.9 to 103%. This method was applied to concurrently determine the levels of GSH, GSSG and HESG in lung samples isolated from mouse after 4-week inhalation exposure to EO at 0, 10, 50, 100 and 200 ppm., (Copyright © 2015 John Wiley & Sons, Ltd.)

Dietary exposure to pronamide resulted in higher incidences of Leydig cell tumors (LCT) at 1000ppm in a 2-year cancer bioassay, but there were no testes effects at 40 or 200ppm, and no testes effects at 12-months at any concentration. A 90-day mode-of-action (MoA) study was conducted at concentrations of 0, 200, 1000 and 2000ppm. Standard parameters and stereological and proliferation analyses of LCs, targeted testis and liver gene expression, in vitro metabolism of testosterone by liver microsomes, and quantification of serum hormones and testosterone metabolites were evaluated. Increased testosterone metabolism due to increases in hepatic microsomal activity, alterations in serum hormone levels, and other data suggest that LCTs were mediated through a perturbation of the HPG-axis. Data suggest that this occurs after a threshold of exposure is reached, indicating a nonlinear/threshold dose-response. Pronamide-induced rat LCTs mediated by alterations to the HPG-axis have low relevance to humans due to quantitative differences in sensitivity between rats and humans to LCTs. Pronamide displayed no genotoxicity or direct endocrine effects. A margin of exposure approach for risk assessment and derivation of the chronic reference dose based on a point of departure of 200ppm is most appropriate and protective of human health., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Pronamide, a selective, systemic, pre- and post-emergence herbicide, caused an increased incidence of thyroid follicular cell adenomas in a rat carcinogenicity study. Thyroid tumors, as well as liver and pituitary changes, were limited only to the high-dose group. The evidence for and against specific potential modes of action (MoAs) for rat thyroid follicular cell adenomas and their relevance to humans is discussed. Pronamide is not mutagenic and therefore, direct DNA reactivity is not relevant as a MoA. The hypothesized MoA for this effect is altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis mediated by the induction of hepatic enzymes, including uridine diphosphate glucuronosyltransferase (UGT). Evaluation of data from a series of regulatory guideline and MoA studies aimed at identifying the causative and associated key events supported a UGT-mediated MoA in the development of thyroid follicular tumors. This MoA for pronamide-induced thyroid tumors in rats, which involves increased thyroid hormone metabolism/clearance, altered thyroid hormone homeostasis and HPT stimulation is not considered relevant to humans based on quantitative species differences, making rats markedly more sensitive than humans to thyroid perturbations., (Copyright © 2015. Published by Elsevier Inc.)

Non-genotoxic carcinogens act by promoting the clonal expansion of preneoplastic cells by directly or indirectly stimulating cell division or inhibiting cell loss in the target organ. The specific mode-of-action (MoA) by which some non-genotoxic carcinogens ultimately cause cancer is not completely understood. To date, there are several proposed MoAs for non-genotoxic carcinogens, and some of these propose inhibition of apoptosis as one of the key events. In general, inhibition of apoptosis is considered a necessary step for cell survival and in theory can occur in combination or in association with other key promotional events, such as cell proliferation, oxidative stress and inhibition of intercellular communication to promote carcinogenesis. However, the evidence supporting the role of inhibition of apoptosis as a necessary step in promoting specific chemically induced tumors is often debated. To address this evidence, we reviewed studies that utilized prototypical nuclear receptor-mediated hepatocarcinogens. Based on this review, it is proposed that the ability to determine the importance of inhibition of apoptosis as a key event in the MoA for tumor promotion is hampered by the limitations of the methods utilized for its detection. This review provides an assessment of the strengths and limitations of the current methodology used for detection of apoptosis and provides suggestions for improving its detection, thereby strengthening the weight of evidence supporting inhibition of apoptosis as a key event in a MoA for tumor promotion.

2-Hydroxyethylated and oxidative DNA nucleosides (DNA adduct biomarkers), such as O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6HEdG), N6-(2-hydroxyethyl)-2'-deoxyadenosine (N6HEdA), 1-(2-hydroxyethyl)-2'-deoxyadenosine (N1HEdA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG), N2,3-etheno-2'-deoxyguanosine (N2,3-ethenodG), α-methyl-γ-hydroxy-1,N2-propano-2'-deoxyguanosine (CrotondG), are important proposed biomarkers for exploring the genotoxicity mechanism of ethylene oxide (EO) in vivo. A liquid chromatography-tandem mass spectrometric method was developed for the simultaneous determination of O6HEdG, N6HEdA, N1HEdA, 8-OHdG, CrotondG, and N2,3-ethenodG together with regular 2'-deoxyguanosine (dG), and 2'-deoxyadenosine (dA) nucleosides in the DNA extracted from mouse lung tissues for the assessment of exposure to EO after inhalation. The lower limits of quantitation for 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, N1HEdA, N6HEdA, dG, and dA were 0.025, 0.00125, 0.025, 0.00125, 0.025, 0.01, 2342, and 2500ng/mL, respectively. The linearity of the calibration curves for all analytes were >0.989. The intra-day assay precision relative standard deviation (RSD) values for quality control (QC) samples for all analytes were ≤13.5% with accuracy values ranging from 86.5% to 111%. The inter-day assay precision (RSD) values for all analytes were ≤18.8% with accuracy values ranging from 87.9% to 119%. This method was used for simultaneous determination of the levels of 8-OHdG, CrotondG, N2,3-EthenodG, O6HEdG, dG, N1HEdA, N6HEdA, and dA in DNA enzymatic hydrolysates from DNA extracted from mouse lung after 12 weeks' inhalation exposure to EO at atmospheric concentrations of 0, 100, and 200ppm. Overall, N2,3-ethenodG was not detected in any samples. 8-OHdG, CrotondG, dG, and dA were all quantifiable in all samples. O6HEdG, N1HEdA, and N6HEdA were quantifiable in most samples and the ratio of the corresponding adduct versus their corresponding DNA base (dG or dA) [×10 (e6)] was increased as the EO exposure concentration increased., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Integrated testing strategies involve the assessment of multiple endpoints within a single toxicity study and represent an important approach for reducing animal use and streamlining testing. The present study evaluated the ability to combine general, immune, and genetic toxicity endpoints into a single study. Specifically, this study evaluated the impact of sheep red blood cell (SRBC) immunization, as part of the T-cell dependent antibody response (TDAR) assay, on organ weights, micronuclei (MN) formation (bone marrow and peripheral blood), and the Comet assay response in the liver of female F344/DuCrl rats treated with cyclophosphamide (CP) a known immunosuppressive chemical and genotoxicant. For the TDAR assay, treatment with CP resulted in a dose-dependent decrease in the antibody response with a suppression of greater than 95% at the high dose. Injection with SRBC had no impact on evaluated organ weights, histopathology, hematology, and clinical chemistry parameters. Analysis of MN formation in bone marrow and peripheral blood revealed a dose-dependent increase in response to CP treatment. Injection with SRBC had no impact on the level of MN in control animals and did not alter the dose response of CP. There was a slight increase in liver DNA damage in response to CP as measured by the Comet assay; however, injection with SRBCs did not alter this endpoint. Overall these data provide strong support for the concurrent assessment of general, immune, and genetic toxicology endpoints within a single study as part of an integrated testing strategy approach., (© 2014 Wiley Periodicals, Inc.)

The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was evaluated in five in vitro screening assays to assess the potential for interaction with the androgen, estrogen and steroidogenesis pathways in the endocrine system. The assays were conducted to meet the requirements of the in vitro component of Tier 1 of the United States Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), and included assays for estrogen receptor (ER) binding (rat uterine cytosol ER binding assay), ER-mediated transcriptional activation (HeLa-9903-ERα transactivation assay), androgen receptor (AR) binding (rat prostate cytosol AR binding assay), aromatase enzymatic activity inhibition (recombinant human CYP19 aromatase inhibition assay), and interference with steroidogenesis (H295R steroidogenesis assay). Results from these five assays demonstrated that 2,4-D does not have the potential to interact in vitro with the estrogen, androgen, or steroidogenesis pathways. These in vitro data are consistent with a corresponding lack of endocrine effects observed in apical in vivo animal studies, and thus provide important supporting data valuable in a comprehensive weight of evidence evaluation indicating a low potential of 2,4-D to interact with the endocrine system., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Sulfoxaflor, a molecule that targets sap-feeding insects, was assessed for carcinogenic potential in groups of 50 Fischer rats fed with diets containing 0, 25, 100, 500 (males), or 750 (females) ppm sulfoxaflor for 2 years according to OECD 453. Sulfoxaflor did not alter the number of rats with Leydig cell tumors (LCTs: 88% of controls and 90-92% in treated groups). The size of LCT was increased at 100 and 500 ppm. The spontaneous incidence of LCT in Fischer rat is 75-100% compared with less than 0.01% in humans. These fundamental interspecies differences in spontaneous incidence of LCT are the result of quantitative and qualitative differences in Leydig cell response to hormonal stimuli. There are nine known modes of actions (MoA) for LCT induction. Analysis sulfoxaflor data suggested a hormone-based dopamine enhancement MoA causing the LCT effect through: 1) increased neuronal dopamine release via specific dopaminergic neuron-based nicotinic acetylcholine receptor (nAChR) agonism, leading to 2) decreased serum prolactin (Prl) levels, 3) downregulation of luteinizing hormone receptor (LHR) gene expression in Leydig cells, 4) transient decreases in serum testosterone, 5) increased serum LH levels, and 6) promotion of LCTs. The analysis suggested that sulfoxaflor promoted LCTs through a subtle stimulation of dopamine release. The MoA for LCT promotion in the carcinogenicity study is considered to have no relevance to humans due to qualitative and quantitative differences between rat and human Leydig cells. Therefore, the Fischer 344 rat LCT promotion associated with lifetime administration of high-dose levels of sulfoxaflor would not pose a cancer hazard to humans.

Sulfoxaflor, a novel active substance that targets sap-feeding insects, induced rodent hepatotoxicity when administered at high dietary doses. Specifically, hepatocellular adenomas and carcinomas increased after 18 months in male and female CD-1 mice at 750 and 1250 ppm, respectively, and hepatocellular adenomas increased after 2 years in male F344 rats at 500 ppm. Studies to determine the mode of action (MoA) for these liver tumors were performed in an integrated and prospective manner as part of the standard battery of toxicology studies such that the MoA data were available prior to, or by the time of, the completion of the carcinogenicity studies. Sulfoxaflor is not genotoxic and the MoA data support the following key events in the etiology of the rodent liver tumors: (1) CAR nuclear receptor activation and (2) hepatocellular proliferation. The MoA data were evaluated in a weight of evidence approach using the Bradford Hill criteria for causation and were found to align with dose and temporal concordance, biological plausibility, coherence, strength, consistency, and specificity for a CAR-mediated MoA while excluding other alternate MoAs. The available data include: activation of CAR, Cyp2b induction, hepatocellular hypertrophy and hyperplasia, absence of liver effects in KO mice, absence of proliferation in humanized mice, and exclusion of other possible mechanisms (e.g., genotoxicity, cytotoxicity, AhR, or PPAR activation), and indicate that the identified rodent liver tumor MoA for sulfoxaflor would not occur in humans. In this case, sulfoxaflor is considered not to be a potential human liver carcinogen.

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17β-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid., (© 2014 Wiley Periodicals, Inc.)

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc., (© 2014 Wiley Periodicals, Inc.)

Chlorpyrifos was selected for EPA's Endocrine Disruptor Screening Program (EDSP) based on widespread use and potential for human and environmental exposures. The purpose of the program is to screen chemicals for their potential to interact with the estrogen, androgen, or thyroid pathways. A battery of 11 assays was completed for chlorpyrifos in accordance with test guidelines developed for EDSP Tier 1 screening. To determine potential endocrine activity, a weight-of-evidence (WoE) evaluation was completed for chlorpyrifos, which included the integration of EDSP assay results with data from regulatory guideline studies and the published literature. This WoE approach was based on the OECD conceptual framework for testing and assessment of potential endocrine-disrupting chemicals and consisted of a systematic evaluation of data, progressing from simple to complex across multiple levels of biological organization. The conclusion of the WoE evaluation is that chlorpyrifos demonstrates no potential to interact with the estrogen, androgen, or thyroid pathways at doses below the dose levels that inhibit cholinesterase. Therefore, regulatory exposure limits for chlorpyrifos, which are based on cholinesterase inhibition, are sufficient to protect against potential endocrine alterations. Based on the results of this WoE evaluation, there is no scientific justification for pursuing additional endocrine testing for chlorpyrifos., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Registration of new plant protection products (e.g., herbicide, insecticide, or fungicide) requires comprehensive mammalian toxicity evaluation including carcinogenicity studies in two species. The outcome of the carcinogenicity testing has a significant bearing on the overall human health risk assessment of the substance and, consequently, approved uses for different crops across geographies. In order to understand the relevance of a specific tumor finding to human health, a systematic, transparent, and hypothesis-driven mode of action (MoA) investigation is, appropriately, an expectation by the regulatory agencies. Here, we describe a novel approach of prospectively generating the MoA data by implementing additional end points to the standard guideline toxicity studies with sulfoxaflor, a molecule in development. This proactive MoA approach results in a more robust integration of molecular with apical end points while minimizing animal use. Sulfoxaflor, a molecule targeting sap-feeding insects, induced liver effects (increased liver weight due to hepatocellular hypertrophy) in an initial palatability probe study for selecting doses for subsequent repeat-dose dietary studies. This finding triggered the inclusion of dose-response investigations of the potential key events for rodent liver carcinogenesis, concurrent with the hazard assessment studies. As predicted, sulfoxaflor induced liver tumors in rats and mice in the bioassays. The MoA data available by the time of the carcinogenicity finding supported the conclusion that the carcinogenic potential of sulfoxaflor was due to CAR/PXR nuclear receptor activation with subsequent hepatocellular proliferation. This MoA was not considered to be relevant to humans as sulfoxaflor is unlikely to induce hepatocellular proliferation in humans and therefore would not be a human liver carcinogen., (Copyright © 2013. Published by Elsevier Inc.)

The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay., (Copyright © 2012 Wiley Periodicals, Inc.)

Introduction: The emerging field of epigenetics has revealed a new layer of gene regulation that is only now being fully explored. Concomitant with the increase in our understanding of epigenetic regulation are questions as to the role environmental factors may play in altering the epigenome. As these correlations between epigenetic changes and toxicity are made, the natural next question is if the current safety assessment paradigm utilizing a no-observed-adverse-effect level (NOAEL) is protective of public health for an epigenetic mechanism., Methods: To begin to answer this question, several case studies were examined where apical end point dose response curves were compared to dose response data on epigenetic end points for 1,3-butadiene, arsenic, and diethylstilbesterol., Results: This limited examination of the available literature for these three molecules revealed that epigenetic alterations largely fell within the dose response curve for apical effects. Perhaps more importantly, this analysis also revealed some key data gaps that should be addressed such as incongruent study designs and limited epigenetic dose response data for only a small subset of known epigenetic marks. Taken together, the answer to the question of whether the current product safety assessment paradigm is protective of epigenetic alterations is "yes, based on our current understanding of epigenetics". That is, this paradigm would be protective of any mechanism that resulted in adverse effects typically observed in guideline studies, because product safety assessment is based upon observed apical effects to drive an overall NOAEL that is the basis to set reference doses for a risk assessment., Discussion: These adverse apical effects are the culmination of all molecular events, regardless of mechanism and may include alterations in the epigenome secondary to the actions of those mechanism(s). The epigenome is in a constant state of flux throughout cellular growth and development, and this dynamic variability is not completely characterized. Thus given the state of our current scientific understanding, a change in itself cannot be contextualized as adverse in the absence of a phenotypic anchor. Clearly, more research is needed in this area to perform additional epigenetic studies that include apical end points with full dose response curves in order to gain a more comprehensive understanding of adverse health outcomes that could be causally linked to epigenetic changes., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Oxyfluorfen is a herbicide that is not genotoxic and produces liver toxicity in rodents, following repeated administration at high dose levels. Lifetime rodent feeding studies reported in 1977 with low-purity oxyfluorfen (85%) showed no increase in any tumor type in rats (800 ppm, high dose) and only a marginally increased incidence of hepatocellular tumors in male CD-1 mice at the highest dose (200 ppm). To evaluate the potential carcinogenicity of the currently registered oxyfluorfen (> 98% purity), we conducted a series of short-term liver mode of action (MOA) toxicology studies in male CD-1 mice administered dietary doses of 0, 40, 200, 800, and 1600 ppm for durations of 3, 7, 10, or 28 days. MOA endpoints examined included liver weight, histopathology, cell proliferation, nuclear receptor-mediated gene expression, and other peroxisome proliferator-specific endpoints and their reversibility. Minimal liver effects were observed in mice administered doses at or below 200 ppm for up to 28 days. Increased liver weight, single-cell necrosis, cell proliferation, and peroxisomal acyl-CoA oxidase (ACO) were observed at 800 ppm after 28 days, but there was no increase in peroxisomes. Expression of Cyp2b10 and Cyp4a10 transcripts, markers of constitutive androstane receptor and peroxisome proliferator activated receptor α nuclear receptor activation, respectively, were increased at 800 and 1600 ppm after 3 or 10 days. Collectively, these data along with the negative genotoxicity demonstrate that oxyfluorfen (> 98% purity) has the potential to induce mouse liver tumors through a nongenotoxic, mitogenic MOA with a clear threshold and is not predicted to be carcinogenic in humans at relevant exposure levels.

The possible interaction of environmental contaminants with the endocrine system has been an environmental concern since the early 1990s. To examine these interactions test guidelines have been introduced by regulatory agencies to screen for possible endocrine active compounds. One of these guidelines is the EPA's OPPTS 890.1550 [Steroidogenesis (Human Cell Line-H295R)]. This guideline requires the quantification of two major biomarkers (testosterone and estradiol) in various biological test systems. Traditional quantitation methodologies such as Radioimmunoassay (RIA) and Enzyme-linked Immunosorbent Assay (ELISA) have been used to quantify low levels of steroids. However, those methodologies have drawbacks such as the radioactive safety, antibody availability, separate assay for each biomarker, and lack of selectivity. In the current study, a rapid and sensitive liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry method (LC/APPI-MS/MS) has been developed and validated for the simultaneous quantitation of testosterone and estradiol in the H295R cell line. Briefly, the media from cultured cells was extracted with dichloromethane (CH(2)Cl(2)) containing internal standards of both testosterone-d(3) and estradiol-(13)C(3); then, the extracted organic layer was concentrated down to dryness. The final residue was derivatized with dansyl chloride solution, and directly analyzed by LC/APPI-MS/MS. The calibration curves, with concentration ranging from 10 to 2500 pg/mL, were linear with coefficient >0.99. The lower limits of quantitation for both testosterone and estradiol were 10 pg/mL. This method was successfully validated to support requirements of the current EPA Steroidogenesis guideline. This type of method may also provide value for rapid and precise measurements of these two hormones in other in vitro or in vivo test systems., (Copyright © 2011 John Wiley & Sons, Ltd.)

There has been a growing concern that epigenetic events, that is, heritable changes in gene expression superimposed on DNA nucleotide sequences, may be involved in chemically and/or nutritionally mediated adverse health outcomes, such as reproductive toxicity and cancer. This concern has been driven by an increasing number of studies reporting toxicant-induced alterations to the epigenome in the form of changes in DNA methylation, histone/chromatin remodeling, and altered expression of non-coding RNAs. These three major mechanisms of epigenetic modifications may have coordinated, independent, or potentially antagonistic influences on gene expression. Complicating this understanding is the incomplete understanding of the normal state and dynamic variation of the epigenome, which differs widely between cells, tissues, developmental state, age, strain, and species. This review serves as a framework to outline characteristics composing an ideal epigenetic screen(s) for hazard identification in product safety assessment. In order to implement such a screen, first there needs to be a better understanding of adaptive versus adverse changes in the epigenome, which includes identification of robust and reproducible causal links between epigenetic changes and adverse apical end points, and second development of improved reporter assay tools to monitor such changes. An ideal screen would be in vitro-based, medium- to high-throughput, and assess all three branches of epigenome control (i.e. methylation, histone modifications, non-coding RNAs), although also being quantitative, objective, portable (i.e. lab to lab), and relevant to humans.

Epigenetics, as it pertains to biology and toxicology, can be defined as heritable changes in gene expression that do not involve mutations and are propagated without continued stimulus. Although potentially reversible, these heritable changes may be classified as mitotic, meiotic, or transgenerational, implicating the wide-ranging impact of epigenetic control in cellular function. A number of biological responses have been classified as being caused by an "epigenetic alteration," sometimes based on sound scientific evidence and often in lieu of an identified genetic mutation. Complicating the understanding and interpretation of perceived epigenetic alterations is an incomplete understanding of the normal state and dynamic variation of the epigenome, which can differ widely between cell and tissue types and stage of development or age. This emerging field is likely to have a profound impact on the study and practice of toxicology in coming years. This document reviews the current state of the science in epigenetic modifications, techniques used to measure these changes, and evaluates the current toxicology testing battery with respect to strengths and potential weaknesses in the identification of epigenetics changes. In addition, case studies implicating transgenerational effects induced by diethylstilbestrol, vinclozolin, and bisphenol A were reviewed to illustrate the application of epigenetics in safety assessment and the strengths and limitations of the study designs. An assessment of toxicology tests currently used in safety evaluation revealed that these tests are expected to identify any potential adverse outcomes resulting from epigenetic changes. Furthermore, in order to increase our understanding of the science of epigenetics in toxicology, this review has revealed that a solid understanding of the biology and variation in the epigenome is essential to contextualize concerns about possible adverse health effects related to epigenetic changes. Finally, the fundamental principles guiding toxicology studies, including relevant doses, dose-rates, routes of exposure, and experimental models, need to be taken into consideration in the design and interpretation of studies within this emerging area of science., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Signal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated., Results: Of several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human BCL10 gene. Chromatin immuno-precipitation reactions confirmed constitutive in vivo STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFkappaB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive in vivo occupancy of BCL10-SBR by STAT5, reduced NFkappaB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFkappaB activity independent of IL-2., Conclusion: These results suggest that the NFkappaB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas.

Prolactin (PRL) receptors are expressed in a broad range of human cell types and in a majority of human breast and prostate cancers. Experimentally, normal and malignant human cells are typically cultured in vitro in media containing bovine PRL (bPRL) from fetal bovine serum or as xenotransplants in vivo in the presence of murine PRL (mPRL). The biological efficacy of bPRL toward hPRL receptors (hPRLR) is controversial, and hPRLR are insensitive to mPRL, but the mechanism is not known. To clarify limitations of current in vitro and in vivo experimental model systems for studies of hPRLR-expressing cells, we tested human and relevant subprimate prolactins in multiple hPRLR bioassays. bPRL and ovine PRL were 10-fold less potent hPRLR agonists than hPRL, although maximal responses at high ligand concentrations (efficacies) equaled that of hPRL. mPRL and rat PRL had greater than 50-fold lower potencies toward hPRLR than hPRL and had 50% reduced efficacies. In fact, mPRL and rat PRL were less effective hPRLR agonists than murine GH. Unexpectedly, mPRL was an effective competitive inhibitor of hPRL binding to hPRLR with an inhibitory constant of 1.3 nm and showed partial antagonist activity, suggesting reduced site-2 binding. Collectively, low bioactivities of bPRL and mPRL toward hPRLR suggest that existing laboratory cancer cell lines grown in 10% bovine serum-supplemented media or in mice are selected for growth under lactogen-depleted conditions. The biology and drug responsiveness of existing human cell lines may therefore not be representative of clinical cancers that are sensitive to circulating PRL.

To identify cell populations directly responsive to prolactin (PRL), GH, erythropoietin, or granulocyte-colony stimulating factor within the physiological setting of an intact mammal, we combined in situ detection of hormone-activated signal transducer and activator of transcription (Stat)-5 in rats with high-throughput tissue array analysis using cutting-edge matrix assembly (CEMA). Inducible activation of Stat5a/b, as judged by levels of nuclear-localized, phosphoTyr694/699-Stat5a/b, served as an immediate and sensitive in situ marker of receptor signaling in rat tissues after injection into male and female rats of a single, receptor-saturating dose of hormone for maximal receptor activation. CEMA tissue arrays facilitated analysis of most tissues, including architecturally complex, thin-walled, and stratified tissues such as gut and skin. In 40 tissues analyzed, 35 PRL-responsive and 32 GH-responsive cell types were detected, of which 22 cell types were responsive to both hormones. Interestingly, PRL but not GH activated Stat5 in nearly all of the endocrine glands. In mammary glands, PRL activated Stat5 in a majority of luminal epithelial cells but not myoepithelial cells, stromal fibroblasts, or adipocytes, whereas GH activated Stat5 in a significant fraction of myoepithelial cells, fibroblasts, and adipocytes but only in a minority of luminal cells. Finally, the organism-wide screening revealed a yet-to-be identified erythropoietin-responsive cell type in connective tissue. CEMA tissue arrays provide cost-effective in situ analysis of large numbers of tissues. Biomarker-based identification of cell populations responsive to individual hormones may shed new light on endocrine disease as well as improve understanding of effects and side effects of hormones and drugs.

Experimental testing of growth, metastatic progression and drug responsiveness of human breast cancer in vivo is performed in immunodeficient mice. Drug candidates need to show promise against human breast cancer in mice before being allowed into clinical trials. Breast cancer growth is under endocrine control by ovarian steroids and the pituitary peptide hormone prolactin. While it is recognized that the most relevant biologic effects of prolactin are achieved with prolactin from the matching species, the biologic efficacy of mouse prolactin for human prolactin receptors has not been recorded. Thus, it is unclear whether the mouse endocrine environment adequately reflects the hormonal environment in breast cancer patients with regard to prolactin. We now show both recombinant and natural pituitary-derived mouse prolactin to be a poor agonist for human prolactin receptors. Mouse prolactin failed to induce human prolactin receptor-mediated biologic responses of cell clustering, proliferation, gene induction and signal transduction, including activation of Stat5, Stat3, Erk1/2 and Akt pathways. Consistent data were derived from human breast cancer lines T-47D, MCF-7 and ZR-75.1, as well as human prolactin receptor-transfected COS-7 and 32D cells. Failure of mouse prolactin to activate human prolactin receptors uncovers a key deficiency of the mouse endocrine environment for human xenotransplant studies. Since most human breast cancers express prolactin receptors, human breast cancer transferred into mice is unnaturally selected for growth in the absence of circulating prolactin. The new insight raises concerns about the validity of analyzing biology and drug responsiveness of human breast cancer in existing mouse xenotransplant models.

Tissue microarrays have become widely adopted for effective parallel in situ analysis of hundreds of tissues placed onto single slides. Traditionally, tissue core punches are transferred into predrilled holes within a scaffold block of paraffin or other material, and sectioned transversely by a microtome to generate array sections. While core-based arraying has greatly advanced tissue analyses, some of the limitations include restricted feature sizes and numbers, variable core depths of unpredictable tissue quality and inability to array thin-walled, stratified tissue samples such as intestines, vessels or skin. Overcoming these limitations, the authors have developed a practical arraying method that combines serial cutting and edge-to-edge bonding of samples to assemble a scaffold-free array matrix -- cutting-edge matrix assembly. Using cutting-edge matrix assembly, the authors have successfully placed more than 10,000 individual tissue pieces on a single histology glass slide. The potential biomedical utility and ongoing efforts to further develop the assembly technology and analysis of high-density cutting-edge matrix assembly tissue microarrays is discussed.

We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.

Background: There is considerable interest in identifying target genes and chromatin binding sites for transcription factors in a genome-wide manner. Such information may become useful in diagnosis and treatment of disease, drug target identification, and for prognostication. In cancer diagnosis, patterns of transcription factor binding to specific regulatory chromatin elements are expected to complement and enhance current diagnostic predictions of tumor behavior based on protein and mRNA analyses. Signal transducer and activator of transcription-5 (Stat5) is a cytokine-activated transcription factor implicated in growth and progression of many malignancies, including hematopoietic, prostate, and breast cancer. We have explored immunoaffinity purification of Stat5-bound chromatin from breast cancer cells to identify Stat5 target sites in an unbiased, genome-wide manner., Results: In this report, we evaluate the efficacy of a Stat5-bound chromatin library to identify valid Stat5 chromatin binding sites within the oncogenome of T-47D human breast cancer cells. A general problem with cloning of immunocaptured, transcription factor-bound chromatin fragments is contamination with non-specific chromatin. However, using an optimized strategy, five out of ten randomly selected clones could be experimentally verified to bind Stat5 both in vitro and in vivo as tested by electrophoretic mobility shift assay and chromatin immunoprecipitation, respectively. While there was no binding to fragments lacking a Stat5 consensus binding sequence, presence of a Stat5 binding sequence did not assure binding., Conclusion: A chromatin library coupled with experimental validation may productively identify novel in vivo Stat5 chromatin binding sites in cancer, including abnormal regulatory sites in tumor-specific neochromatin.