Systems Modeling of Developmental Vascular Toxicity.

The more than 80,000 chemicals in commerce present a challenge for hazard assessments that toxicity testing in the 21st century strives to address through high-throughput screening (HTS) assays. Assessing chemical effects on human development adds an additional layer of complexity to the screening, with a need to capture complex and dynamic events essential for proper embryo-fetal development. HTS data from ToxCast/Tox21 informs systems toxicology models, which incorporate molecular targets and biological pathways into mechanistic models describing the effects of chemicals on human cells, 3D organotypic culture models, and small model organisms. Adverse Outcome Pathways (AOPs) provide a useful framework for integrating the evidence derived from these in silico and in vitro systems to inform chemical hazard characterization. To illustrate this formulation, we have built an AOP for developmental toxicity through a mode of action linked to embryonic vascular disruption (Aop43). Here, we review the model for quantitative prediction of developmental vascular toxicity from ToxCast HTS data and compare the HTS results to functional vascular development assays in complex cell systems, virtual tissues, and small model organisms. ToxCast HTS predictions from several published and unpublished assays covering different aspects of the angiogenic cycle were generated for a test set of 38 chemicals representing a range of putative vascular disrupting compounds (pVDCs). Results boost confidence in the capacity to predict adverse developmental outcomes from HTS in vitro data and model computational dynamics for in silico reconstruction of developmental systems biology. Finally, we demonstrate the integration of the AOP and developmental systems toxicology to investigate the unique modes of action of two angiogenesis inhibitors.

Clinical Strategies for Sampling Word Recognition Performance.

Purpose: Computer simulation was used to estimate the statistical properties of searches for maximum word recognition ability (PB max). These involve presenting multiple lists and discarding all scores but that of the 1 list that produced the highest score. The simulations, which model limitations inherent in the precision of word recognition scores, were done to inform clinical protocols. A secondary consideration was a derivation of 95% confidence intervals for significant changes in score from phonemic scoring of a 50-word list. Method: The PB max simulations were conducted on a "client" with flat performance intensity functions. The client's performance was assumed to be 60% initially and 40% for a second assessment. Thousands of estimates were obtained to examine the precision of (a) single lists and (b) multiple lists using a PB max procedure. This method permitted summarizing the precision for assessing a 20% drop in performance. Results: A single 25-word list could identify only 58.4% of the cases in which performance fell from 60% to 40%. A single 125-word list identified 99.8% of the declines correctly. Presenting 3 or 5 lists to find PB max produced an undesirable finding: an increase in the word recognition score. Conclusions: A 25-word list produces unacceptably low precision for making clinical decisions. This finding holds in both single and multiple 25-word lists, as in a search for PB max. A table is provided, giving estimates of 95% critical ranges for successive presentations of a 50-word list analyzed by the number of phonemes correctly identified.

Building a developmental toxicity ontology.

BACKGROUND: As more information is generated about modes of action for developmental toxicity and more data are generated using high-throughput and high-content technologies, it is becoming necessary to organize that information. This report discussed the need for a systematic representation of knowledge about developmental toxicity (i.e., an ontology) and proposes a method to build one based on knowledge of developmental biology and mode of action/ adverse outcome pathways in developmental toxicity. METHODS: This report is the result of a consensus working group developing a plan to create an ontology for developmental toxicity that spans multiple levels of biological organization. RESULTS: This report provide a description of some of the challenges in building a developmental toxicity ontology and outlines a proposed methodology to meet those challenges. As the ontology is built on currently available web-based resources, a review of these resources is provided. Case studies on one of the most well-understood morphogens and developmental toxicants, retinoic acid, are presented as examples of how such an ontology might be developed. DISCUSSION: This report outlines an approach to construct a developmental toxicity ontology. Such an ontology will facilitate computer-based prediction of substances likely to induce human developmental toxicity.

Embryonic vascular disruption adverse outcomes: Linking high throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Embryonic vascular disruption adverse outcomes: Linking high-throughput signaling signatures with functional consequences.

Embryonic vascular disruption is an important adverse outcome pathway (AOP) as chemical disruption of cardiovascular development induces broad prenatal defects. High-throughput screening (HTS) assays aid AOP development although linking in vitro data to in vivo apical endpoints remains challenging. This study evaluated two anti-angiogenic agents, 5HPP-33 and TNP-470, across the ToxCastDB HTS assay platform and anchored the results to complex in vitro functional assays: the rat aortic explant assay (AEA), rat whole embryo culture (WEC), and the zebrafish embryotoxicity (ZET) assay. Both were identified as putative vascular disruptive compounds (pVDCs) in ToxCastDB and disrupted angiogenesis and embryogenesis in the functional assays. Differences were observed in potency and adverse effects: 5HPP-33 was embryolethal (WEC and ZET); TNP-470 produced caudal defects at lower concentrations. This study demonstrates how a tiered approach using HTS signatures and complex functional in vitro assays might be used to prioritize further in vivo developmental toxicity testing.

Pure-Tone-Spondee Threshold Relationships in Functional Hearing Loss: A Test of Loudness Contribution.

Purpose: The purpose of this article is to examine explanations for pure-tone average-spondee threshold differences in functional hearing loss. Method: Loudness magnitude estimation functions were obtained from 24 participants for pure tones (0.5 and 1.0 kHz), vowels, spondees, and speech-shaped noise as a function of level (20-90 dB SPL). Participants listened monaurally through earphones. Loudness predictions were obtained for the same stimuli by using a computational, dynamic loudness model. Results: When evaluated at the same SPL, speech-shaped noise was judged louder than vowels/spondees, which were judged louder than tones. Equal-loudness levels were inferred from fitted loudness functions for the group. For the clinical application, the 2.1-dB difference between spondees and tones at equal loudness became a 12.1-dB difference when the stimuli were converted from SPL to HL. Conclusions: Nearly all of the pure-tone average-spondee threshold differences in functional hearing loss are attributable to references for calibration for 0 dB HL for tones and speech, which are based on detection and recognition, respectively. The recognition threshold for spondees is roughly 9 dB higher than the speech detection threshold; persons feigning a loss, who base loss magnitude on loudness, do not consider this difference. Furthermore, the dynamic loudness model was more accurate than the static model.

Tiered application of the neutral red release and EpiOcular™ assays for evaluating the eye irritation potential of agrochemical formulations.

Agrochemical formulations have been underrepresented in validation efforts for implementing alternative eye irritation approaches but represent a significant opportunity to reduce animal testing. This study assesses the utility of the neutral red release assay (NRR) and EpiOcular™ assay (EO) for predicting the eye irritation potential of 64 agrochemical formulations relative to Draize data. In the NRR, formulations with an NRR50 value ≤ 50 mg/mL were categorized as UN GHS Cat 1 and those >250 mg/mL were classified as UN GHS Non Classified (NC). The accuracy, sensitivity, and specificity were 78, 85 and 76% and 73, 85 and 61% for identifying UN GHS 1 and NC formulations, respectively. Specificity was poor for formulations with NRR50 > 50 to ≤250 mg/mL. The EO (ET-40 method) was explored to differentiate formulations that were UN GHS 1/2 and UN GHS NC. The EO resulted in accuracy, sensitivity, and specificity of 65%, 58% and 75% for identifying UN GHS NC formulations. To improve the overall performance, the assays were implemented using a tiered-approach where the NRR was run as a first-tier followed by the EO. The tiered-approach resulted in improved accuracy (75%) and balanced sensitivity (73%) and specificity (77%) for distinguishing between irritating and non-irritating agrochemical formulations.

Dietary Route of Exposure for Rabbit Developmental Toxicity Studies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Evaluation of OASIS QSAR Models Using ToxCast™ in Vitro Estrogen and Androgen Receptor Binding Data and Application in an Integrated Endocrine Screening Approach.

BACKGROUND: Integrative testing strategies (ITSs) for potential endocrine activity can use tiered in silico and in vitro models. Each component of an ITS should be thoroughly assessed. OBJECTIVES: We used the data from three in vitro ToxCast™ binding assays to assess OASIS, a quantitative structure-activity relationship (QSAR) platform covering both estrogen receptor (ER) and androgen receptor (AR) binding. For stronger binders (described here as AC50 < 1 µM), we also examined the relationship of QSAR predictions of ER or AR binding to the results from 18 ER and 10 AR transactivation assays, 72 ER-binding reference compounds, and the in vivo uterotrophic assay. METHODS: NovaScreen binding assay data for ER (human, bovine, and mouse) and AR (human, chimpanzee, and rat) were used to assess the sensitivity, specificity, concordance, and applicability domain of two OASIS QSAR models. The binding strength relative to the QSAR-predicted binding strength was examined for the ER data. The relationship of QSAR predictions of binding to transactivation- and pathway-based assays, as well as to in vivo uterotrophic responses, was examined. RESULTS: The QSAR models had both high sensitivity (> 75%) and specificity (> 86%) for ER as well as both high sensitivity (92-100%) and specificity (70-81%) for AR. For compounds within the domains of the ER and AR QSAR models that bound with AC50 < 1 µM, the QSAR models accurately predicted the binding for the parent compounds. The parent compounds were active in all transactivation assays where metabolism was incorporated and, except for those compounds known to require metabolism to manifest activity, all assay platforms where metabolism was not incorporated. Compounds in-domain and predicted to bind by the ER QSAR model that were positive in ToxCast™ ER binding at AC50 < 1 µM were active in the uterotrophic assay. CONCLUSIONS: We used the extensive ToxCast™ HTS binding data set to show that OASIS ER and AR QSAR models had high sensitivity and specificity when compounds were in-domain of the models. Based on this research, we recommend a tiered screening approach wherein a) QSAR is used to identify compounds in-domain of the ER or AR binding models and predicted to bind; b) those compounds are screened in vitro to assess binding potency; and c) the stronger binders (AC50 < 1 µM) are screened in vivo. This scheme prioritizes compounds for integrative testing and risk assessment. Importantly, compounds that are not in-domain, that are predicted either not to bind or to bind weakly, that are not active in in vitro, that require metabolism to manifest activity, or for which in vivo AR testing is in order, need to be assessed differently. CITATION: Bhhatarai B, Wilson DM, Price PS, Marty S, Parks AK, Carney E. 2016. Evaluation of OASIS QSAR models using ToxCast™ in vitro estrogen and androgen receptor binding data and application in an integrated endocrine screening approach. Environ Health Perspect 124:1453-1461; http://dx.doi.org/10.1289/EHP184.

Grouping 34 Chemicals Based on Mode of Action Using Connectivity Mapping.

Connectivity mapping is a method used in the pharmaceutical industry to find connections between small molecules, disease states, and genes. The concept can be applied to a predictive toxicology paradigm to find connections between chemicals, adverse events, and genes. In order to assess the applicability of the technique for predictive toxicology purposes, we performed gene array experiments on 34 different chemicals: bisphenol A, genistein, ethinyl-estradiol, tamoxifen, clofibrate, dehydorepiandrosterone, troglitazone, diethylhexyl phthalate, flutamide, trenbolone, phenobarbital, retinoic acid, thyroxine, 1α,25-dihydroxyvitamin D3, clobetasol, farnesol, chenodeoxycholic acid, progesterone, RU486, ketoconazole, valproic acid, desferrioxamine, amoxicillin, 6-aminonicotinamide, metformin, phenformin, methotrexate, vinblastine, ANIT (1-naphthyl isothiocyanate), griseofulvin, nicotine, imidacloprid, vorinostat, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) at the 6-, 24-, and 48-hour time points for 3 different concentrations in the 4 cell lines: MCF7, Ishikawa, HepaRG, and HepG2 GEO (super series accession no.: GSE69851). The 34 chemicals were grouped in to predefined mode of action (MOA)-based chemical classes based on current literature. Connectivity mapping was used to find linkages between each chemical and between chemical classes. Cell line-specific linkages were compared with each other and to test whether the method was platform and user independent, a similar analysis was performed against publicly available data. The study showed that the method can group chemicals based on MOAs and the inter-chemical class comparison alluded to connections between MOAs that were not predefined. Comparison to the publicly available data showed that the method is user and platform independent. The results provide an example of an alternate data analysis process for high-content data, beneficial for predictive toxicology, especially when grouping chemicals for read across purposes.

Evaluation of TOPKAT, Toxtree, and Derek Nexus in Silico Models for Ocular Irritation and Development of a Knowledge-Based Framework To Improve the Prediction of Severe Irritation.

Assessment of ocular irritation is an essential component of any risk assessment. A number of (Q)SARs and expert systems have been developed and are described in the literature. Here, we focus on three in silico models (TOPKAT, BfR rulebase implemented in Toxtree, and Derek Nexus) and evaluate their performance using 1644 in-house and 123 European Centre for Toxicology and Ecotoxicology of Chemicals (ECETOC) compounds with existing in vivo ocular irritation classification data. Overall, the in silico models performed poorly. The best consensus predictions of severe ocular irritants were 52 and 65% for the in-house and ECETOC compounds, respectively. The prediction performance was improved by designing a knowledge-based chemical profiling framework that incorporated physicochemical properties and electrophilic reactivity mechanisms. The utility of the framework was assessed by applying it to the same test sets and three additional publicly available in vitro irritation data sets. The prediction of severe ocular irritants was improved to 73-77% if compounds were filtered on the basis of AlogP_MR (hydrophobicity with molar refractivity). The predictivity increased to 74-80% for compounds capable of preferentially undergoing hard electrophilic reactions, such as Schiff base formation and acylation. This research highlights the need for reliable ocular irritation models to be developed that take into account mechanisms of action and individual structural classes. It also demonstrates the value of profiling compounds with respect to their chemical reactivity and physicochemical properties that, in combination with existing models, results in better predictions for severe irritants.

Acute Toxicity Prediction in Multiple Species by Leveraging Mechanistic ToxCast Mitochondrial Inhibition Data and Simulation of Oral Bioavailability.

There is great interest in assessing the in vivo toxicity of chemicals using nonanimal alternatives. However, acute mammalian toxicity is not adequately predicted by current in silico or in vitro approaches. Mechanisms of acute toxicity are likely conserved across invertebrate, aquatic, and mammalian species, suggesting that dose-response concordance would be high and in vitro mechanistic data could predict responses in multiple species under conditions of similar bioavailability. We tested this hypothesis by comparing acute toxicity between rat, daphnia, and fish and by comparing their respective acute data to inhibition of mitochondria membrane potential (MMP) using U.S. Environmental Protection Agency ToxCast in vitro high-throughput screening data. Logarithmic scatter plots of acute toxicity data showed a clear relationship between fish, daphnia, and intravenous rat but not oral rat data. Similar plots versus MMP showed a well-delineated upper boundary for fish, daphnia, and intravenous data but were scattered without an upper boundary for rat oral data. Adjustments of acute oral rat toxicity values by simulating fractional absorption and CYP-based metabolism as well as removing compounds with hydrolyzable linkages or flagged as substrates for glucuronidation delineated an upper boundary for rat oral toxicity versus MMP. Mitochondrial inhibition at low concentrations predicted highly acutely toxic chemicals for fish and daphnia but not the rat where toxicity was often attenuated. This use of a single high-throughput screening assay to predict acute toxicity in multiple species represents a milestone and highlights the promise of such approaches but also the need for refined tools to address systemic bioavailability and the impact of limited absorption and first pass metabolism.

Chemical Safety Assessment Using Read-Across: Assessing the Use of Novel Testing Methods to Strengthen the Evidence Base for Decision Making.

BACKGROUND: Safety assessment for repeated dose toxicity is one of the largest challenges in the process to replace animal testing. This is also one of the proof of concept ambitions of SEURAT-1, the largest ever European Union research initiative on alternative testing, co-funded by the European Commission and Cosmetics Europe. This review is based on the discussion and outcome of a workshop organized on initiative of the SEURAT-1 consortium joined by a group of international experts with complementary knowledge to further develop traditional read-across and include new approach data. OBJECTIVES: The aim of the suggested strategy for chemical read-across is to show how a traditional read-across based on structural similarities between source and target substance can be strengthened with additional evidence from new approach data--for example, information from in vitro molecular screening, "-omics" assays and computational models--to reach regulatory acceptance. METHODS: We identified four read-across scenarios that cover typical human health assessment situations. For each such decision context, we suggested several chemical groups as examples to prove when read-across between group members is possible, considering both chemical and biological similarities. CONCLUSIONS: We agreed to carry out the complete read-across exercise for at least one chemical category per read-across scenario in the context of SEURAT-1, and the results of this exercise will be completed and presented by the end of the research initiative in December 2015.

Application of the KeratinoSens™ assay for assessing the skin sensitization potential of agrochemical active ingredients and formulations.

Assessment of skin sensitization potential is an important component of the safety evaluation process for agrochemical products. Recently, non-animal approaches including the KeratinoSens™ assay have been developed for predicting skin sensitization potential. Assessing the utility of the KeratinoSens™ assay for use with multi-component mixtures such as agrochemical formulations has not been previously evaluated and is a significant need. This study was undertaken to evaluate the KeratinoSens™ assay prediction potential for agrochemical formulations. The assay was conducted for 8 agrochemical active ingredients (AIs) including 3 sensitizers (acetochlor, meptyldinocap, triclopyr), 5 non-sensitizers (aminopyralid, clopyralid, florasulam, methoxyfenozide, oxyfluorfen) and 10 formulations for which in vivo sensitization data were available. The KeratinoSens™ correctly predicted the sensitization potential of all the AIs. For agrochemical formulations it was necessary to modify the standard assay procedure whereby the formulation was assumed to have a common molecular weight. The resultant approach correctly predicted the sensitization potential for 3 of 4 sensitizing formulations and all 6 non-sensitizing formulations when compared to in vivo data. Only the meptyldinocap-containing formulation was misclassified, as a result of high cytotoxicity. These results demonstrate the promising utility of the KeratinoSens™ assay for evaluating the skin sensitization potential of agrochemical AIs and formulations.

Predicting the future: opportunities and challenges for the chemical industry to apply 21st-century toxicity testing.

Interest in applying 21st-century toxicity testing tools for safety assessment of industrial chemicals is growing. Whereas conventional toxicology uses mainly animal-based, descriptive methods, a paradigm shift is emerging in which computational approaches, systems biology, high-throughput in vitro toxicity assays, and high-throughput exposure assessments are beginning to be applied to mechanism-based risk assessments in a time- and resource-efficient fashion. Here we describe recent advances in predictive safety assessment, with a focus on their strategic application to meet the changing demands of the chemical industry and its stakeholders. The opportunities to apply these new approaches is extensive and include screening of new chemicals, informing the design of safer and more sustainable chemical alternatives, filling information gaps on data-poor chemicals already in commerce, strengthening read-across methodology for categories of chemicals sharing similar modes of action, and optimizing the design of reduced-risk product formulations. Finally, we discuss how these predictive approaches dovetail with in vivo integrated testing strategies within repeated-dose regulatory toxicity studies, which are in line with 3Rs principles to refine, reduce, and replace animal testing. Strategic application of these tools is the foundation for informed and efficient safety assessment testing strategies that can be applied at all stages of the product-development process.

Human illnesses and animal deaths associated with freshwater harmful algal blooms-Kansas.

Freshwater harmful algal bloom (FHAB) toxins can cause morbidity and mortality in both humans and animals, and the incidence of FHABs in the United States and Kansas has increased. In 2010, the Kansas Department of Health and Environment (KDHE) developed a FHAB policy and response plan. We describe the epidemiology of FHAB-associated morbidity and mortality in humans and animals in Kansas. Healthcare providers and veterinarians voluntarily reported FHAB-associated cases to KDHE. An investigation was initiated for each report to determine the source of exposure and to initiate public health mitigation actions. There were 38 water bodies with a confirmed FHAB in 2011. There were 34 reports of human and animal FHAB-associated health events in 2011, which included five dog deaths and hospitalization of two human case patients. Five confirmed human illnesses, two dog illnesses and five dog deaths were associated with one lake. Four human and seven dog cases were exposed to the lake after a public health alert was issued. Public health officials and FHAB partners must ensure continued awareness of the risks to the public, educate healthcare providers and veterinarians on FHAB-related health events and encourage timely reporting to public health authorities.

Comparative response of rat and rabbit conceptuses in vitro to inhibitors of histiotrophic nutrition.

Histiotrophic nutrition via the visceral yolk sac is an essential nutritional pathway of the rodent conceptus, and inhibition of this pathway may cause growth retardation, malformations, and death in rodent embryos. Morphologic differences among species during early development indicate that the visceral yolk sac histiotrophic nutrition pathway may be of lesser importance in nonrodent species, including humans. Here, comparative studies were conducted with inhibitors of different steps in the visceral yolk sac histiotrophic nutrition pathway to determine whether the rabbit is similarly responsive to the rat. Early somite stage New Zealand White rabbit and Crl:CD(SD) rat conceptuses (gestation day 9, rabbits; gestation day 10, rats) were exposed for 48 hr to three different histiotrophic nutrition pathway inhibitors using whole embryo culture techniques, after which they were evaluated for growth and malformations. Cubilin antibody, an inhibitor of endocytosis, reduced growth and development and increased malformations in both rat and rabbit embryos, although the rabbit appeared more sensitive. Leupeptin, a lysosomal cysteine protease inhibitor, also impaired growth and development and increased malformations in rat embryos, while in the rabbit it induced malformations and a slight decrease in morphology score but had no effect upon growth. Trypan blue, an inhibitor of endocytosis and endosome maturation, affected all measures in both species to a similar degree at the highest concentration (2500 µg/ml), but rat embryos responded to a greater extent at lower concentrations. Although the specific adverse outcomes appear to be different, these results demonstrate that rabbits, like rats, are sensitive to inhibitors of the histiotrophic nutrition pathway.

A critical assessment of the methodologies to investigate the role of inhibition of apoptosis in rodent hepatocarcinogenesis.

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.

FutureTox II: in vitro data and in silico models for predictive toxicology.

FutureTox II, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in January, 2014. The meeting goals were to review and discuss the state of the science in toxicology in the context of implementing the NRC 21st century vision of predicting in vivo responses from in vitro and in silico data, and to define the goals for the future. Presentations and discussions were held on priority concerns such as predicting and modeling of metabolism, cell growth and differentiation, effects on sensitive subpopulations, and integrating data into risk assessment. Emerging trends in technologies such as stem cell-derived human cells, 3D organotypic culture models, mathematical modeling of cellular processes and morphogenesis, adverse outcome pathway development, and high-content imaging of in vivo systems were discussed. Although advances in moving towards an in vitro/in silico based risk assessment paradigm were apparent, knowledge gaps in these areas and limitations of technologies were identified. Specific recommendations were made for future directions and research needs in the areas of hepatotoxicity, cancer prediction, developmental toxicity, and regulatory toxicology.

Exposure-based validation list for developmental toxicity screening assays.

Validation of alternative assays requires comparison of the responses to toxicants in the alternative assay with in vivo responses. Chemicals have been classified as "positive" or "negative" in vivo, despite the fact that developmental toxicity is conditional on magnitude of exposure. We developed a list of positive and negative developmental exposures, with exposure defined by toxicokinetic data, specifically maternal plasma Cmax . We selected a series of 20 chemicals that caused developmental toxicity and for which there were appropriate toxicokinetic data. Where possible, we used the same chemical for both positive and negative exposures, the positive being the Cmax at a dose level that produced significant teratogenicity or embryolethality, the negative being the Cmax at a dose level not causing developmental toxicity. It was not possible to find toxicokinetic data at the no-effect level for all positive compounds, and the negative exposure list contains Cmax values for some compounds that do not have developmental toxicity up to the highest dose level tested. This exposure-based reference list represents a fundamentally different approach to the evaluation of alternative tests and is proposed as a step toward application of alternative tests in quantitative risk assessment.