DARTpaths, an in silico platform to investigate molecular mechanisms of compounds.

SUMMARY: Xpaths is a collection of algorithms that allow for the prediction of compound-induced molecular mechanisms of action by integrating phenotypic endpoints of different species; and proposes follow-up tests for model organisms to validate these pathway predictions. The Xpaths algorithms are applied to predict developmental and reproductive toxicity (DART) and implemented into an in silico platform, called DARTpaths. AVAILABILITY AND IMPLEMENTATION: All code is available on GitHub https://github.com/Xpaths/dartpaths-app under Apache license 2.0, detailed overview with demo is available at https://www.vivaltes.com/dartpaths/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Applying genomics in regulatory toxicology: a report of the ECETOC workshop on omics threshold on non-adversity.

In a joint effort involving scientists from academia, industry and regulatory agencies, ECETOC's activities in Omics have led to conceptual proposals for: (1) A framework that assures data quality for reporting and inclusion of Omics data in regulatory assessments; and (2) an approach to robustly quantify these data, prior to interpretation for regulatory use. In continuation of these activities this workshop explored and identified areas of need to facilitate robust interpretation of such data in the context of deriving points of departure (POD) for risk assessment and determining an adverse change from normal variation. ECETOC was amongst the first to systematically explore the application of Omics methods, now incorporated into the group of methods known as New Approach Methodologies (NAMs), to regulatory toxicology. This support has been in the form of both projects (primarily with CEFIC/LRI) and workshops. Outputs have led to projects included in the workplan of the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) group of the Organisation for Economic Co-operation and Development (OECD) and to the drafting of OECD Guidance Documents for Omics data reporting, with potentially more to follow on data transformation and interpretation. The current workshop was the last in a series of technical methods development workshops, with a sub-focus on the derivation of a POD from Omics data. Workshop presentations demonstrated that Omics data developed within robust frameworks for both scientific data generation and analysis can be used to derive a POD. The issue of noise in the data was discussed as an important consideration for identifying robust Omics changes and deriving a POD. Such variability or "noise" can comprise technical or biological variation within a dataset and should clearly be distinguished from homeostatic responses. Adverse outcome pathways (AOPs) were considered a useful framework on which to assemble Omics methods, and a number of case examples were presented in illustration of this point. What is apparent is that high dimension data will always be subject to varying processing pipelines and hence interpretation, depending on the context they are used in. Yet, they can provide valuable input for regulatory toxicology, with the pre-condition being robust methods for the collection and processing of data together with a comprehensive description how the data were interpreted, and conclusions reached.

A new paradigm for regulatory sciences.

Regulatory science, rooted in legal requirements, provides a mechanism for identifying, assessing, and managing harm to humans and the environment from exposure to hazardous substances. A challenge for regulatory authorities is that many governing laws reflect the scientific paradigm of the mid-20th century. By the nature of legislative processes, most laws are not able to readily adapt to incorporate scientific advances that are inherent in an ever-evolving paradigm. Consequently, the issue of rigid legal frameworks has become prominent in global discussions related to the incorporation of reliable and relevant modern technology to fulfill regulatory needs. To explore this issue, we apply Thomas Kuhn's The Structure of Scientific Revolutions as a conceptual framework to help understand the natural progression of scientific paradigms (from normal science, to anomaly, to crisis, to revolution, and finally to a new normal), identify where we are now in the paradigm cycle, and to explore a path towards a revolution that enables timely implementation of the best available science to fulfil legal requirements.

Exposure considerations in human safety assessment: Report from an EPAA Partners' Forum.

Understanding and estimating the exposure to a substance is one of the fundamental requirements for safe manufacture and use. Many approaches are taken to determine exposure to substances, mainly driven by potential use and regulatory need. There are many opportunities to improve and optimise the use of exposure information for chemical safety. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a Partners' Forum (PF) to explore exposure considerations in human safety assessment of industrial products to agree key conclusions for the regulatory acceptance of exposure assessment approaches and priority areas for further research investment. The PF recognised the widescale use of exposure information across industrial sectors with the possibilities of creating synergies between different sectors. Further, the PF acknowledged that the EPAA could make a significant contribution to promote the use of exposure data in human safety assessment, with an aim to address specific regulatory needs. To achieve this, research needs, as well as synergies and areas for potential collaboration across sectors, were identified.

A mathematical model of the role of aggregation in sonic hedgehog signalling.

Effective regulation of the sonic hedgehog (Shh) signalling pathway is essential for normal development in a wide variety of species. Correct Shh signalling requires the formation of Shh aggregates on the surface of producing cells. Shh aggregates subsequently diffuse away and are recognised in receiving cells located elsewhere in the developing embryo. Various mechanisms have been postulated regarding how these aggregates form and what their precise role is in the overall signalling process. To understand the role of these mechanisms in the overall signalling process, we formulate and analyse a mathematical model of Shh aggregation using nonlinear ordinary differential equations. We consider Shh aggregate formation to comprise of multimerisation, association with heparan sulfate proteoglycans (HSPG) and binding with lipoproteins. We show that the size distribution of the Shh aggregates formed on the producing cell surface resembles an exponential distribution, a result in agreement with experimental data. A detailed sensitivity analysis of our model reveals that this exponential distribution is robust to parameter changes, and subsequently, also to variations in the processes by which Shh is recruited by HSPGs and lipoproteins. The work demonstrates the time taken for different sized Shh aggregates to form and the important role this likely plays in Shh diffusion.

An assay for screening xenobiotics for inhibition of rat thyroid gland peroxidase activity.

1. A number of chemicals have been shown to produce disruption of the thyroid gland, resulting in reduced thyroid hormone synthesis, by a mechanism involving inhibition of thyroid peroxidase (TPO) activity (EC 1.11.1.8).2. An assay was developed for rat thyroid gland microsomal TPO activity, employing L-tyrosine as the physiological substrate, with analysis of the formation of the 3-iodo-L-tyrosine (3MIT) metabolite by ultra-performance liquid chromatography-mass spectrometry-mass spectrometry.3. Formation of 3MIT was linear with respect to both rat thyroid gland microsomal protein concentration and incubation time, whereas only small quantities of 3,5-diodo-L-tyrosine were formed.4. Studies were performed with nine known TPO inhibitors. The most potent inhibitors were 3-amino-1,2,4-triazole, ethylene thiourea, methimazole and 6-propyl-2-thiouracil which had IC50 values (i.e. concentration to produce a 50% inhibition of enzyme activity) of 0.059, 0.791, 1.07 and 1.96 µM, respectively, whereas the least potent inhibitor was sodium perchlorate which had an IC50 value of 13,800 µM.5. For five inhibitors, where literature data were available, the observed IC50 values obtained in this study employing rat thyroid gland microsomes and L-tyrosine as substrate were similar to those previously reported using the spectrophotometric guaiacol oxidation assay.

Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish.

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

Developing prototypes of a modernized approach to assess crop protection chemical safety.

In 2019, the US EPA Administrator issued a directive directing the agency away from reliance on vertebrate tests by 2035, whilst maintaining high-quality human health and environmental risk assessments. There is no accepted approach to achieve this. The decade-long duration of the crop protection (CP) chemical R&D process therefore requires both the invention and application of a modernized approach to those CP chemical projects entering corporate research portfolios by the mid-2020s. We conducted problem formulation discussions with regulatory agency scientists which created the problem statement: "Develop, demonstrate, and implement a modern scientifically sound and robust strategy that applies appropriate and flexible exposure and effects characterization without chemical specific vertebrate tests to reliably address risk, uncertainties, and deficiencies in data and its interpretation with equivalent confidence as do the currently accepted test guidelines and meet the regulatory needs of the agencies". The solution must provide the knowledge needed to confidently conclude human health and environmental protective risk assessments. Exploring this led to a conceptual model involving the creation and parallel submission of a new approach without reliance on chemical-specific vertebrate tests. Assessment in parallel to a traditional package will determine whether it supports some, or all, of the necessary risk management actions. Analysis of any deficiencies will provide valuable feedback to focus development of tools or approaches for subsequent iterations. When found to provide sufficient information, it will form the technical foun­dation of stakeholder engagement to explore acceptance of a new approach to CP chemical risk assessment.

The US EPA, and other regulatory agencies, aim to reduce the use of vertebrate animal tests for assessing risks of crop protection chemicals. There is currently no accepted way to do this. We outline a proposal to perform both the assessment using traditional vertebrate testing and a set of new non-animal methods. These data sets must each be combined with a calculated estimate of user exposure to the pesticide based on its intended use. Comparing the outcome of these two assess­ments will show whether the set of non-animal methods needs to be improved further. When the new approach appears to reliably predict the risks, the different stakeholders must be brought together to assess whether the non-animal methods package is acceptable and can replace the tests on vertebrate animals while maintaining the same level of protection of human health and the environment.

New approach methodologies (NAMs): identifying and overcoming hurdles to accelerated adoption.

New approach methodologies (NAMs) can deliver improved chemical safety assessment through the provision of more protective and/or relevant models that have a reduced reliance on animals. Despite the widely acknowledged benefits offered by NAMs, there continue to be barriers that prevent or limit their application for decision-making in chemical safety assessment. These include barriers related to real and perceived scientific, technical, legislative and economic issues, as well as cultural and societal obstacles that may relate to inertia, familiarity, and comfort with established methods, and perceptions around regulatory expectations and acceptance. This article focuses on chemical safety science, exposure, hazard, and risk assessment, and explores the nature of these barriers and how they can be overcome to drive the wider exploitation and acceptance of NAMs. Short-, mid- and longer-term goals are outlined that embrace the opportunities provided by NAMs to deliver improved protection of human health and environmental security as part of a new paradigm that incorporates exposure science and a culture that promotes the use of protective toxicological risk assessments.

A metabolomics investigation of non-genotoxic carcinogenicity in the rat.

Non-genotoxic carcinogens (NGCs) promote tumor growth by altering gene expression, which ultimately leads to cancer without directly causing a change in DNA sequence. As a result NGCs are not detected in mutagenesis assays. While there are proposed biomarkers of carcinogenic potential, the definitive identification of non-genotoxic carcinogens still rests with the rat and mouse long-term bioassay. Such assays are expensive and time-consuming and require a large number of animals, and their relevance to human health risk assessments is debatable. Metabolomics and lipidomics in combination with pathology and clinical chemistry were used to profile perturbations produced by 10 compounds that represented a range of rat non-genotoxic hepatocarcinogens (NGC), non-genotoxic non-hepatocarcinogens (non-NGC), and a genotoxic hepatocarcinogen. Each compound was administered at its maximum tolerated dose level for 7, 28, and 91 days to male Fisher 344 rats. Changes in liver metabolite concentration differentiated the treated groups across different time points. The most significant differences were driven by pharmacological mode of action, specifically by the peroxisome proliferator activated receptor alpha (PPAR-α) agonists. Despite these dominant effects, good predictions could be made when differentiating NGCs from non-NGCs. Predictive ability measured by leave one out cross validation was 87% and 77% after 28 days of dosing for NGCs and non-NGCs, respectively. Among the discriminatory metabolites we identified free fatty acids, phospholipids, and triacylglycerols, as well as precursors of eicosanoid and the products of reactive oxygen species linked to processes of inflammation, proliferation, and oxidative stress. Thus, metabolic profiling is able to identify changes due to the pharmacological mode of action of xenobiotics and contribute to early screening for non-genotoxic potential.

Predictive liver lipid biomarker signature of Acetyl-coenzyme A carboxylase inhibitor related developmental toxicity in non-pregnant female Han Wistar rats - Lipidomics biomarker discovery and validation.

INTRODUCTION: Acetyl-coenzyme A carboxylase (ACCase) inhibition is an attractive herbicide target. However, issues with fetal developmental toxicity identified at the late stages of the development process can halt progression of previously promising candidates. OBJECTIVES: To select and verify predictive lipid biomarkers of ACCase inhibition activity in vivo using liver samples obtained from early stage 7 day repeat dose studies in non-pregnant female Han Wistar rats that could be translated to developmental toxicity endpoints discovered during late-stage studies to provide an early screening tool. METHODS: Liver samples from eight rat repeat dose studies, exposed to six ACCase inhibitors from three different chemistries and one alternative mode of action (MoA) that also perturbs lipid biochemistry, were analysed using liquid chromatography - high resolution accurate mass - mass spectrometry. Multivariate and univariate data analysis methods were used for biomarker discovery and validation. RESULTS: A biomarker signature consisting of sixteen lipids biomarkers were selected. Verification of the signature as indicative of ACCase inhibition was established by demonstrating consistent perturbations in the biomarkers using two different ACCase inhibitor chemistries and the lack thereof with an alternate MoA. The fold change profile pattern was predictive of which test substance doses would or would not cause developmental toxicity. CONCLUSION: A strategy for selecting and verifying a robust signature of lipid biomarkers for predicting a toxicological end point has been described and demonstrated. Differences in lipidomic profiles correlated with developmental toxicity suggesting that indicators of a molecular initiation event resulting in pup developmental toxicity can be predicted from short term, toxicity studies conducted in non-pregnant adult female Han Wistar rats.

Challenges and opportunities for overcoming dog use in agrochemical evaluation and registration.

Progress in developing new tools, assays, and approaches to assess human hazard and health risk provides an opportunity to re-evaluate the necessity of dog studies for the safety evaluation of agrochemicals. A workshop was held where partic­ipants discussed the strengths and limitations of past use of dogs for pesticide evaluations and registrations. Opportunities were identified to support alternative approaches to answer human safety questions without performing the required 90-day dog study. Development of a decision tree for determining when the dog study might not be necessary to inform pesticide safety and risk assessment was proposed. Such a process will require global regulatory authority participation to lead to its acceptance. The identification of unique effects in dogs that are not identified in rodents will need further evaluation and determination of their relevance to humans. The establishment of in vitro and in silico approaches that can provide critical data on relative species sensitivity and human relevance will be an important tool to advance the decision process. Promising novel tools including in vitro comparative metabolism studies, in silico models, and high-throughput assays able to identify metabolites and mechanisms of action leading to development of adverse outcome pathways will need further development. To replace or eliminate the 90-day dog study, a collaborative, multidisciplinary, international effort that transcends organi­zations and regulatory agencies will be needed in order to develop guidance on when the study would not be necessary for human safety and risk assessment.

Toxicogenomics: the challenges and opportunities to identify biomarkers, signatures and thresholds to support mode-of-action.

Toxicogenomics (TGx) can be defined as the application of "omics" techniques to toxicology and risk assessment. By identifying molecular changes associated with toxicity, TGx data might assist hazard identification and investigate causes. Early technical challenges were evaluated and addressed by consortia (e.g. ISLI/HESI and the Microarray Quality Control consortium), which demonstrated that TGx gave reliable and reproducible information. The MAQC also produced "best practice on signature generation" after conducting an extensive evaluation of different methods on common datasets. Two findings of note were the need for methods that control batch variability, and that the predictive ability of a signature changes in concert with the variability of the endpoint. The key challenge remaining is data interpretation, because TGx can identify molecular changes that are causal, associated with or incidental to toxicity. Application of Bradford Hill's tests for causation, which are used to build mode of action (MOA) arguments, can produce reasonable hypotheses linking altered pathways to phenotypic changes. However, challenges in interpretation still remain: are all pathway changes equal, which are most important and plausibly linked to toxicity? Therefore the expert judgement of the toxicologist is still needed. There are theoretical reasons why consistent alterations across a metabolic pathway are important, but similar changes in signalling pathways may not alter information flow. At the molecular level thresholds may be due to the inherent properties of the regulatory network, for example switch-like behaviours from some network motifs (e.g. positive feedback) in the perturbed pathway leading to the toxicity. The application of systems biology methods to TGx data can generate hypotheses that explain why a threshold response exists. However, are we adequately trained to make these judgments? There is a need for collaborative efforts between regulators, industry and academia to properly define how these technologies can be applied using appropriate case-studies.

Transforming the evaluation of agrochemicals.

The present agrochemical safety evaluation paradigm is long-standing and anchored in well-established testing and evaluation procedures. However, it does not meet the present-day challenges of rapidly growing populations, food insecurity, and pressures from climate change. To transform the current framework and apply modern evaluation strategies that better support sustainable agriculture, the Health and Environmental Sciences Institute (HESI) assembled a technical committee to reframe the safety evaluation of crop-protection products. The committee is composed of international experts from regulatory agencies, academia, industry and nongovernmental organizations. Their mission is to establish a framework that supports the development of fit-for-purpose agrochemical safety evaluation that is applicable to changing global, as well as local needs and regulatory decisions, and incorporates relevant evolving science. This will be accomplished through the integration of state-of-the-art scientific methods, technologies and data sources, to inform safety and risk decisions, and adapt them to evolving local and global needs. The project team will use a systems-thinking approach to develop the tools that will implement a problem formulation and exposure driven approach to create sustainable, safe and effective crop protection products, and reduce, replace and refine animal studies with fit-for-purpose assays. A new approach necessarily will integrate the most modern tools and latest advances in chemical testing methods to guarantee the robust human and environmental safety and risk assessment of agrochemicals. This article summarizes the challenges associated with the modernization of agrochemical safety evaluation, proposes a potential roadmap, and seeks input and engagement from the broader community to advance this effort. © 2022 Health and Environmental Sciences Institute (HESI). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A Transformative Vision for an Omics-Based Regulatory Chemical Testing Paradigm.

Use of molecular data in human and ecological health risk assessments of industrial chemicals and agrochemicals has been anticipated by the scientific community for many years; however, these data are rarely used for risk assessment. Here, a logic framework is proposed to explore the feasibility and future development of transcriptomic methods to refine and replace the current apical endpoint-based regulatory toxicity testing paradigm. Four foundational principles are outlined and discussed that would need to be accepted by stakeholders prior to this transformative vision being realized. Well-supported by current knowledge, the first principle is that transcriptomics is a reliable tool for detecting alterations in gene expression that result from endogenous or exogenous influences on the test organism. The second principle states that alterations in gene expression are indicators of adverse or adaptive biological responses to stressors in an organism. Principle 3 is that transcriptomics can be employed to establish a benchmark dose-based point of departure (POD) from short-term, in vivo studies at a dose level below which a concerted molecular change (CMC) is not expected. Finally, Principle 4 states that the use of a transcriptomic POD (set at the CMC dose level) will support a human health-protective risk assessment. If all four principles are substantiated, this vision is expected to transform aspects of the industrial chemical and agrochemical risk assessment process that are focused on establishing safe exposure levels for mammals across numerous toxicological contexts resulting in a significant reduction in animal use while providing equal or greater protection of human health. Importantly, these principles and approaches are also generally applicable for ecological safety assessment.

Exploration of the DARTable Genome- a Resource Enabling Data-Driven NAMs for Developmental and Reproductive Toxicity Prediction.

The identification of developmental and reproductive toxicity (DART) is a critical component of toxicological evaluations of chemical safety. Adverse Outcome Pathways (AOPs) provide a framework to describe biological processes leading to a toxic effect and can provide insights in understanding the mechanisms underlying toxicological endpoints and aid the development of new approach methods (NAMs). Integrated approaches to testing and assessment (IATA) can be developed based on AOP knowledge and can serve as pragmatic approaches to chemical hazard characterization using NAMs. However, DART effects remain difficult to predict given the diversity of biological mechanisms operating during ontogenesis and consequently, the considerable number of potential molecular initiating events (MIEs) that might trigger a DART Adverse Outcome (DART AO). Consequently, two challenges that need to be overcome to create an AOP-based DART IATA are having sufficient knowledge of relevant biology and using this knowledge to determine the appropriate selection of cell systems that provide sufficient coverage of that biology. The wealth of modern biological and bioinformatics data can be used to provide this knowledge. Here we demonstrate the utility of bioinformatics analyses to address these questions. We integrated known DART MIEs with gene-developmental phenotype information to curate the hypothetical human DARTable genome (HDG, ∼5 k genes) which represents the comprehensive set of biomarkers for DART. Using network analysis of the human interactome, we show that HDG genes have distinct connectivity compared to other genes. HDG genes have higher node degree with lower neighborhood connectivity, betweenness centralities and average shortest path length. Therefore, HDG is highly connected to itself and to the wider network and not only to their local community. Also, by comparison with the Druggable Genome we show how the HDG can be prioritized to identify potential MIEs based on potential to interact with small molecules. We demonstrate how the HDG in combination with gene expression data can be used to select a panel of relevant cell lines (RD-1, OVCAR-3) for inclusion in an IATA and conclude that bioinformatic analyses can provide the necessary insights and serve as a resource for the development of a screening panel for a DART IATA.

An evaluation of carcinogenicity predictors from short-term and sub chronic repeat-dose studies of agrochemicals in rats: Opportunities to refine and reduce animal use.

The aim of this study was to examine whether short term, repeat dose, rat studies provide sufficient information about potential carcinogenicity to enable predictions about the carcinogenic potential of agrochemicals to be made earlier in compound development. This study aimed to identify any correlations between toxicity findings obtained for short term rat studies (28 day and 90 day) and neoplastic findings obtained from 24 month rat carcinogenicity studies for agrochemical compounds (18 compounds) tested in Han Wistar and Sprague Dawley rats. The macroscopic pathology, microscopic pathology, hematology, biochemistry, organ weights, estrogen receptor activation and genotoxicity results were examined. Seven out of 18 non genotoxic compounds developed tumors in treated rats in the carcinogenicity study and of these, two compounds showed no preneoplastic findings in the affected tissues (false negatives). Of the remaining five true positives, correlations were noted between corneal opacity and keratitis (90 day study) as early indicators of squamous cell carcinoma and papilloma of the cornea of the eye (compound 1, a hydroxyphenylpyruvate dioxygenase inhibitor) and inflammation of the stomach and kidney (90 day study) and gastric squamous cell papilloma and squamous cell carcinoma and renal tubular adenoma and carcinoma, respectively (compound 12, a fungicide with multisite activity). Minor decreases in uterine weight and increases in estradiol hydroxylation activity at 28 days were associated with endometrial adenocarcinoma (compound 18, a mitochondrial complex II electron transport inhibitor). Early liver weight increases and hepatocellular centrilobular hypertrophy (28 day study) were associated with thyroid follicular adenomas (compound 11, a succinate dehydrogenase inhibitor) in female animals only. Hepatic centrilobular hypertrophy (28 day studies) correlated with thyroid adenomas in males in carcinogenicity studies (compound 2, a hydroxyphenylpyruvate dioxygenase inhibitor). In contrast, treatment related, nasopharynx tumors (compound 3, an elongase inhibitor) and uterine adenocarcinoma (compound 9, a succinate dehydrogenase inhibitor) could not be correlated with findings from the short term studies examined. Eleven compounds displayed preneoplastic findings with no tumors (false positives) and there were no compounds with no preneoplastic findings and no tumors (true negatives). This work indicates the value of examining historical, short term studies for specific, nonneoplastic findings which correlate with tumors in carcinogenicity studies, which may obviate the need for further animal carcinogenicity studies.

Comparison of the predictive nature of the Genomic Allergen Rapid Detection (GARD) assay with mammalian assays in determining the skin sensitisation potential of agrochemical active ingredients.

Alternatives to mammalian testing are highly desirable to predict the skin sensitisation potential of agrochemical active ingredients (AI). The GARD assay, a stimulated, dendritic cell-like, cell line measuring genomic signatures, was evaluated using twelve AIs (seven sensitisers and five non-sensitisers) and the results compared with historical results from guinea pig or local lymph node assay (LLNA) studies. Initial GARD results suggested 11/12 AIs were sensitisers and six concurred with mammalian data. Conformal predictions changed one AI to a non-sensitiser. An AI identified as non-sensitising in the GARD assay was considered a potent sensitiser in the LLNA. In total 7/12 GARD results corresponded with mammalian data. AI chemistries might not be comparable to the GARD training set in terms of applicability domains. Whilst the GARD assay can replace mammalian tests for skin sensitisation evaluation for compounds including cosmetic ingredients, further work in agrochemical chemistries is needed for this assay to be a viable replacement to animal testing. The work conducted here is, however, considered exploratory research and the methodology needs further development to be validated for agrochemicals. Mammalian and other alternative assays for regulatory safety assessments of AIs must provide confidence to assign the appropriate classification for human health protection.

Species differences in phenobarbital-mediated UGT gene induction in rat and human liver microtissues.

Species differences in hepatic metabolism of thyroxine (T4) by uridine diphosphate glucuronosyl transferase (UGT) and susceptibility to thyroid hormone imbalance could underlie differences in thyroid carcinogenesis caused by hepatic enzyme inducers in rats and humans. To investigate this hypothesis we examined profiles of hepatic UGT induction by the prototypical CAR activator phenobarbital (PB) in rat and human liver 3D microtissues. The rationale for this approach was that 3D microtissues would generate data more relevant to humans. Rat and human liver 3D microtissues were exposed to PB over a range of concentrations (500 u M - 2000 u M) and times (24-96 hr). Microarray and proteomics analyses were performed on parallel samples to generate integrated differentially expressed gene (DEG) datasets. Bioinformatics analysis of DEG data, including CAR response element (CRE) sequence analysis of UGT promoters, was used to assess species differences in UGT induction relative to CAR-mediated transactivation potential. A higher proportion of human UGT promoters were found to contain consensus CREs compared to the rat homologs. UGTs 1a6, 2b17 and 2b37 were upregulated by PB in rat liver 3D microtissues, but unaltered in human liver 3D microtissues. By contrast, human UGTs 1A8, 1A10 and 2B10 showed higher levels of induction (RNA and /or protein) compared to the rat homologs. There was general concordance between the presence of CREs and the induction of UGT RNA. As UGT1A and 2B isoforms metabolise T4, these results suggest that differences in UGT induction could contribute to differential susceptibility to CAR-mediated thyroid carcinogenesis in rats and humans.

An integrated functional genomic study of acute phenobarbital exposure in the rat.

BACKGROUND: Non-genotoxic carcinogens are notoriously difficult to identify as they do not damage DNA directly and have diverse modes of action, necessitating long term in vivo studies. The early effects of the classic rodent non-genotoxic hepatocarcinogen phenobarbital have been investigated in the Fisher rat using a combination of metabolomics and transcriptomics, to investige early stage mechanistic changes that are predictive of longer term pathology. RESULTS: Liver and blood plasma were profiled across 14 days, and multivariate statistics used to identify perturbed pathways. Both metabolomics and transcriptomics detected changes in the liver which were dose dependent, even after one day of exposure. Integration of the two datasets associated perturbations with specific pathways. Hepatic glycogen was decreased due to a decrease in synthesis, and plasma triglycerides were decreased due to an increase in fatty acid uptake by the liver. Hepatic succinate was increased and this was associated with increased heme biosynthesis. Glutathione synthesis was also increased, presumably in response to oxidative stress. Liquid Chromatography Mass Spectrometry demonstrated a remodeling of lipid species, possibly resulting from proliferation of the smooth endoplasmic reticulum. CONCLUSIONS: The data fusion of metabolomic and transcriptomic changes proved to be a highly sensitive approach for monitoring early stage changes in altered hepatic metabolism, oxidative stress and cytochrome P450 induction simultaneously. This approach is particularly useful in interpreting changes in metabolites such as succinate which are hubs of metabolism.