Recommendations on dose level selection for repeat dose toxicity studies.

Prior to registering and marketing any new pharmaceutical, (agro)chemical or food ingredient product manufacturers must, by law, generate data to ensure human safety. Safety testing requirements vary depending on sector, but generally repeat-dose testing in animals form the basis for human health risk assessments. Dose level selection is an important consideration when designing such studies, to ensure that exposure levels that lead to relevant hazards are identified. Advice on dose level selection is provided in test guidelines and allied guidance documents, but it is not well harmonised, particularly for selection of the highest dose tested. This paper further builds on concepts developed in a technical report by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) which recommends pragmatic approaches to dose selection considering regulatory requirements, animal welfare and state of the art scientific approaches. Industry sectors have differing degrees of freedom to operate regarding dose level selection, depending on the purpose of the studies and the regulatory requirements/legislation, and this is reflected in the overall recommended approaches. An understanding of systemic exposure should be utilised where possible (e.g., through toxicokinetic approaches) and used together with apical endpoints from existing toxicity studies to guide more appropriate dose level selection. The highest dose should be limited to a reasonable level, causing minimal but evident toxicity to the test animals without significantly compromising their well-being. As the science of predictive human exposure further develops and matures, this will provide exciting and novel opportunities for more human-relevant approaches to dose level selection.

New ideas for non-animal approaches to predict repeated-dose systemic toxicity: Report from an EPAA Blue Sky Workshop.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a 'Blue Sky Workshop' on new ideas for non-animal approaches to predict repeated-dose systemic toxicity. The aim of the Workshop was to formulate strategic ideas to improve and increase the applicability, implementation and acceptance of modern non-animal methods to determine systemic toxicity. The Workshop concluded that good progress is being made to assess repeated dose toxicity without animals taking advantage of existing knowledge in toxicology, thresholds of toxicological concern, adverse outcome pathways and read-across workflows. These approaches can be supported by New Approach Methodologies (NAMs) utilising modern molecular technologies and computational methods. Recommendations from the Workshop were based around the needs for better chemical safety assessment: how to strengthen the evidence base for decision making; to develop, standardise and harmonise NAMs for human toxicity; and the improvement in the applicability and acceptance of novel techniques. "Disruptive thinking" is required to reconsider chemical legislation, validation of NAMs and the opportunities to move away from reliance on animal tests. Case study practices and data sharing, ensuring reproducibility of NAMs, were viewed as crucial to the improvement of non-animal test approaches for systemic toxicity.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Assessment of health risks resulting from early-life exposures: Are current chemical toxicity testing protocols and risk assessment methods adequate?

Abstract Over the last couple of decades, the awareness of the potential health impacts associated with early-life exposures has increased. Global regulatory approaches to chemical risk assessment are intended to be protective for the diverse human population including all life stages. However, questions persist as to whether the current testing approaches and risk assessment methodologies are adequately protective for infants and children. Here, we review physiological and developmental differences that may result in differential sensitivity associated with early-life exposures. It is clear that sensitivity to chemical exposures during early-life can be similar, higher, or lower than that of adults, and can change quickly within a short developmental timeframe. Moreover, age-related exposure differences provide an important consideration for overall susceptibility. Differential sensitivity associated with a life stage can reflect the toxicokinetic handling of a xenobiotic exposure, the toxicodynamic response, or both. Each of these is illustrated with chemical-specific examples. The adequacy of current testing protocols, proposed new tools, and risk assessment methods for systemic noncancer endpoints are reviewed in light of the potential for differential risk to infants and young children.

SEURAT: Safety Evaluation Ultimately Replacing Animal Testing--recommendations for future research in the field of predictive toxicology.

The development of non-animal methodology to evaluate the potential for a chemical to cause systemic toxicity is one of the grand challenges of modern science. The European research programme SEURAT is active in this field and will conclude its first phase, SEURAT-1, in December 2015. Drawing on the experience gained in SEURAT-1 and appreciating international advancement in both basic and regulatory science, we reflect here on how SEURAT should evolve and propose that further research and development should be directed along two complementary and interconnecting work streams. The first work stream would focus on developing new 'paradigm' approaches for regulatory science. The goal here is the identification of 'critical biological targets' relevant for toxicity and to test their suitability to be used as anchors for predicting toxicity. The second work stream would focus on integration and application of new approach methods for hazard (and risk) assessment within the current regulatory 'paradigm', aiming for acceptance of animal-free testing strategies by regulatory authorities (i.e. translating scientific achievements into regulation). Components for both work streams are discussed and may provide a structure for a future research programme in the field of predictive toxicology.

A strategy for safety assessment of chemicals with data gaps for developmental and/or reproductive toxicity.

Alternative methods for full replacement of in vivo tests for systemic endpoints are not yet available. Read across methods provide a means of maximizing utilization of existing data. A limitation for the use of read across methods is that they require analogs with test data. Repeat dose data are more frequently available than are developmental and/or reproductive toxicity (DART) studies. There is historical precedent for using repeat dose data in combination with a database uncertainty factor (UF) to account for missing DART data. We propose that use of the DART decision tree (Wu et al., 2013), in combination with a database UF, provides a path forward for DART data gap filling that better utilizes all of the data. Our hypothesis was that chemical structures identified by the DART tree as being related to structures with known DART toxicity would potentially have lower DART NOAELs compared to their respective repeat dose NOAELs than structures that lacked this association. Our analysis supports this hypothesis and as a result also supports that the DART decision tree can be used as part of weight of evidence in the selection of an appropriate DART database UF factor.

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.

Framework for identifying chemicals with structural features associated with the potential to act as developmental or reproductive toxicants.

Developmental and reproductive toxicity (DART) end points are important hazard end points that need to be addressed in the risk assessment of chemicals to determine whether or not they are the critical effects in the overall risk assessment. These hazard end points are difficult to predict using current in silico tools because of the diversity of mechanisms of action that elicit DART effects and the potential for narrow windows of vulnerability. DART end points have been projected to consume the majority of animals used for compliance with REACH; thus, additional nonanimal predictive tools are urgently needed. This article presents an empirically based decision tree for determining whether or not a chemical has receptor-binding properties and structural features that are consistent with chemical structures known to have toxicity for DART end points. The decision tree is based on a detailed review of 716 chemicals (664 positive, 16 negative, and 36 with insufficient data) that have DART end-point data and are grouped into defined receptor binding and chemical domains. When tested against a group of chemicals not included in the training set, the decision tree is shown to identify a high percentage of chemicals with known DART effects. It is proposed that this decision tree could be used both as a component of a screening system to identify chemicals of potential concern and as a component of weight-of-evidence decisions based on structure-activity relationships (SAR) to fill data gaps without generating additional test data. In addition, the chemical groupings generated could be used as a starting point for the development of hypotheses for in vitro testing to elucidate mode of action and ultimately in the development of refined SAR principles for DART that incorporate mode of action (adverse outcome pathways).

Identifying chemicals based on receptor binding/bioactivation/mechanistic explanation associated with potential to elicit hepatotoxicity and to support structure activity relationship-based read-across.

The liver is the most common target organ in toxicology studies. The development of chemical structural alerts for identifying hepatotoxicity will play an important role in in silico model prediction and help strengthen the identification of analogs used in structure activity relationship (SAR)- based read-across. The aim of the current study is development of an SAR-based expert-system decision tree for screening of hepatotoxicants across a wide range of chemistry space and proposed modes of action for clustering of chemicals using defined core chemical categories based on receptor-binding or bioactivation. The decision tree is based on âˆ¼ 1180 different chemicals that were reviewed for hepatotoxicity information. Knowledge of chemical receptor binding, metabolism and mechanistic information were used to group these chemicals into 16 different categories and 102 subcategories: four categories describe binders to 9 different receptors, 11 categories are associated with possible reactive metabolites (RMs) and there is one miscellaneous category. Each chemical subcategory has been associated with possible modes of action (MOAs) or similar key structural features. This decision tree can help to screen potential liver toxicants associated with core structural alerts of receptor binding and/or RMs and be used as a component of weight of evidence decisions based on SAR read-across, and to fill data gaps.

Structure-activity relationship read-across and transcriptomics for branched carboxylic acids.

The purpose of this study was to use chemical similarity evaluations, transcriptional profiling, in vitro toxicokinetic data, and physiologically based pharmacokinetic (PBPK) models to support read-across for a series of branched carboxylic acids using valproic acid (VPA), a known developmental toxicant, as a comparator. The chemicals included 2-propylpentanoic acid (VPA), 2-ethylbutanoic acid, 2-ethylhexanoic acid (EHA), 2-methylnonanoic acid, 2-hexyldecanoic acid, 2-propylnonanoic acid (PNA), dipentyl acetic acid or 2-pentylheptanoic acid, octanoic acid (a straight chain alkyl acid), and 2-ethylhexanol. Transcriptomics was evaluated in 4 cell types (A549, HepG2, MCF7, and iCell cardiomyocytes) 6 h after exposure to 3 concentrations of the compounds, using the L1000 platform. The transcriptional profiling data indicate that 2- or 3-carbon alkyl substituents at the alpha position of the carboxylic acid (EHA and PNA) elicit a transcriptional profile similar to the one elicited by VPA. The transcriptional profile is different for the other chemicals tested, which provides support for limiting read-across from VPA to much shorter and longer acids. Molecular docking models for histone deacetylases, the putative target of VPA, provide a possible mechanistic explanation for the activity cliff elucidated by transcriptomics. In vitro toxicokinetic data were utilized in a PBPK model to estimate internal dosimetry. The PBPK modeling data show that as the branched chain increases, predicted plasma Cmax decreases. This work demonstrates how transcriptomics and other mode of action-based methods can improve read-across.

Effects of transplacental 17-α-ethynyl estradiol or bisphenol A on the developmental profile of steroidogenic acute regulatory protein in the rat testis.

Previous research from our laboratory has determined the transcript profiles for developing fetal rat female and male reproductive tracts following transplacental exposure to estrogens. Prenatal exposure to bisphenol A (BPA) or 17-α-ethynyl estradiol (EE) significantly affects steroidogenic acute regulatory (StAR) protein transcript levels in the developing male rat reproductive tract. The purpose of this study was to establish the intratesticular distribution and temporal expression pattern of StAR, a key gene involved in steroidogenesis. Beginning on gestation day (GD) 11, pregnant Sprague-Dawley rats were exposed daily to 10µg/kg/day EE and fetal testes were harvested at GD16, 18, or 20. Quantitative reverse transcriptase PCR (QRT-PCR) demonstrated no significant difference in StAR transcript levels present at GD16. However, at GD18, StAR transcripts were significantly decreased following exposure. Immunohistochemistry demonstrated similar StAR protein levels in interstitial region of GD16 testes and an obvious decrease in StAR protein levels in the interstitial region of GD18 testes. Moreover, starting at GD11 additional dams were dosed with 0.001 or 0.1 µg/kg/day EE or 0.02, 0.5, 400 mg/kg/day BPA via subcutaneous injections. QRT-PCR validated previous microarray dose-related decreases in StAR transcripts at GD20, whereas immunohistochemistry results demonstrated decreases in StAR protein levels in the interstitial region at the highest EE and BPA doses only. Neither EE nor BPA exposure caused morphological changes in the developing seminiferous cords, Sertoli cells, gonocytes, or the interstitial region or Leydig cells at GD16-20. High levels of estrogens decrease StAR expression in the fetal rat testis during late gestation.

Correlation of chemical structure with reproductive and developmental toxicity as it relates to the use of the threshold of toxicological concern.

In the absence of toxicological data on a chemical, the threshold of toxicological concern (TTC) approach provides a system to estimate a conservative exposure below which there is a low probability of risk for adverse health effects. The original toxicology dataset underlying the TTC was based on NOELs from repeat dose studies. Subsequently there have been several efforts to assess whether or not these limits are also protective for reproductive/developmental effects. This work expands the database of chemicals with reproductive and developmental data, presents these data in a comprehensive and transparent format and groups the chemicals according to the TTC "Cramer Class" rules. Distributions of NOAELs from each of these classes were used to assess whether the previously proposed TTC values based on repeat dose data are protective for reproductive/developmental toxicity endpoints as well. The present analysis indicates that, for each Cramer Class, the reproductive and developmental endpoints would be protected at the corresponding general TTC tiers derived by Munro et al. (1996).

Article title: Transcriptional profiling efficacy to define biological activity similarity for cosmetic ingredients' safety assessment based on next-generation read-across.

The objective of this work was to use transcriptional profiling to assess the biological activity of structurally related chemicals to define their biological similarity and with that, substantiate the validity of a read-across approach usable in risk assessment. Two case studies are presented, one with 4 short alkyl chain parabens: methyl (MP), ethyl (EP), butyl (BP), and propylparaben (PP), as well as their main metabolite, p-hydroxybenzoic acid (pHBA) with the assumption that propylparaben was the target chemical; and a second one with caffeine and its main metabolites theophylline, theobromine and paraxanthine where CA was the target chemical. The comprehensive transcriptional response of MCF7, HepG2, A549 and ICell cardiomyocytes was evaluated (TempO-Seq) after exposure to vehicle-control, each paraben or pHBA, CA or its metabolites, at 3 non-cytotoxic concentrations, for 6 h. Differentially expressed genes (FDR ≥0.05, and fold change ±1.2≥) were identified for each chemical, at each concentration, and used to determine similarities. Each of the chemicals is able to elicit changes in the expression of a number of genes, as compared to controls. Importantly, the transcriptional profile elicited by each of the parabens shares a high degree of similarity across the group. The highest number of genes commonly affected was between butylparaben and PP. The transcriptional profile of the parabens is similar to the one elicited by estrogen receptor agonists, with BP being the closest structural and biological analogue for PP. In the CA case, the transcriptional profile elicited of all four methylxanthines had a high degree of similarity across the cell types, with CA and theophylline being the most active. The most robust response was obtained in the cardiomyocytes with the highest transcriptional profile similarity between CA and TP. The transcriptional profile of the methylxanthines is similar to the one elicited by inhibitors of phosphatidylinositol 3-kinase as well as other kinase inhibitors. Overall, our results support the approach of incorporating transcriptional profiling in well-designed in vitro tests as one robust stream of data to support biological similarity driven read-across procedures and strengthening the traditional structure-based approaches useful in risk assessment.

A New Approach Methodology (NAM) Based Assessment of Butylated hydroxytoluene (BHT) for Endocrine Disruption Potential.

Butylated hydroxytoluene (BHT) is a synthetic antioxidant widely used in many industrial sectors. BHT is a well-studied compound for which there are many favorable regulatory decisions. However, a recent opinion by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) hypothesizes a role for BHT in endocrine disruption (ANSES (2021). This opinion is based on observations in mostly rat studies where changes to thyroid physiology are observed. Enzymatic induction of Cytochrome P450-mediated thyroid hormone catabolism has been proposed as a mechanism for these observations, however, a causal relationship has not been proven. Other evidence proposed in the document includes a read across argument to butylated hydroxyanisole (BHA), another Community Rolling Action Plan (CoRAP)-listed substance with endocrine disruption concerns. We tested the hypothesis that BHT is an endocrine disruptor by using a Next Generation Risk Assessment (NGRA) method. Four different cell lines: A549, HCC1428, HepG2, and MCF7 were treated with BHT and a series of BHT analogs at 5 different concentrations, RNA was isolated from cell extracts and run on the L1000 gene array platform. A toxicogenomics-based assessment was performed by comparing BHT's unique genomic signature to a large external database containing signatures of other compounds (including many known endocrine disruptors) to identify if any endocrine disruption-related modes of action (MoAs) are prevalent among BHT and other compounds with similar genomic signatures. In addition, we performed a toxicogenomics-based structure activity relationship (SAR) assessment of BHT and a series of structurally similar analogs to understand if endocrine disruption is a relevant MoA for chemicals that are considered suitable analogs to BHT using the P&G read across framework (Wu et al., 2010). Neither BHT nor any of its analogs connected to compounds that had endocrine activity for estrogens, androgens, thyroid, or steroidogenesis.

Pluripotent stem cell assays: Modalities and applications for predictive developmental toxicity.

This manuscript provides a review focused on embryonic stem cell-based models and their place within the landscape of alternative developmental toxicity assays. Against the background of the principles of developmental toxicology, the wide diversity of alternative methods using pluripotent stem cells developed in this area over the past half century is reviewed. In order to provide an overview of available models, a systematic scoping review was conducted following a published protocol with inclusion criteria, which were applied to select the assays. Critical aspects including biological domain, readout endpoint, availability of standardized protocols, chemical domain, reproducibility and predictive power of each assay are described in detail, in order to review the applicability and limitations of the platform in general and progress moving forward to implementation. The horizon of innovative routes of promoting regulatory implementation of alternative methods is scanned, and recommendations for further work are given.

Laboratory models and their role in assessing teratogenesis.

Laboratory models have an important role in identifying exposures with teratogenic potential, determining mechanisms of abnormal development, and supporting or refuting the biological plausibility of associations identified in human studies. Laboratory animals are the most widely used models, but are rapidly being supplemented by in vitro tools. Testing paradigms that have been in place since the mid-1960s for pharmaceuticals, and soon thereafter for industrial chemicals and pesticides, have been used to evaluate the potential developmental toxicity of thousands of agents. These models have served as the principal basis for regulatory decisions about acceptable exposure levels and restrictions on use of certain drugs during pregnancy.

Alternative (non-animal) methods for cosmetics testing: current status and future prospects-2010.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commission's Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 7-9 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 5-7 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Developmental toxicity testing for safety assessment: new approaches and technologies.

The ILSI Health and Environmental Sciences Institute's Developmental and Reproductive Toxicology Technical Committee held a 2-day workshop entitled "Developmental Toxicology-New Directions" in April 2009. The fourth session of this workshop focused on new approaches and technologies for the assessment of developmental toxicology. This session provided an overview of the application of genomics technologies for developmental safety assessment, the use of mouse embryonic stem cells to capture data on developmental toxicity pathways, dynamical cell imaging of zebrafish embryos, the use of computation models of development pathways and systems, and finally, high-throughput in vitro approaches being utilized by the EPA ToxCast program. Issues discussed include the challenges of anchoring in vitro predictions to relevant in vivo endpoints and the need to validate pathway-based predictions with targeted studies in whole animals. Currently, there are 10,000 to 30,000 chemicals in world-wide commerce in need of hazard data for assessing potential health risks. The traditional animal study designs for assessing developmental toxicity cannot accommodate the evaluation of this large number of chemicals, requiring that alternative technologies be utilized. Though a daunting task, technologies are being developed and utilized to make that goal reachable.

Case studies to test: A framework for using structural, reactivity, metabolic and physicochemical similarity to evaluate the suitability of analogs for SAR-based toxicological assessments.

A process for evaluating analogs for use in SAR (Structure-Activity Relationship) assessments was previously published (Wu et al. 2010). Subsequently, this process has been updated to include a decision tree for estrogen binding (from US EPA) and flags for developmental and reproductive toxicity (DART). This paper presents the results of blinded case studies designed to test this updated framework. The results of these case studies support the conclusion that the process outlined by Wu et al. (2010) can be successfully applied to develop surrogate values for risk assessment. The read across results generated by the process were shown to be protective when compared to the actual toxicity data. Successful application of the approach requires significant expertise as well as discipline to not overstep the boundaries of the defined analogs and the rating system. The end result of this rigor can be the inability to read across all endpoints for all chemicals resulting in data gaps that cannot be filled using read across, however, this reflects the current state of the science and is preferable to making non-protective decisions. Future work will be targeted towards expanding read across capabilities. Two examples of a broader category approach are also shown.

An ontology for developmental processes and toxicities of neural tube closure.

In recent years, the development and implementation of animal-free approaches to chemical and pharmaceutical hazard and risk assessment has taken off. Alternative approaches are being developed starting from the perspective of human biology and physiology. Neural tube closure is a vital step that occurs early in human development. Correct closure of the neural tube depends on a complex interplay between proteins along a number of protein concentration gradients. The sensitivity of neural tube closure to chemical disturbance of signalling pathways such as the retinoid pathway, is well known. To map the pathways underlying neural tube closure, literature data on the molecular regulation of neural tube closure were collected. As the process of neural tube closure is highly conserved in vertebrates, the extensive literature available for the mouse was used whilst considering its relevance for humans. Thus, important cell compartments, regulatory pathways, and protein interactions essential for neural tube closure under physiological circumstances were identified and mapped. An understanding of aberrant processes leading to neural tube defects (NTDs) requires detailed maps of neural tube embryology, including the complex genetic signals and responses underlying critical cellular dynamical and biomechanical processes. The retinoid signaling pathway serves as a case study for this ontology because of well-defined crosstalk with the genetic control of neural tube patterning and morphogenesis. It is a known target for mechanistically-diverse chemical structures that disrupt neural tube closure The data presented in this manuscript will set the stage for constructing mathematical models and computer simulation of neural tube closure for human-relevant AOPs and predictive toxicology.