A Novel Transgenic Model to Study Thyroid Axis Activity in Early Life Stage Medaka.

Identifying endocrine disrupting chemicals in order to limit their usage is a priority and required according to the European Regulation. There are no Organization for Economic Co-operation and Development (OECD) test guidelines based on fish available for the detection of Thyroid axis Active Chemicals (TACs). This study aimed to fill this gap by developing an assay at eleuthero-embryonic life stages in a novel medaka (Oryzias latipes) transgenic line. This transgenic line expresses green fluorescent protein (GFP) in thyrocytes, under the control of the medaka thyroglobulin gene promoter. The fluorescence expressed in the thyrocytes is inversely proportional to the thyroid axis activity. When exposed for 72 h to activators (triiodothyronine (T3) and thyroxine (T4)) or inhibitors (6-N-propylthiouracil (PTU), Tetrabromobisphenol A (TBBPA)) of the thyroid axis, the thyrocytes can change their size and express lower or higher levels of fluorescence, respectively. This reflects the regulation of thyroglobulin by the negative feedback loop of the Hypothalamic-Pituitary-Thyroid axis. T3, T4, PTU, and TBBPA induced fluorescence changes with the lowest observable effect concentrations (LOECs) of 5 µg/L, 1 µg/L, 8 mg/L, and 5 mg/L, respectively. This promising tool could be used as a rapid screening assay and also to help decipher the mechanisms by which TACs can disrupt the thyroid axis in medaka.

Development of an adverse outcome pathway network for nephrotoxicity.

Adverse outcome pathways (AOPs) were introduced in modern toxicology to provide evidence-based representations of the events and processes involved in the progression of toxicological effects across varying levels of the biological organisation to better facilitate the safety assessment of chemicals. AOPs offer an opportunity to address knowledge gaps and help to identify novel therapeutic targets. They also aid in the selection and development of existing and new in vitro and in silico test methods for hazard identification and risk assessment of chemical compounds. However, many toxicological processes are too intricate to be captured in a single, linear AOP. As a result, AOP networks have been developed to aid in the comprehension and placement of associated events underlying the emergence of related forms of toxicity-where complex exposure scenarios and interactions may influence the ultimate adverse outcome. This study utilised established criteria to develop an AOP network that connects thirteen individual AOPs associated with nephrotoxicity (as sourced from the AOP-Wiki) to identify several key events (KEs) linked to various adverse outcomes, including kidney failure and chronic kidney disease. Analysis of the modelled AOP network and its topological features determined mitochondrial dysfunction, oxidative stress, and tubular necrosis to be the most connected and central KEs. These KEs can provide a logical foundation for guiding the selection and creation of in vitro assays and in silico tools to substitute for animal-based in vivo experiments in the prediction and assessment of chemical-induced nephrotoxicity in human health.

Hepatotoxicity of silver nanoparticles: Benchmark concentration modeling of an in vitro transcriptomics study in human iPSC-derived hepatocytes.

Despite two decades of research on silver nanoparticle (AgNP) toxicity, a safe threshold for exposure has not yet been established, albeit being critically needed for risk assessment and regulatory decision-making. Traditionally, a point-of-departure (PoD) value is derived from dose response of apical endpoints in animal studies using either the no-observed-adverse-effect level (NOAEL) approach, or benchmark dose (BMD) modeling. To develop new approach methodologies (NAMs) to inform human risk assessment of AgNPs, we conducted a concentration response modeling of the transcriptomic changes in hepatocytes derived from human induced pluripotent stem cells (iPSCs) after being exposed to a wide range concentration (0.01-25 µg/ml) of AgNPs for 24 h. A plausible transcriptomic PoD of 0.21 µg/ml was derived for a pathway related to the mode-of-action (MOA) of AgNPs, and a more conservative PoD of 0.10 µg/ml for a gene ontology (GO) term not apparently associated with the MOA of AgNPs. A reference dose (RfD) could be calculated from either of the PoDs as a safe threshold for AgNP exposure. The current study illustrates the usefulness of in vitro transcriptomic concentration response study using human cells as a NAM for toxicity study of chemicals that lack adequate toxicity data to inform human risk assessment.

A new approach methodology using kinetically-derived maximum dose levels in risk assessment - A case study with afidopyropen.

We present a case study for afidopyropen (AF; insecticide) to characterize chronic dietary human health risk using a Risk 21-based approach. Our objective is to use a well-tested pesticidal active ingredient (AF) to show how a new approach methodology (NAM), using the kinetically-derived maximum dose (KMD) and with far less animal testing, can reliably identify a health-protective point of departure (PoD) for chronic dietary human health risk assessments (HHRA). Chronic dietary HHRA involves evaluation of both hazard and exposure information to characterize risk. Although both are important, emphasis has been placed on a checklist of required toxicological studies for hazard characterization, with human exposure information only considered after evaluation of hazard data. Most required studies are not used to define the human endpoint for HHRA. The information presented demonstrates a NAM that uses the KMD determined by saturation of a metabolic pathway, which can be used as an alternative POD. In these cases, the full toxicological database may not need to be generated. Demonstration that the compound is not genotoxic and that the KMD is protective of adverse effects in 90-day oral rat and reproductive/developmental studies is sufficient to support the use of the KMD as an alternative POD.

Improvements to in silico skin sensitisation predictions through privacy-preserving data sharing.

In silico models are often built solely on publicly available data which may mean that they are less predictive for proprietary chemical space. Data sharing initiatives can improve the performance of such models, but organisations are often unable to share their data due to the need to protect their business interests and maintain the confidentiality of the chemicals in their research and development programmes. In silico models like Derek Nexus, which use expert knowledge to develop structural alerts based on chemical toxicity, can use proprietary data to identify new areas of chemical space and/or refine existing alerts whilst still preserving the privacy of the confidential data. Five hundred and thirty seven proprietary chemicals with skin sensitisation data were shared which led to the implementation of 7 new alerts and 5 modified alerts, with a concomitant 19% increase in sensitivity and 3% increase in specificity of the model.

Towards a future regulatory framework for chemicals in the European Union - Chemicals 2.0.

The body of EU chemicals legislation has evolved since the 1960s, producing the largest knowledge base on chemicals worldwide. Like any evolving system, however, it has become increasingly diverse and complex, resulting in inefficiencies and potential inconsistencies. In the light of the EU Chemicals Strategy for Sustainability, it is therefore timely and reasonable to consider how aspects of the system could be simplified and streamlined, without losing the hard-earned benefits to human health and the environment. In this commentary, we propose a conceptual framework that could be the basis of Chemicals 2.0 - a future safety assessment and management approach that is based on the application of New Approach Methodologies (NAMs), mechanistic reasoning and cost-benefit considerations. Chemicals 2.0 is designed to be a more efficient and more effective approach for assessing chemicals, and to comply with the EU goal to completely replace animal testing, in line with Directive 2010/63/EU. We propose five design criteria for Chemicals 2.0 to define what the future system should achieve. The approach is centered on a classification matrix in which NAMs for toxicodynamics and toxicokinetics are used to classify chemicals according to their level of concern. An important principle is the need to ensure an equivalent, or higher, protection level.

Physico-Chemical Approaches to Investigate Surface Hydroxyls as Determinants of Molecular Initiating Events in Oxide Particle Toxicity.

The study of molecular recognition patterns is crucial for understanding the interactions between inorganic (nano)particles and biomolecules. In this review we focus on hydroxyls (OH) exposed at the surface of oxide particles (OxPs) which can play a key role in molecular initiating events leading to OxPs toxicity. We discuss here the main analytical methods available to characterize surface OH from a quantitative and qualitative point of view, covering thermogravimetry, titration, ζ potential measurements, and spectroscopic approaches (NMR, XPS). The importance of modelling techniques (MD, DFT) for an atomistic description of the interactions between membranes/proteins and OxPs surfaces is also discussed. From this background, we distilled a new approach methodology (NAM) based on the combination of IR spectroscopy and bioanalytical assays to investigate the molecular interactions of OxPs with biomolecules and membranes. This NAM has been already successfully applied to SiO2 particles to identify the OH patterns responsible for the OxPs' toxicity and can be conceivably extended to other surface-hydroxylated oxides.

A framework for chemical safety assessment incorporating new approach methodologies within REACH.

The long-term investment in new approach methodologies (NAMs) within the EU and other parts of the world is beginning to result in an emerging consensus of how to use information from in silico, in vitro and targeted in vivo sources to assess the safety of chemicals. However, this methodology is being adopted very slowly for regulatory purposes. Here, we have developed a framework incorporating in silico, in vitro and in vivo methods designed to meet the requirements of REACH in which both hazard and exposure can be assessed using a tiered approach. The outputs from each tier are classification categories, safe doses, and risk assessments, and progress through the tiers depends on the output from previous tiers. We have exemplified the use of the framework with three examples. The outputs were the same or more conservative than parallel assessments based on conventional studies. The framework allows a transparent and phased introduction of NAMs in chemical safety assessment and enables science-based safety decisions which provide the same level of public health protection using fewer animals, taking less time, and using less financial and expert resource. Furthermore, it would also allow new methods to be incorporated as they develop through continuous selective evolution rather than periodic revolution.

Use of New Approach Methodologies (NAMs) in regulatory decisions for chemical safety: Report from an EPAA Deep Dive Workshop.

New Approach Methodologies (NAMs) are considered to include any in vitro, in silico or chemistry-based method, as well as the strategies to implement them, that may provide information that could inform chemical safety assessment. Current chemical legislation in the European Union is limited in its acceptance of the widespread use of NAMs. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a 'Deep Dive Workshop' to explore the use of NAMs in chemical safety assessment, the aim of which was to support regulatory decisions, whilst intending to protect human health. The workshop recognised that NAMs are currently used in many industrial sectors, with some considered as fit for regulatory purpose. Moreover, the workshop identified key discussion points that can be addressed to increase the use and regulatory acceptance of NAMs. These are based on the changes needed in frameworks for regulatory requirements and the essential needs in education, training and greater stakeholder engagement as well the gaps in the scientific basis of NAMs.

A 10-step framework for use of read-across (RAX) in next generation risk assessment (NGRA) for cosmetics safety assessment.

This paper presents a 10-step read-across (RAX) framework for use in cases where a threshold of toxicological concern (TTC) approach to cosmetics safety assessment is not possible. RAX builds on established approaches that have existed for more than two decades using chemical properties and in silico toxicology predictions, by further substantiating hypotheses on toxicological similarity of substances, and integrating new approach methodologies (NAM) in the biological and kinetic domains. NAM include new types of data on biological observations from, for example, in vitro assays, toxicogenomics, metabolomics, receptor binding screens and uses physiologically-based kinetic (PBK) modelling to inform about systemic exposure. NAM data can help to substantiate a mode/mechanism of action (MoA), and if similar chemicals can be shown to work by a similar MoA, a next generation risk assessment (NGRA) may be performed with acceptable confidence for a data-poor target substance with no or inadequate safety data, based on RAX approaches using data-rich analogue(s), and taking account of potency or kinetic/dynamic differences.

Rethinking chronic toxicity and carcinogenicity assessment for agrochemicals project (ReCAAP): A reporting framework to support a weight of evidence safety assessment without long-term rodent bioassays.

Rodent cancer bioassays have been long-required studies for regulatory assessment of human cancer hazard and risk. These studies use hundreds of animals, are resource intensive, and certain aspects of these studies have limited human relevance. The past 10 years have seen an exponential growth of new technologies with the potential to effectively evaluate human cancer hazard and risk while reducing, refining, or replacing animal use. To streamline and facilitate uptake of new technologies, a workgroup comprised of scientists from government, academia, non-governmental organizations, and industry stakeholders developed a framework for waiver rationales of rodent cancer bioassays for consideration in agrochemical safety assessment. The workgroup used an iterative approach, incorporating regulatory agency feedback, and identifying critical information to be considered in a risk assessment-based weight of evidence determination of the need for rodent cancer bioassays. The reporting framework described herein was developed to support a chronic toxicity and carcinogenicity study waiver rationale, which includes information on use pattern(s), exposure scenario(s), pesticidal mode-of-action, physicochemical properties, metabolism, toxicokinetics, toxicological data including mechanistic data, and chemical read-across from similar registered pesticides. The framework could also be applied to endpoints other than chronic toxicity and carcinogenicity, and for chemicals other than agrochemicals.

The Evolution of Regulatory Toxicology: Where is the Gardener?

There is a need for paradigm change in the methodology employed for toxicological testing and assessment. It could be said that this change is well on its way, through an evolutionary progress analogous to that of natural selection. Darwin's Theory of Evolution has defined the idea of evolution and descendancy since the last third of the 19th century. Increasingly, this concept of 'evolution' is being applied beyond the field of biology. This Comment article discusses the progress of toxicological testing in the context of 'evolutionary pressure' and deliberates how this process can help foster the development, implementation and acceptance of mechanistic and human-relevant methods in this field. By comparing the current regulatory landscape in toxicity testing and assessment to specific elements in Charles Darwin's evolutionary theory, we aim to better understand the needs and requirements for the future.

Integration of data across toxicity endpoints for improved safety assessment of chemicals: the example of carcinogenicity assessment.

In view of the need to enhance the assessment of consumer products called for in the EU Chemicals Strategy for Sustainability, we developed a methodology for evaluating hazard by combining information across different systemic toxicity endpoints and integrating the information with new approach methodologies. This integrates mechanistic information with a view to avoiding redundant in vivo studies, minimising reliance on apical endpoint tests and ultimately devising efficient testing strategies. Here, we present the application of our methodology to carcinogenicity assessment, mapping the available information from toxicity test methods across endpoints to the key characteristics of carcinogens. Test methods are deconstructed to allow the information they provide to be organised in a systematic way, enabling the description of the toxicity mechanisms leading to the adverse outcome. This integrated approach provides a flexible and resource-efficient means of fully exploiting test methods for which test guidelines are available to fulfil regulatory requirements for systemic toxicity assessment as well as identifying where new methods can be integrated.

Incorporating lines of evidence from New Approach Methodologies (NAMs) to reduce uncertainties in a category based read-across: A case study for repeated dose toxicity.

A group of triazole compounds was selected to investigate the confidence that may be associated with read-across of a complex data gap: repeated dose toxicity. The read-across was evaluated using Assessment Elements (AEs) from the European Chemicals Agency's (ECHA's) Read-Across Assessment Framework (RAAF), alongside appraisal of associated uncertainties. Following an initial read-across based on chemical structure and properties, uncertainties were reduced by the integration of data streams such as those from New Approach Methodologies (NAM) and other existing data. In addition, addressing the findings of the ECHA RAAF framework, complemented with specific questions concerning uncertainties, increased the confidence that can be placed in read-across. Although a data rich group of compounds with a strong mechanistic basis was analysed, it was clearly demonstrated that NAM data available from publicly available resources could be applied to support read-across. It is acknowledged that most read-across studies will not be so data rich or mechanistically robust, therefore some targeted experimentation may be required to fill the data gaps. In this sense, NAMs should constitute new experimental tests performed with the specific goal of reducing the uncertainties and demonstrating the read-across hypothesis.

The SCCS Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation, 11th revision, 30-31 March 2021, SCCS/1628/21.

The "SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation, 11 th Revision" (SCCS/1628/21) contains relevant and updated information on the different aspects of testing and safety evaluation of cosmetic substances in Europe. The emphasis is on cosmetic ingredients for which a concern has been expressed for human health. Indirectly, the Guidance also provides some advice on the safety of finished products. A general aim is to improve harmonised compliance with the current cosmetic EU legislation, Regulation (EC) No 1223/2009, for which animal testing and marketing bans fully apply from 2013 onwards. This means that no in vivo testing of ingredients or finished products is allowed in Europe for the purpose of cosmetics. For this reason, the SCCS has closely followed the progress made in regard to the development and validation of alternative replacement methods, also referred to as new approach methodology (NAM). The "SCCS Notes of Guidance" are regularly revised and updated in order to incorporate progress made and experience gained over time, in particular on the use of NAMs, and the new methods and data that became available since previous revision (SCCS/1602/18) formed the basis of the current (11 th) Revision.

Acute toxicity "six-pack" studies supporting approved new drug applications in the U.S., 2015-2018.

The acute toxicity "six-pack" is a battery of animal tests used to evaluate acute systemic toxicity by three routes of exposure, skin and eye irritation/corrosion, and skin sensitization. A perception exists that these tests are not required for pharmaceuticals. For the four years from 2015 through 2018, we tallied the number of corresponding tests submitted by sponsors in approved, original new drug applications, as reported by the U.S. Food and Drug Administration (FDA) in publicly available reviews. In 125 reviews, we identified 228 single dose acute toxicity studies, 62 in vivo local tolerance studies, and 32 in vivo skin sensitization studies, as well as 37 in vitro or ex vivo local tolerance studies. A total of 4798 animals were used in these studies; however, FDA's reporting was inconsistent, and we estimate the actual number of animals used to be 8998. For the evaluation of single dose acute toxicity, we accessed two guidance documents with conflicting recommendations regarding routes of administration and number of species to be used. For the evaluation of local tolerance and skin sensitization, most studies examined were conducted by routes other than that intended for human administration. Non-animal methods used to evaluate skin sensitization were not reported.

Exploring current read-across applications and needs among selected U.S. Federal Agencies.

Read-across is a well-established data gap-filling technique applied for regulatory purposes. In US Environmental Protection Agency's New Chemicals Program under TSCA, read-across has been used extensively for decades, however the extent of application and acceptance of read-across among U.S. federal agencies is less clear. In an effort to build read-across capacity, raise awareness of the state of the science, and work towards a harmonization of read-across approaches across U.S. agencies, a new read-across workgroup was established under the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM). This is one of several ad hoc groups ICCVAM has convened to implement the ICCVAM Strategic Roadmap. In this article, we outline the charge and scope of the workgroup and summarize the current applications, tools used, and needs of the agencies represented on the workgroup for read-across. Of the agencies surveyed, the Environmental Protection Agency had the greatest experience in using read-across whereas other agencies indicated that they would benefit from gaining a perspective of the landscape of the tools and available guidance. Two practical case studies are also described to illustrate how the read-across approaches applied by two agencies vary on account of decision context.

Predicting estrogen receptor activation by a group of substituted phenols: An integrated approach to testing and assessment case study.

Traditional approaches for chemical risk assessment cannot keep pace with the number of substances requiring assessment. Thus, in a global effort to expedite and modernize chemical risk assessment, New Approach Methodologies (NAMs) are being explored and developed. Included in this effort is the OECD Integrated Approaches for Testing and Assessment (IATA) program, which provides a forum for OECD member countries to develop and present case studies illustrating the application of NAM in various risk assessment contexts. Here, we present an IATA case study for the prediction of estrogenic potential of three target phenols: 4-tert-butylphenol, 2,4-di-tert-butylphenol and octabenzone. Key features of this IATA include the use of two computational approaches for analogue selection for read-across, data collected from traditional and NAM sources, and a workflow to generate predictions regarding the targets' ability to bind the estrogen receptor (ER). Endocrine disruption can occur when a chemical substance mimics the activity of natural estrogen by binding to the ER and, if potency and exposure are sufficient, alters the function of the endocrine system to cause adverse effects. The data indicated that of the three target substances that were considered herein, 4-tert-butylphenol is a potential endocrine disruptor. Further, this IATA illustrates that the NAM approach explored is health protective when compared to in vivo endpoints traditionally used for human health risk assessment.

Alternative methods go green! Green toxicology as a sustainable approach for assessing chemical safety and designing safer chemicals

Green toxicology is marching chemistry into the 21st century. This emerging framework will transform how chemical safety is evaluated by incorporating evaluation of the hazards, exposures, and risks associated with chemicals into early product development in a way that minimizes adverse impacts on human and environmental health. The goal is to minimize toxic threats across entire supply chains through smarter designs and policies. Traditional animal testing methods are replaced by faster, cutting-edge innovations like organs-on-chips and artificial intelligence predictive models that are also more cost-effective. Core principles of green toxicology include utilizing alternative test methods, applying the precautionary principle, considering lifetime impacts, and emphasizing risk prevention over reaction. This paper provides an overview of these foundational concepts and describes current initiatives and future opportunities to advance the adoption of green toxicology approaches. Chal-lenges and limitations are also discussed. Green shoots are emerging with governments offering carrots like the European Green Deal to nudge industry. Noteworthy, animal rights and environ-mental groups have different ideas about the needs for testing and their consequences for animal use. Green toxicology represents the way forward to support both these societal needs with sufficient throughput and human relevance for hazard information and minimal animal suffering. Green toxi-cology thus sets the stage to synergize human health and ecological values. Overall, the integration of green chemistry and toxicology has potential to profoundly shift how chemical risks are evaluated and managed to achieve safety goals in a more ethical, ecologically-conscious manner.

Green toxicology aims to make chemicals safer by design. It focuses on preventing toxicity issues early during development instead of testing after products are developed. Green toxicology uses modern non-animal methods like computer models and lab tests with human cells to predict if chem­icals could be hazardous. Benefits are faster results, lower costs, and less animal testing. The principles of green toxicology include using alternative tests, applying caution even with uncertain data, con­sidering lifetime impacts across global supply chains, and emphasizing prevention over reaction. The article highlights European and US policy efforts to spur sustainable chemistry innovation which will necessitate greener approaches to assess new materials and drive adoption. Overall, green toxi­cology seeks to integrate safer design concepts so that human and environmental health are valued equally with functionality and profit. This alignment promises safer, ethical products but faces chal­lenges around validating new methods and overcoming institutional resistance to change.