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.

New supporting data to guide the use of evident toxicity in acute oral toxicity studies (OECD TG 420).

Currently there are three test guidelines (TG) for acute oral toxicity studies of substances or mixtures from the Organisation for Economic Co-operation and Development (OECD). TG 423 and TG 425 use lethality as an endpoint, while TG 420 replaces death with 'evident toxicity', defined as clear signs that exposure to a higher dose would result in death. However, the perceived subjectivity of 'evident toxicity' may be preventing wider use of TG 420. To address this, the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) and the European Partnership for Alternative Approaches to Animal Testing (EPAA) collaborated to provide recommendations on the recognition of 'evident toxicity'. Historical data from acute oral toxicity studies were analysed for clinical signs at the lower dose that could have predicted death at the higher dose. Several signs including ataxia, laboured respiration, and eyes partially closed, alone or in combination, are highly predictive. Others such as lethargy, decreased respiration, and loose faeces have lower but still appreciable positive predictive value (PPV). The data has been used to develop recommendations to promote use of TG 420 and thus reduce the suffering and numbers of animals used in acute oral toxicity studies.

An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

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.

The use of recovery animals in nonclinical safety assessment studies with monoclonal antibodies: further 3Rs opportunities remain.

Assessment of reversibility from nonclinical toxicity findings in animals with potential adverse clinical impact is required during pharmaceutical development, but there is flexibility around how and when this is performed and if recovery animals are necessary. For monoclonal antibodies (mAbs) and in accordance with ICH S6(R1) if inclusion of recovery animals is warranted, this need only occur in one study. Data on study designs for first-in-human (FIH)-enabling and later-development toxicity studies were shared from a recent collaboration between the NC3Rs, EPAA, Netherlands Medicines Evaluation Board (MEB) and 14 pharmaceutical companies. This enabled a review of practices on recovery animal use during mAb development and identification of opportunities to reduce research animal use. Recovery animals were included in 68% of FIH-enabling and 69% of later-development studies, often in multiple studies in the same program. Recovery groups were commonly in control plus one test article-dosed group or in all dose groups (45% of studies, each design). Based on the shared data review and conclusions, limiting inclusion of recovery to a single nonclinical toxicology study and species, study design optimisation and use of existing knowledge instead of additional recovery groups provide opportunities to further reduce animal use within mAb development programs.

Re-evaluating the need for chronic toxicity studies with therapeutic monoclonal antibodies, using a weight of evidence approach.

To support registration of monoclonal antibodies (mAbs) for chronic indications, 6-month toxicity studies have historically been conducted. Experience with mAb development has shown a relatively benign and well-understood safety profile for this class, with most toxicity findings anticipated based on pharmacology. We evaluated whether a 6-month toxicity study is necessary to assess the long-term safety of mAbs. Data on First-in-Human (FIH)-enabling and chronic toxicity studies were shared for 142 mAbs submitted by 11 companies. Opportunities to further optimize study designs to reduce animal usage were identified. For 71% of mAbs, no toxicities or no new toxicities were noted in chronic studies compared to FIH-enabling study findings. New toxicities of potential concern for human safety or that changed trial design were identified in 13.5% of cases, with 7% being considered critical and 2% leading to program termination. An iterative, weight-of-evidence model which considers factors that influence the overall risk for a mAb to cause toxicity was developed. This model enables an evidence-based justification, suggesting when 3-month toxicity studies are likely sufficient to support late-stage clinical development and registration for some mAbs.

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.

Exploring the Definition of "Similar Toxicities": Case Studies Illustrating Industry and Regulatory Interpretation of ICH S6(R1) for Long-Term Toxicity Studies in One or Two Species.

ICH S6 (R1) states that safety evaluation of biotherapeutics should normally include 2 relevant species when available (i.e., a rodent and non-rodent species in which the test material is pharmacologically active), at least for short-term toxicology studies (generally supporting Phase I trials). For subsequent long-term toxicology studies (e.g., chronic studies up to 6 months dosing duration), there are options to reduce to only one species when justified, including when the mechanism of action of the biologic is well-understood or the toxicity findings in the short-term studies are "similar" in both the rodent and non-rodent species. Across the industry, around 25 to 33% of biologics assess multiple species within short-term toxicity studies but it is often unclear how different companies and regulators are applying the ICH S6 (R1) principles of "similar toxicity profiles" to progress with either 1 or 2 species in the long-term studies, in particular whether the absence of toxicities is considered within this definition. Sponsors may potentially continue to use 2 species to avoid regulatory risk and potential delays in development timelines, representing missed opportunities for reducing animal use, particularly of non-human primates, during drug development.This article summarizes presentations from a symposium at the 41st Annual meeting of the American College of Toxicology (ACT) in November 2020, in which industry case studies and regulatory perspectives addressed considerations and decisions for using 1 or 2 species for long-term toxicity studies, highlighting any common themes or experience that could be applicable for use in future decision-making.

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.

Investigating endocrine-disrupting properties of chemicals in fish and amphibians: Opportunities to apply the 3Rs.

Many regulations are beginning to explicitly require investigation of a chemical's endocrine-disrupting properties as a part of the safety assessment process for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher-tier in vivo assays. Higher-tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher-tier assays are animal- and resource-intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop ("Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs"). Over 50 delegates attended from North America and Europe, across academia, laboratories, and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement and/or new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow-up. Integr Environ Assess Manag 2022;18:442-458. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Refining Procedures within Regulatory Toxicology Studies: Improving Animal Welfare and Data.

During the development of potential new medicines or agrochemicals, an assessment of the safety profile to humans and environmental species is conducted using a range of different in silico and in vitro techniques in conjunction with metabolism and toxicity studies using animals. The required studies are outlined within international regulatory guidelines which acknowledge and support the application of the 3Rs to reduce the number of animals used or to refine the procedures performed when these studies are deemed to be necessary. The continued development of new technologies and adoption of best-practice approaches to laboratory animal housing and study procedures has generated a series of refinements that can be incorporated into animal studies throughout the package. These refinements benefit the welfare of fish, mice, rats, rabbits, dogs, minipigs, and non-human primates (NHPs) whilst maintaining or improving data quality within general toxicology, metabolism, and other studies and can also bring efficiencies to processes that benefit study costs and timings. Examples are shared which cover the following topics: social housing of dogs and NHPs, surgical refinements in the rat bile duct cannulation model for collection of data for metabolism studies, whether fasting is really required prior to clinical pathology sampling, and the use of microsampling for toxicokinetics.

Rethinking agrochemical safety assessment: A perspective.

Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in the 1980s. However, since then, there have been significant advances in the toxicological sciences that have improved our understanding of mechanisms underpinning adverse human health effects. The time is ripe to 'rethink' approaches used for human safety assessments of agrochemicals to ensure they reflect current scientific understanding and increasingly embrace new opportunities to improve human relevance and predictivity, and to reduce the reliance on animals. Although the ultimate aim is to enable a paradigm shift and an overhaul of global regulatory data requirements, there is much that can be done now to ensure new opportunities and approaches are adopted and implemented within the current regulatory frameworks. This commentary reviews current initiatives and emerging opportunities to embrace new approaches to improve agrochemical safety assessment for humans, and considers various endpoints and initiatives (including acute toxicity, repeat dose toxicity studies, carcinogenicity, developmental and reproductive toxicity, exposure-driven approaches, inhalation toxicity, and data modelling). Realistic aspirations to improve safety assessment, incorporate new technologies and reduce reliance on animal testing without compromising protection goals are discussed.

Opportunities and challenges related to saturation of toxicokinetic processes: Implications for risk assessment.

Top dose selection for repeated dose animal studies has generally focused on identification of apical endpoints, use of the limit dose, or determination of a maximum tolerated dose (MTD). The intent is to optimize the ability of toxicity tests performed in a small number of animals to detect effects for hazard identification. An alternative approach, the kinetically derived maximum dose (KMD), has been proposed as a mechanism to integrate toxicokinetic (TK) data into the dose selection process. The approach refers to the dose above which the systemic exposures depart from being proportional to external doses. This non-linear external-internal dose relationship arises from saturation or limitation of TK process(es), such as absorption or metabolism. The importance of TK information is widely acknowledged when assessing human health risks arising from exposures to environmental chemicals, as TK determines the amount of chemical at potential sites of toxicological responses. However, there have been differing opinions and interpretations within the scientific and regulatory communities related to the validity and application of the KMD concept. A multi-stakeholder working group, led by the Health and Environmental Sciences Institute (HESI), was formed to provide an opportunity for impacted stakeholders to address commonly raised scientific and technical issues related to this topic and, more specifically, a weight of evidence approach is recommended to inform design and dose selection for repeated dose animal studies. Commonly raised challenges related to the use of TK data for dose selection are discussed, recommendations are provided, and illustrative case examples are provided to address these challenges or refute misconceptions.

Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint.

The fish acute toxicity test (TG203; OECD, 2019) is frequently used and highly embedded in hazard and risk assessment globally. The test estimates the concentration of a chemical that kills 50% of the fish (LC50) over a 96 h exposure and is considered one of the most severe scientific procedures undertaken. Over the years, discussions at the Organisation for Economic Co-operation and Development (OECD) have resulted in changes to the test which reduce the number of fish used, as well as the development of a (potential) replacement test (TG236, OECD, 2013). However, refinement of the mortality endpoint with an earlier (moribundity) endpoint was not considered feasible during the Test Guideline's (TG) last update in 2019. Several stakeholders met at a UK-based workshop to discuss how TG203 can be refined, and identified two key opportunities to reduce fish suffering: (1) application of clinical signs that predict mortality and (2) shortening the test duration. However, several aspects need to be addressed before these refinements can be adopted. TG203 has required recording of major categories of sublethal clinical signs since its conception, with the option to record more detailed signs introduced in the 2019 update. However, in the absence of guidance, differences in identification, recording and reporting of clinical signs between technicians and laboratories is likely to have generated piecemeal data of varying quality. Harmonisation of reporting templates, and training in clinical sign recognition and recording are needed to standardise clinical sign data. This is critical to enable robust data-driven detection of clinical signs that predict mortality. Discussions suggested that the 96 h duration of TG203 cannot stand up to scientific scrutiny. Feedback and data from UK contract research organisations (CROs) conducting the test were that a substantial proportion of mortalities occur in the first 24 h. Refinement of TG203 by shortening the test duration would reduce suffering (and test failure rate) but requires a mechanism to correct new results to previous 96 h LC50 data. The actions needed to implement both refinement opportunities are summarised here within a roadmap. A shift in regulatory assessment, where the 96 h LC50 is a familiar base for decisions, will also be critical.

Preclinical screening for antidepressant activity - shifting focus away from the Forced Swim Test to the use of translational biomarkers.

Depression is the world's predominant mental health problem and a leading cause of disability. Neuropharmacological research has not yet advanced treatments to sufficiently meet clinical need, largely due to the failure of animal models to predict clinical efficacy. The forced swim test (FST) has been extensively used in the field of antidepressant research but has been under scrutiny due to its perceived severity to animals. Any use of animals in experiments and testing must have a scientific or regulatory purpose and researchers need to ensure that there is no scientifically valid alternative. However, regulatory requirements have been incorrectly cited as a reason to support the use of the FST. More research is required on tests that do not involve stressing animals as replacements for the FST. Non-behavioural neurochemical measures might provide a means to advance neuropharmacological developments while reducing animal suffering. For example, brain-derived neurotrophic factor (BDNF) may be promising.

Opportunities and Challenges for Integrating New In Vitro Methodologies in Hazard Testing and Risk Assessment.

Nanomaterials are defined as materials with at least one dimension of 100 nm or less. Their small size confers unique properties that may alter the toxicity profile when compared to larger forms of the same material, requiring additional considerations for safety assessment. There has been a rise in the development of nanomaterials for many applications, and although traditional approaches for toxicity testing may address some of the new toxicity concerns, many may not be directly applicable to nanomaterials and new tools or approaches may need to be developed. Since nanomaterials can exist in many different forms, each of which may cause different adverse biological effects, reliance on traditional in vivo models for safety assessment will simply not be feasible or sustainable, given the volume of materials that may need to be tested. It is essential to consider and develop new in vitro methods that can be applied for hazard identification and risk assessment. Many challenges are associated with using alternative approaches to ensure they are as robust and reliable as traditional in vivo approaches, but by overcoming these issues and adopting new testing strategies there are opportunities to improve safety assessments and reduce the reliance on animal-based toxicity testing strategies.

A comprehensive view on mechanistic approaches for cancer risk assessment of non-genotoxic agrochemicals.

Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.

Opportunities for use of one species for longer-term toxicology testing during drug development: A cross-industry evaluation.

An international expert working group representing 37 organisations (pharmaceutical/biotechnology companies, contract research organisations, academic institutions and regulatory bodies) collaborated in a data sharing exercise to evaluate the utility of two species within regulatory general toxicology studies. Anonymised data on 172 drug candidates (92 small molecules, 46 monoclonal antibodies, 15 recombinant proteins, 13 synthetic peptides and 6 antibody-drug conjugates) were submitted by 18 organisations. The use of one or two species across molecule types, the frequency for reduction to a single species within the package of general toxicology studies, and a comparison of target organ toxicities identified in each species in both short and longer-term studies were determined. Reduction to a single species for longer-term toxicity studies, as used for the development of biologicals (ICHS6(R1) guideline) was only applied for 8/133 drug candidates, but might have been possible for more, regardless of drug modality, as similar target organ toxicity profiles were identified in the short-term studies. However, definition and harmonisation around the criteria for similarity of toxicity profiles is needed to enable wider consideration of these principles. Analysis of a more robust dataset would be required to provide clear, evidence-based recommendations for expansion of these principles to small molecules or other modalities where two species toxicity testing is currently recommended.