Lessons for the Future of NAMs from History, Philosophy and Social Studies of Science.

This paper explores what we can learn from the humanities and social sciences about how standards operate in and around science, in order to understand more about how 'the gold standard' can be shifted away from the use of animals in research and testing, and toward New Approach Methodologies (NAMs). These fields allow us to consider potential futures of NAMs as alternatives, replacements, or complements to animal use in testing and research. As we demonstrate, the questions that we pose and how they are framed are as important as the answers that result. Rather than asking how to 'redefine the gold standard', norms and expectations for NAMs must be actively debated and transparently defined. These considerations would be based, in part, on what has been learned in the past from non-human animal models and systems, but also use the norms within the fields from which the NAMs derive in light of the rich broader contexts within which they are being developed. As we argue, notions such as 'a gold standard' are limited and must be replaced by contextualised standards that depend on the scientific, sociocultural and other factors that contribute to our understanding of a particular method (new or otherwise) as 'good' for a particular purpose.

International regulatory uses of acute systemic toxicity data and integration of new approach methodologies.

Chemical regulatory authorities around the world require systemic toxicity data from acute exposures via the oral, dermal, and inhalation routes for human health risk assessment. To identify opportunities for regulatory uses of non-animal replacements for these tests, we reviewed acute systemic toxicity testing requirements for jurisdictions that participate in the International Cooperation on Alternative Test Methods (ICATM): Brazil, Canada, China, the European Union, Japan, South Korea, Taiwan, and the USA. The chemical sectors included in our review of each jurisdiction were cosmetics, consumer products, industrial chemicals, pharmaceuticals, medical devices, and pesticides. We found acute systemic toxicity data were most often required for hazard assessment, classification, and labeling, and to a lesser extent quantitative risk assessment. Where animal methods were required, animal reduction methods were typically recommended. For many jurisdictions and chemical sectors, non-animal alternatives are not accepted, but several jurisdictions provide guidance to support the use of test waivers to reduce animal use for specific applications. An understanding of international regulatory requirements for acute systemic toxicity testing will inform ICATM's strategy for the development, acceptance, and implementation of non-animal alternatives to assess the health hazards and risks associated with acute toxicity.

Alternatives to Fish Acute Whole Effluent Toxicity (WET) Testing: Predictability of RTgill-W1 Cells and Fathead Minnow Embryos with Actual Wastewater Samples.

Toxicity assays using fish cells and embryos continue to gain momentum as a more ethical and informative alternative to fish acute toxicity testing. The goal of our study was to test the accuracy of RTgill-W1 cells and the fathead minnow (Pimephales promelas) embryos to predict actual whole effluent toxicity (WET) in the fathead minnow larvae. The three models were compared concurrently using samples of various origins and treatment types. Additionally, the toxicity of reference toxicants (Cd, Cu, NH3-N, 3,4-dichloraniline, and benzalkonium chloride) spiked into a nontoxic wastewater was compared. The toxicity of reference toxicants was tested in isosmotic and hypoosmotic exposure media in RTgill-W1 cells. Of the 28 wastewater samples, 14 induced a toxic response in fish larvae. Embryos predicted 11 of the 14 wastewater samples toxic to the larvae, whereas RTgill-W1 cells predicted the toxicity of all 14 toxic samples to the larvae. In addition, embryos and RTgill-W1 cells predicted toxicity in two and six additional samples, respectively, that were nontoxic to larvae. Exposures in hypoosmotic medium significantly increased sensitivity of RTgill-W1 cells to all reference toxicants, excluding benzalkonium chloride, compared to exposures in isosmotic medium and showed toxicity levels similar to that in larvae. Thus, hypoosmotic exposure medium should be considered for aquatic toxicity testing applications. Overall, both gill cell and embryo models predicted toxicity in the majority of wastewater samples toxic to larvae and demonstrated their applicability for regulatory WET testing.

Use of the Allium cepa Model to Assess the Cytogenotoxicity of Luffariella herdmani Marine Sponge Extract.

Marine sponge extracts are known to contain potentially toxic compounds that have biological activities of possible pharmacological interest. Thus, it is vital that biological models are used for the preliminary toxicity screening of such extracts. The present study reports the use of Allium cepa, a low-cost plant-based in vivo model, to assess the cytotoxicity and genotoxicity of Luffariella herdmani marine sponge crude extract (SCE). Pre-germinated onion bulbs, exposed for 96 hours to different concentrations of SCE (ranging from 0.3125 to 20 µg/ml), were used to determine general cytotoxicity. Root length as well as morphological abnormalities were recorded. Genotoxicity was assessed by exposing the root tips to SCE (0.3125-20 µg/ml) and the appropriate controls for 48 hours, and then staining with acetocarmine. The Mitotic Index (MI), Mitotic Phase Indices (MPIs) and chromosomal aberrations were evaluated and recorded. SCE inhibited A. cepa root growth (EC50 = 10.34 µg/ml) and elicited a mitodepressive effect (LC50 = 1.95 µg/ml) in a dose-dependent and significant manner. In addition, macroscopic alterations as well as chromosomal aberrations were detected. Overall, our findings indicate that L. herdmani crude extract exhibits cytotoxic and genotoxic activity, suggesting that it might contain substances with anti-proliferative/anticancer potential that could be subject to further characterisation.

Decision making in next generation risk assessment for skin allergy: Using historical clinical experience to benchmark risk.

Our aim is to develop and apply next generation approaches to skin allergy risk assessment that do not require new animal test data and better quantify uncertainties. Quantitative risk assessment for skin sensitisation uses safety assessment factors to extrapolate from the point of departure to an acceptable human exposure level. It is currently unclear whether these safety assessment factors are appropriate when using non-animal test data to derive a point-of departure. Our skin allergy risk assessment model Defined Approach uses Bayesian statistics to infer a human-relevant metric of sensitiser potency with explicit quantification of uncertainty, using any combination of human repeat insult patch test, local lymph node assay, direct peptide reactivity assay, KeratinoSens™, h-CLAT or U-SENS™ data. Here we describe the incorporation of benchmark exposures pertaining to use of consumer products with clinical data supporting a high/low risk categorisation for skin sensitisation. Margins-of-exposure (potency estimate to consumer exposure level ratio) are regressed against the benchmark risk classifications, enabling derivation of a risk metric defined as the probability that an exposure is low risk. This approach circumvents the use of safety assessment factors and provides a simple and transparent mechanism whereby clinical experience can directly feed-back into risk assessment decisions.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

A hypothetical skin sensitisation next generation risk assessment for coumarin in cosmetic products.

Next generation Risk Assessment (NGRA) is an exposure-led, hypothesis-driven approach which integrates new approach methodologies (NAMs) to assure safety without generating animal data. This hypothetical skin allergy risk assessment of two consumer products - face cream containing 0.1% coumarin and deodorant containing 1% coumarin - demonstrates the application of our skin allergy NGRA framework which incorporates our Skin Allergy Risk Assessment (SARA) Model. SARA uses Bayesian statistics to provide a human relevant point of departure and risk metric for a given chemical exposure based upon input data that can include both NAMs and historical in vivo studies. Regardless of whether NAM or in vivo inputs were used, the model predicted that the face cream and deodorant exposures were low and high risk respectively. Using only NAM data resulted in a minor underestimation of risk relative to in vivo. Coumarin is a predicted pro-hapten and consequently, when applying this mechanistic understanding to the selection of NAMs the discordance in relative risk could be minimized. This case study demonstrates how integrating a computational model and generating bespoke NAM data in a weight of evidence framework can build confidence in safety decision making.

2018 Lush Science Prize.

The Lush Prize supports animal-free testing by awarding money prizes of up to £350,000 per year to the most effective projects and individuals who have been working towards the goal of replacing animals in product or ingredient safety testing. Since its inception in 2012, the Lush Prize has distributed almost £2 million. Prizes are awarded for developments in five strategic areas: Science; Lobbying; Training; Public Awareness; and Young Researchers. In 2015, the judges also awarded a Black Box prize for the development of the skin sensitisation Adverse Outcome Pathway and its associated in vitro assays. The Science Prize is awarded to researchers whose work the judging panel believe to have made the most significant contribution, in the preceding year, to the replacement of animal testing. This 2018 Science Background paper outlines the research projects that were presented to the Prize judges as potential candidates for the 2018 Lush Science Prize award. To obtain an overview of developments in the field of animal replacement in toxicity research, recent work by the relevant scientific institutions and projects in this area, including the OECD, CAAT, ECVAM, UK NC3Rs, US Tox21 Programme, the ToxCast programme and EU-ToxRisk, was reviewed. Recent developments in toxicity testing research were investigated by searching the relevant literature. Abstracts from conferences focusing on animal replacement in toxicity testing that were held in the preceding 12 months, were also analysed, including those from the 2017 10th World Congress on Alternatives and Animals in the Life Sciences and the 2018 Society of Toxicology annual conference.

The Implementation of the Three Rs in Regulatory Toxicity and Biosafety Assessment: The Indian Perspective.

Animal models have long served as a basis for scientific experimentation, biomedical research, drug development and testing, disease modelling and toxicity studies, as they are widely thought to provide meaningful, human-relevant predictions. However, many of these systems are resource intensive and time-consuming, have low predictive value and are associated with great social and ethical dilemmas. Often drugs appear to be effective and safe in these classical animal models, but later prove to be ineffective and/or unsafe in clinical trials. These issues have paved the way for a paradigm shift from the use of in vivo approaches, toward the 'science of alternatives'. This has fuelled several research and regulatory initiatives, including the ban on the testing of cosmetics on animals. The new paradigm has been shifted toward increasing the relevance of the models for human predictivity and translational efficacy, and this has resulted in the recent development of many new methodologies, from 3-D bio-organoids to bioengineered 'human-on-a-chip' models. These improvements have the potential to significantly advance medical research globally. This paper offers a stance on the existing strategies and practices that utilise alternatives to animals, and outlines progress on the incorporation of these models into basic and applied research and education, specifically in India. It also seeks to provide a strategic roadmap to streamline the future directions for the country's policy changes and investments. This strategic roadmap could be a useful resource to guide research institutions, industries, regulatory agencies, contract research organisations and other stakeholders in transitioning toward modern approaches to safety and risk assessment that could replace or reduce the use of animals without compromising the safety of humans or the environment.

Importance of in vitro conditions for modeling the in vivo dose in humans by in vitro-in vivo extrapolation (IVIVE).

In vitro studies are increasingly proposed to replace in vivo toxicity testing of substances. We set out to apply physiologically based pharmacokinetic (PBPK) modeling to predict the in vivo dose of amiodarone that leads to the same concentration-time profile in the supernatant and the cell lysate of cultured primary human hepatic cells (PHH). A PBPK human model was constructed based on the structure and tissue distribution of amiodarone in a rat model and using physiological human parameters. The predicted concentration-time profile in plasma was in agreement with human experimental data with the unbound fraction of amiodarone in plasma crucially affecting the goodness-of-fit. Using the validated kinetic model, we subsequently described the in vitro concentration-time data of amiodarone in PHH culture. However, this could be only appropriately modeled under conditions of zero protein binding and the very low clearance of the in vitro system in PHH culture. However, these represent unphysiological conditions and, thus, the main difference between the in vivo and the in vitro systems. Our results reveal that, for meaningful quantitative extrapolation from in vitro to in vivo conditions in PBPK studies, it is essential to avoid non-intended differences between these conditions. Specifically, clearance and protein binding, as demonstrated in our analysis of amiodarone modeling, are important parameters to consider.

Identifying sublethal endpoints for evaluating neurotoxic compounds utilizing the fish embryo toxicity test.

Fish embryos are increasingly being utilized in aquatic toxicity testing, as evidenced by the Organisation for Economic Co-operation and Development's approval of the fish embryo acute toxicity (FET) test. However, the FET test only allows for the estimation of acute toxicity, whereas other test methods such as the larval growth and survival (LGS) test allow for the estimation of both acute and chronic toxicity. Additionally, it has been demonstrated that the FET test is less sensitive than other test methods for some neurotoxic compounds. To address these limitations, efforts to identify sublethal endpoints that increase FET test sensitivity and allow for the prediction of sublethal adverse effects have begun. As such, the objectives of the current study were 1) to compare estimated LC50 values from the FET and LGS test for three known neurotoxicants: fluoride (F), nickel (Ni), and cadmium (Cd) and 2) to evaluate the responsiveness of potential sublethal endpoints for the FET test related to growth (i.e., wet weight and snout-vent length), neurological development (i.e., spontaneous contraction frequency and eye size), and cardiovascular function (i.e., heart rate and pericardial area). The calculated LC50 values from the F and Cd FET test were significantly higher than those from the LGS test, demonstrating that the FET test is less sensitive than the LGS test for neurotoxic compounds. Only Cd exposure resulted in alterations in any of the sublethal endpoints investigated. Embryos/eleutheroembryos exposed to Cd displayed alterations in length, eye size, and pericardial area at concentrations five-fold less than the estimated LC50 value, suggesting that for Cd the inclusion of these sublethal endpoints would improve the sensitivity of the FET test. Overall, these results provide evidence that for some neurotoxicants, the inclusion of sublehtal endpoints may improve the utility of the FET test; however, further research utilizing a broader range of neurotoxicants with differing mechanisms of action, is needed to fully establish such endpoints in the context of routine FET test.

Enhancing the fathead minnow fish embryo toxicity test: Optimizing embryo production and assessing the utility of additional test endpoints.

The fathead minnow fish embryo toxicity (FET) test has been identified as a potential alternative to toxicity test methods that utilize older fish. However, several challenges have been identified with the fathead minnow FET test, including: 1) difficulties in obtaining appropriately-staged embryos for FET test initiation, 2) a paucity of data comparing fathead minnow FET test performance to the fathead minnow larval growth and survival (LGS) test and 3) a lack of sublethal endpoints that could be used to estimate chronic toxicity and/or predict adverse effects. These challenges were addressed through three study objectives. The first objective was to optimize embryo production by assessing the effect of breeding group composition (number of males and females) on egg production. Results showed that groups containing one male and four females produced the largest clutches, enhancing the likelihood of procuring sufficient numbers of embryos for FET test initiation. The second study objective was to compare the performance of the FET test to that of the fathead minnow LGS test using three reference toxicants. The FET and LGS tests were similar in their ability to predict the acute toxicity of sodium chloride and ethanol, but the FET test was found to be more sensitive than the LGS test for sodium dodecyl sulfate. The last objective of the study was to evaluate the utility and practicality of several sublethal metrics (i.e., growth, developmental abnormalities and growth- and stress-related gene expression) as FET test endpoints. Developmental abnormalities, including pericardial edema and hatch success, were found to offer the most promise as additional FET test endpoints, given their responsiveness, potential for predicting adverse effects, ease of assessment and low cost of measurement.

Quantifying the benefits of using read-across and in silico techniques to fulfill hazard data requirements for chemical categories.

Substantial benefits are realized through the use of read-across and in silico techniques to fill data gaps for structurally similar substances. Considerable experience in applying these techniques was gained under two voluntary high production volume (HPV) chemical programs - the International Council of Chemical Associations' (ICCA) Cooperative Chemicals Assessment Programme (with the cooperation of the Organization of Economic Cooperation and Development) and the U.S. Environmental Protection Agency's HPV Challenge Program. These programs led to the compilation and public availability of baseline sets of health and environmental effects data for thousands of chemicals. The American Cleaning Institute's (ACI) contribution to these national and global efforts included the compilation of these datasets for 261 substances. Chemicals that have structural similarities are likely to have similar environmental fate, physical-chemical and toxicological properties, which was confirmed by examining available data from across the range of substances within categories of structurally similar HPV chemicals. These similarities allowed the utilization of read-across, trend analysis techniques and qualitative structure activity relationship ((Q)SAR) tools to fill data gaps. This paper presents the first quantification of actual benefits resulting from avoided testing through the use of read-across and in silico tools. Specifically, in the evaluation of these 261 noted substances, the use of 100,000-150,000 test animals and the expenditures of $50,000,000 to $70,000,000 (US) were avoided.

The use of non-animal alternatives in the safety evaluations of cosmetics ingredients by the Scientific Committee on Consumer Safety (SCCS).

In Europe, the safety evaluation of cosmetics is based on the safety evaluation of each individual ingredient. Article 3 of the Cosmetics Regulation specifies that a cosmetic product made available on the market is to be safe for human health when used normally or under reasonably foreseeable conditions. For substances that cause some concern with respect to human health (e.g., colourants, preservatives, UV-filters), safety is evaluated at the Commission level by a scientific committee, presently called the Scientific Committee on Consumer Safety (SCCS). According to the Cosmetics Regulations, in the EU, the marketing of cosmetics products and their ingredients that have been tested on animals for most of their human health effects, including acute toxicity, is prohibited. Nevertheless, any study dating from before this prohibition took effect is accepted for the safety assessment of cosmetics ingredients. The in vitro methods reported in the dossiers submitted to the SCCS are here evaluated from the published reports issued by the scientific committee of the Directorate General of Health and Consumers (DG SANCO); responsible for the safety of cosmetics ingredients. The number of studies submitted to the SCCS that do not involve animals is still low and in general the safety of cosmetics ingredients is based on in vivo studies performed before the prohibition.

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.

Exploring Alternatives for Marine Toxicity Testing: Initial Evaluation of Fish Embryo and Mysid Tests.

Current regulations require that toxicity assessments be performed using standardized toxicity testing methods, often using fish. Recent legislation in both the European Union and United States has mandated that toxicity testing alternatives implement the 3Rs of animal research (replacement, reduction, and refinement) whenever possible. There have been advances in the development of alternatives for freshwater assessments, but there is a lack of analogous developments for marine assessments. One potential alternative testing method is the fish embryo toxicity (FET) test, which uses fish embryos rather than older fish. In the present study, FET methods were applied to two marine model organisms, the sheepshead minnow and the inland silverside. Another potential alternative is the mysid shrimp survival and growth test, which uses an invertebrate model. The primary objective of the present study was to compare the sensitivity of these three potential alternative testing methods to two standardized fish-based tests using 3,4-dichloroaniline (DCA), a common reference toxicant. A secondary objective was to characterize the ontogeny of sheepshead minnows and inland silversides. This provided a temporal and visual guide that can be used to identify appropriately staged embryos for inclusion in FET tests and delineate key developmental events (e.g., somite development, eyespot formation, etc.). Comparison of the testing strategies for assessing DCA indicated that: (1) the standardized fish tests possessed comparable sensitivity to each other; (2) the mysid shrimp tests possessed comparable sensitivity to the standardized fish tests; (3) the sheepshead minnow and inland silverside FET tests were the least sensitive testing strategies employed; and (4) inclusion of sublethal endpoints (i.e., hatchability and pericardial edema) in the marine FETs increased their sensitivity. Environ Toxicol Chem 2024;43:1285-1299. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Development of a human liver microphysiological coculture system for higher throughput chemical safety assessment.

Chemicals in the systemic circulation can undergo hepatic xenobiotic metabolism, generate metabolites, and exhibit altered toxicity compared with their parent compounds. This article describes a 2-chamber liver-organ coculture model in a higher-throughput 96-well format for the determination of toxicity on target tissues in the presence of physiologically relevant human liver metabolism. This 2-chamber system is a hydrogel formed within each well consisting of a central well (target tissue) and an outer ring-shaped trough (human liver tissue). The target tissue chamber can be configured to accommodate a three-dimensional (3D) spheroid-shaped microtissue, or a 2-dimensional (2D) cell monolayer. Culture medium and compounds freely diffuse between the 2 chambers. Human-differentiated HepaRG liver cells are used to form the 3D human liver microtissues, which displayed robust protein expression of liver biomarkers (albumin, asialoglycoprotein receptor, Phase I cytochrome P450 [CYP3A4] enzyme, multidrug resistance-associated protein 2 transporter, and glycogen), and exhibited Phase I/II enzyme activities over the course of 17 days. Histological and ultrastructural analyses confirmed that the HepaRG microtissues presented a differentiated hepatocyte phenotype, including abundant mitochondria, endoplasmic reticulum, and bile canaliculi. Liver microtissue zonation characteristics could be easily modulated by maturation in different media supplements. Furthermore, our proof-of-concept study demonstrated the efficacy of this coculture model in evaluating testosterone-mediated androgen receptor responses in the presence of human liver metabolism. This liver-organ coculture system provides a practical, higher-throughput testing platform for metabolism-dependent bioactivity assessment of drugs/chemicals to better recapitulate the biological effects and potential toxicity of human exposures.

Characterization of RTgill-W1 cells epithelial development on transwell inserts: Evaluation of osmotic and toxic challenges.

RTgill-W1 cells cannot be directly exposed to freshwater (FW) or seawater (SW) due to osmotic stress. Adjustments of exposure solutions are needed, but these might reduce the bioavailability and toxicity of pollutants. To facilitate cell polarization and allow direct exposure of water samples, cells were cultured on transwell inserts. Monolayer formation was measured by trans-epithelial electrical resistance (TEER) and an apparent permeability (Papp) assay. At 14 days both TEER and Papp indicated the lowest permeability. Cell viability showed that cells can tolerate apical FW with complete medium (L-15/FBS) in the basolateral compartment but SW reduced cell viability. However, when reference toxicants, silver nitrate and sodium dodecyl benzene sulfonate, were added no toxicity was detected. Increased osmolality in the apical side and presence of proteins indicated diffusion from the basolateral to the apical side. Thus, reduced toxicity was likely caused by complexation with media salts and amino acids. A protein and amino acid free exposure medium (L-15/ex) was applied in the basolateral compartment. However, FW exposures with basolateral L-15/ex resulted in reduced cell viability. To reduce osmotic stress, mannitol was added to apical FW maintaining basolateral L-15/ex which improved cell viability and allowed detection of silver toxicity. Finally, RTgill-W1 cells did not show normal tight junction protein (ZO-1) immunocytochemical staining, which fits with the formation of a leaky epithelium. Overall, culturing of RTgill-W1 cells on transwell inserts allowed direct exposure to mannitol FW medium but showed a reduced sensitivity to toxicants. Thus, exposure on flat bottom wells is recommended for routine toxicity testing.

Beyond AOPs: A Mechanistic Evaluation of NAMs in DART Testing.

New Approach Methodologies (NAMs) promise to offer a unique opportunity to enable human-relevant safety decisions to be made without the need for animal testing in the context of exposure-driven Next Generation Risk Assessment (NGRA). Protecting human health against the potential effects a chemical may have on embryo-foetal development and/or aspects of reproductive biology using NGRA is particularly challenging. These are not single endpoint or health effects and risk assessments have traditionally relied on data from Developmental and Reproductive Toxicity (DART) tests in animals. There are numerous Adverse Outcome Pathways (AOPs) that can lead to DART, which means defining and developing strict testing strategies for every AOP, to predict apical outcomes, is neither a tenable goal nor a necessity to ensure NAM-based safety assessments are fit-for-purpose. Instead, a pragmatic approach is needed that uses the available knowledge and data to ensure NAM-based exposure-led safety assessments are sufficiently protective. To this end, the mechanistic and biological coverage of existing NAMs for DART were assessed and gaps to be addressed were identified, allowing the development of an approach that relies on generating data relevant to the overall mechanisms involved in human reproduction and embryo-foetal development. Using the knowledge of cellular processes and signalling pathways underlying the key stages in reproduction and development, we have developed a broad outline of endpoints informative of DART. When the existing NAMs were compared against this outline to determine whether they provide comprehensive coverage when integrated in a framework, we found them to generally cover the reproductive and developmental processes underlying the traditionally evaluated apical endpoint studies. The application of this safety assessment framework is illustrated using an exposure-led case study.

Understanding Ecotoxicological Responses of Fish Embryos and Gill Cells to Cationic Polymers.

Cationic polymers are considered by the scientific and regulatory communities as a group of greater interest amongst the polymers in commerce. As a category, relatively little hazard information is available in the public literature. Very few examples exist of published, high-quality polymer characterization and quantification of exposure. In the present study we describe a series of fish embryo toxicity (FET) and fish gill cytotoxicity assays used to establish a baseline understanding of several representative polyquaternium categories (PQ-6, PQ-10, PQ-16) in animal alternative models, accompanied by high-quality analytical characterization. Materials were chosen to encompass a range of molecular weights and charge densities to determine the influence of test material characteristics on toxicity. Both chorionated and dechorionated FET assays were generally similar to published acute fish toxicity data. Toxicity was correlated with cationic polymer charge density, and not with molecular weight, and was a combination of physical effects and likely toxicity at the site of action. Toxicity could be ameliorated by humic acid in a dose-dependent manner. Fish gill cytotoxicity results were orders of magnitude less sensitive than FET test responses. Environ Toxicol Chem 2022;41:2259-2272. © 2022 SETAC.