Non-animal Test Research Articles

Linking EDC-laden Food Consumption and Modern Lifestyle Habits with Preeclampsia: A Non-Animal Approach to Identifying Early Diagnostic Biomarkers Through Biochemical Alterations

Preeclampsia, a pregnancy complication characterized by new-onset hypertension with or without proteinuria and/or end-organ damage, may be influenced by exposure to endocrine-disrupting chemicals present in processed foods and associated with modern lifestyles. This study explores this potential link using a non-animal approach to identify early diagnostic biomarkers for preeclampsia. Seventy pregnant women aged 21-41 years participated, completing questionnaires assessing socio-demographic factors, Suboptimal Health Status Questionnaire scores for fatigue, digestive, cardiovascular, immune, and mental health issues, and exposure to endocrine-disrupting chemicals from processed food consumption and daily product use. Peripheral blood samples were analyzed for hormone profiles, complete blood count, and liver function tests. Statistical analysis revealed that mothers above 27 years old, with a Body Mass Index exceeding 32.25 Kg/m2, and a Mean Arterial Pressure of 108.4 ±1.1 mmHg exhibited a potential obesogenic effect on preeclampsia development. Socio-demographic factors like lower economic class, housewife status, primiparous pregnancy, non-graduate education, and rural residence were significantly associated. Analysis of biochemical parameters revealed that serum creatinine, blood urea, total protein, platelet count, blood urea nitrogen, bilirubin profile, LFT profile, and thyroid profile showed potential detrimental effects on kidney, liver, muscle, and thyroid function in preeclampsia patients. Notably, PC, serum urea, bilirubin, total protein, SGOT, ALP, and TSH levels were significantly associated with preeclampsia in individuals reporting higher exposure to EDCs. Minor biochemical alterations were also observed with dairy product consumption. SHS-25 analysis indicated a significant increase in fatigue, and digestive, cardiovascular, immune, and mental health-related issues in patients. Probably, biochemical alterations due to EDC exposure from processed foods and modern lifestyle habits contribute to organ dysfunction in preeclampsia. Identifying these potential biomarkers may pave the way for the development of non-invasive, early diagnostic tools for improved preeclampsia management. This research emphasizes the importance of non-animal testing methods for assessing EDC-related health risks in pregnancy and contributes to the advancement of early PE diagnosis strategies.

Chemicals regulation and non-animal methods: displacing the gold standard.

Regulating industrial chemicals in foodstuffs and consumer products is a major aspect of protecting populations against health risks. Non-animal testing methods are an essential part of the radical change to the framework for toxicity testing that is long overdue in global economies. This paper discusses reasons why the drive to reduce animal testing for chemical safety testing is so difficult to achieve, as perceived by those who are closely involved in chemicals regulations in different capacities. Progress is slow, despite the fact that the ethico-legal conditions for a move away from animal testing are largely in place, and despite scientific arguments for a radical change in the paradigm of toxicity testing, away from reliance on animal studies. I present empirical data drawn from two studies in a European Commission context promoting non-animal methods. The aim of the paper is modest. It is to foreground the voices of those who deal with the science and regulation of chemicals on a day-to-day basis, rather than to offer a theoretical framework for what I heard from them. I offer a synthesis of the main challenges faced by non-animal alternatives, as these are perceived by people in different stakeholder groups dealing with chemicals regulation. I show where there are pockets of agreement between different stakeholders, and where the main disagreements lie. In particular there is dispute and disagreement over what counts as validation of these alternative tests, and by implication of the traditional 'gold standard' of animal testing. Finally, I suggest that the shift to non-animal methods in chemicals regulation demonstrates the need for the concept of validation to be broadened from a purely techno-scientific definition, and be more explictly understood as a demand for trust and acceptance, with more attention given to the complex social, institutional and economic settings in which it operates.

Effects of novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) on function and homeostasis in human and rat pancreatic beta-cell lines

Despite the fact that environmental pollution has been implicated in the global rise of diabetes, the research on the impact of emerging pollutants such as novel flame retardants remains limited. In line with the shift towards the use of non-animal approaches in toxicological testing, this study aimed to investigate the effects of two novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) in rat (INS1E) and human (NES2Y) pancreatic beta-cell lines. One-week exposure to 1 μM and 10 μM TDCIPP and TPhP altered intracellular insulin and proinsulin levels, but not the levels of secreted insulin (despite the presence of a statistically insignificant trend). The exposures also altered the protein expression of several factors involved in beta-cell metabolic pathways and signaling, including ATP citrate lyase, isocitrate dehydrogenase 1, perilipins, glucose transporters, ER stress-related factors, and antioxidant enzymes. This study has brought new and valuable insights into the toxicity of TDCIPP and TPhP on beta-cell function and revealed alterations that might impact insulin secretion after more extended exposure. It also adds to the scarce studies using in vitro pancreatic beta-cells models in toxicological testing, thereby promoting the development of non-animal testing strategy for identifying pro-diabetic effects of chemical pollutants.

The dimer effect: A refinement approach towards skin sensitization assessment in-chemico using Amino acid Derivative Reactivity Assay.

Skin sensitization is a key endpoint for safety assessment, especially for cosmetics and personal care products. The adverse outcome pathway for skin sensitization and the chemical and biological events driving the induction of human skin sensitization are now well understood. Several non-animal test methods have been developed to predict sensitizer potential by measuring the impact of chemical sensitizers on these key events. In this work, we have focused on Key Event 1 (the molecular initiating step), which is based on formation of a covalent adduct between skin sensitizers and endogenous proteins and/or peptides in the skin. There exists three in-chemico assays approved by the Organization for Economic Co-operation and Development-(1) Direct Peptide Reactivity Assay (DPRA), (2) Amino Acid Derivative Reactivity Assay (ADRA), and (3) Kinetic Direct Peptide Reactivity Assay (kDPRA) to quantify peptide/amino acid derivative depletion after incubation with test chemicals. However, overestimated depletion of the cysteine-based peptide/amino acid derivatives is known in such assays because of the dimerization of the thiol group. In this present work, we report the synthesis and structural confirmation of the dimer of N-(2-[1-naphthyl]acetyl)-L-cysteine (NAC) from the ADRA assay to allow simultaneous determination of (a) peptide depletion by quantifying NAC monomer and (b) peptide dimerization by quantifying NAC dimer thereby eliminating the overestimation. We present a case study with three chemicals to demonstrate the importance of this approach. Thus, this simultaneous assay gives a more informed view of the peptide reactivity of chemicals to better identify skin sensitizers.

Development of a non-animal testing method using genetically engineered biosensors for assessing skin sensitizers

Development of a non-animal testing method using genetically engineered biosensors for assessing skin sensitizers

Feasibility of Using a Type I IFN-Based Non-Animal Approach to Predict Vaccine Efficacy and Safety Profiles.

Animal-based tests are used for the control of vaccine quality. However, because highly purified and safe vaccines are now available, alternative approaches that can replace or reduce animal use for the assessment of vaccine outcomes must be established. In vitro tests for vaccine quality control exist and have already been implemented. However, these tests are specifically designed for some next-generation vaccines, and this makes them not readily available for testing other vaccines. Therefore, universal non-animal tests are still needed. Specific signatures of the innate immune response could represent a promising approach to predict the outcome of vaccines by non-animal methods. Type I interferons (IFNs) have multiple immunomodulatory activities, which are exerted through effectors called interferon stimulated genes (ISGs), and are one of the most important immune signatures that might provide potential candidate molecular biomarkers for this purpose. This paper will mainly examine if this idea might be feasible by analyzing all relevant published studies that have provided type I IFN-related biomarkers for evaluating the safety and efficacy profiles of vaccines using an advanced transcriptomic approach as an alternative to the animal methods. Results revealed that such an approach could potentially provide biomarkers predictive of vaccine outcomes after addressing some limitations.

AOPWIKI-EXPLORER: An interactive graph-based query engine leveraging large language models

Adverse Outcome Pathways (AOPs) provide a basis for non-animal testing, by outlining the cascade of molecular and cellular events initiated upon stressor exposure, leading to adverse effects. In recent years, the scientific community has shown interest in developing AOPs through crowdsourcing, with the results archived in the AOP-Wiki: a centralized repository coordinated by the OECD, hosting nearly 512 AOPs (April, 2023). However, the AOP-Wiki platform currently lacks a versatile querying system, which hinders developers' exploration of the AOP network and impedes its practical use in risk assessment. This work proposes to unleash the full potential of the AOP-Wiki archive by adapting its data into a Labelled Property Graph (LPG) schema. Additionally, the tool offers a visual network query interface for both database-specific and natural language queries, facilitating the retrieval and analysis of graph data. The multi-query interface allows non-technical users to construct flexible queries, thereby enhancing the potential for AOP exploration. By reducing the time and technical requirements, the present query engine enhances the practical utilization of the valuable data within AOP-Wiki. To evaluate the platform, a case study is presented with three levels of use-case scenarios (simple, moderate, and complex queries). AOPWIKI-EXPLORER is freely available on GitHub (https://github.com/Crispae/AOPWiki_Explorer) for wider community reach and further enhancement.

Chemicals regulation and non-animal methods: displacing the gold standard

Regulating industrial chemicals in foodstuffs and consumer products is a major aspect of protecting populations against health risks. Non-animal testing methods are an essential part of the radical change to the framework for toxicity testing that is long overdue in global economies. This paper discusses reasons why the drive to reduce animal testing for chemical safety testing is so difficult to achieve, as perceived by those who are closely involved in chemicals regulations in different capacities. Progress is slow, despite the fact that the ethico-legal conditions for a move away from animal testing are largely in place, and despite scientific arguments for a radical change in the paradigm of toxicity testing, away from reliance on animal studies. I present empirical data drawn from two studies in a European Commission context promoting non-animal methods. The aim of the paper is modest. It is to foreground the voices of those who deal with the science and regulation of chemicals on a day-to-day basis, rather than to offer a theoretical framework for what I heard from them. I offer a synthesis of the main challenges faced by non-animal alternatives, as these are perceived by people in different stakeholder groups dealing with chemicals regulation. I show where there are pockets of agreement between different stakeholders, and where the main disagreements lie. In particular there is dispute and disagreement over what counts as validation of these alternative tests, and by implication of the traditional ‘gold standard’ of animal testing. Finally, I suggest that the shift to non-animal methods in chemicals regulation demonstrates the need for the concept of validation to be broadened from a purely techno-scientific definition, and be more explictly understood as a demand for trust and acceptance, with more attention given to the complex social, institutional and economic settings in which it operates.

Computational application of internationally harmonized defined approaches to skin sensitization: DASS App.

Chemically induced skin sensitization, or allergic contact dermatitis, is a common occupational and public health issue. Regulatory authorities require an assessment of potential to cause skin sensitization for many chemical products. Defined approaches for skin sensitization (DASS) identify potential chemical skin sensitizers by integrating data from multiple non-animal tests based on human cells, molecular targets, and computational model predictions using standardized data interpretation procedures. While several DASS are internationally accepted by regulatory agencies, the data interpretation procedures vary in logical complexity, and manual application can be time-consuming or prone to error. We developed the DASS App, an open-source web application, to facilitate user application of three regulatory testing strategies for skin sensitization assessment: the Two-out-of-Three (2o3), the Integrated Testing Strategy (ITS), and the Key Event 3/1 Sequential Testing Strategy (KE 3/1 STS) without the need for software downloads or computational expertise. The application supports upload and analysis of user-provided data, includes steps to identify inconsistencies and formatting issues, and provides predictions in a downloadable format. This open-access web-based implementation of internationally harmonized regulatory guidelines for an important public health endpoint is designed to support broad user uptake and consistent, reproducible application. The DASS App is freely accessible via https://ntp.niehs.nih.gov/go/952311 and all scripts are available on GitHub ( https://github.com/NIEHS/DASS ).

Rational arguments for regulatory acceptance of consistency testing: benefits of non-animal testing over in vivo release testing of vaccines

ABSTRACTIntroductionThere are rational arguments to replace existing in vivo potency and safety assays for batch release testing of vaccines with more advanced non-animal techniques to measure critical quality attributes. However, the introduction of in vitro alternatives to replace in vivo release assays of authorized vaccines is challenging.Areas coveredThis report describes the hurdles encountered in substituting in vivo assays and ways to overcome these and provides arguments why more advanced in vitro alternatives are superior, not only as a tool to monitor the quality of vaccines but also from a practical, economical, and ethical point of view. The rational arguments provided for regulatory acceptance can support a strategy to replace/substitute any in vivo batch release test if an appropriate non-animal testing strategy is available.Expert opinionFor several vaccines, in vivo release assays have been replaced leading to an optimized control strategy. For other vaccines, new assays are being developed that can expect to be introduced within 5–10 years. From a scientific, logistical, and animal welfare perspective, it would be beneficial to substitute all existing in vivo batch release assays for vaccines. Given the challenges related to development, validation, and acceptance of new methods, and considering the relatively low prices of some legacy vaccines, this cannot be done without government incentives and supportive regulatory authorities from all regions.

SciRAPnano: a pragmatic and harmonized approach for quality evaluation of in vitro toxicity data to support risk assessment of nanomaterials.

Large amounts of nanotoxicity data from alternative non-animal (in vitro) test methods have been generated, but there is a lack of harmonized quality evaluation approaches for these types of data. Tools for scientifically sound and structured evaluation of the reliability and relevance of in vitro toxicity data to effectively inform regulatory hazard assessment of nanomaterials (NMs), are needed. Here, we present the development of a pragmatic approach to facilitate such evaluation. The tool was developed based on the Science in Risk Assessment and Policy (SciRAP) tool currently applicable to quality evaluation of chemical toxicity studies. The approach taken to develop the tool, referred to as SciRAPnano, included refinement of the original SciRAP in vitro tool through implementation of identified NM-relevant criteria, and further refined based on a set of case studies involving evaluation of 11 studies investigating in vitro toxicity of nano-sized titanium dioxide. Parameters considered cover key physicochemical properties as well as assay-specific aspects that impact NM toxicity, including NM interference with test methods and NM transformation. The final SciRAPnano tool contains 38 criteria for reporting quality, 19 criteria for methodological quality, and 4 guidance items to evaluate relevance. The approach covers essential parameters for pragmatic and harmonized evaluation of NM in vitro toxicity studies and allows for structured use of in vitro data in regulatory hazard assessment of NMs, including transparency on data quality.

Effectively scaling assessment efforts in the evaluation of environmental risks of offshore produced water discharges using a tiered approach

For offshore oil and gas production facilities, overboard disposal of treated produced water (PW) remains a principal waste management option, and regulatory frameworks have been established to manage these discharges. Risk-based assessment (RBA) of PW, which aims to quantify potential impacts on the environment, has been adopted in some regulatory systems, but is also applied by oil and gas operators as part of their risk management systems. In 2020, the International Association of Oil and Gas Producers published a guidance document, including a four-tiered assessment framework aiming for a broader understanding and consistent application of PW RBA globally. The current study reviewed case studies and categorised them along the proposed assessment tiers. Regulatory developments are shown to be key drivers for the application of PW RBA globally, but required tools are often to resource intense for early risk screening. The proposed framework promotes the use of existing data, read-across techniques, and non-animal testing in two initial screening tiers, characterised by little to no sampling. In addition, it allows to refine higher tier assessments through the application of species sensitivity distributions and the inclusion of exposure time to avoid conservative assessment factors. This enables assessments to better approximate observations from field verification, included as a final tier to determine the level of discernible effects. The proposed tiered assessment approach can be considered a novel way to scale assessment efforts to the situation especially in cases where resources are limited, priorities for further investigations need to be set, rapid evaluation of hazard levels is required, sampling logistics and transport of large volumes is complicated and/or there is an opportunity for reduced animal testing. This makes early assessments more effective and ensures logical and efficient actions are triggered for understanding and managing environmental risks of produced water discharges.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of National Control Laboratories and National Regulatory Authorities

The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) is reviewing World Health Organization (WHO) manuals, guidelines and recommendations for vaccines and biotherapeutics to identify the extent to which animal-based testing methods are described. The aim is to recommend where updates to these documents can lead to an increased and more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction and Refinement of animal tests) in the quality control and batch release testing requirements for vaccines and biotherapeutics. Improved adoption of 3Rs principles and non-animal testing strategies will help to reduce the delays and costs associated with product release testing. Developing recommendations that are widely applicable by both the manufacturers and national regulatory authorities for vaccines and biological therapeutics globally requires a detailed understanding of how different organisations view the opportunities and barriers to better integration of the 3Rs. To facilitate this, we developed and distributed a survey aimed at individuals who work for national regulatory authorities (NRAs) and/or national control laboratories (NCLs). In this paper, we present the key findings from this survey and how these will help inform the recommendations for wider integration of 3Rs approaches by WHO in their guidance documents applicable to the quality control and batch release testing of vaccines and biotherapeutics.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

P16-15: Non-animal testing methods in the development of novel drug carriers targeting organs of enterohepatic circulation

P16-15: Non-animal testing methods in the development of novel drug carriers targeting organs of enterohepatic circulation

Advanced human iPSC-based preclinical model for Parkinson's disease with optogenetic alpha-synuclein aggregation.

Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models invitro and invivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.

Integrating Computational Data Science in University Curriculum for the New Generation of Scientists

Integration of computational data science (CDS) into the university curriculum offers several advantages for students, faculty and the institution. This article discusses the benefits to students of introducing CDS into the university curriculum with a focus on developing skills in cheminformatics, data analysis, structure–activity relationships, modelling and simulation. Moreover, CDS can enable students to engage with complex chemical and toxicological data in new and dynamic ways, helping them to develop a more nuanced understanding of the potential hazards and risks associated with different chemicals and substances. On the other hand, it can foster greater collaboration between students and faculty and with external partners in industry and government. This can lead to the development of more effective and efficient toxicological testing methods and tools to screen chemicals for potential hazards and aid the development of environmentally friendly chemicals. Overall, the integration of CDS into the university curriculum will help prepare the next generation of scientists giving them a competitive edge to make considerable contributions to green chemistry, designing safer chemicals and non-animal testing methods. It will enable them to tackle modern challenges facing society including identifying safer and more sustainable chemicals and predicting the health and environmental impacts of novel chemical substances.

Hi-MGT: A hybrid molecule graph transformer for toxicity identification

Conventional toxicity testing methods that rely on animal experimentation are resource-intensive, time-consuming, and ethically controversial. Therefore, the development of alternative non-animal testing approaches is crucial. This study proposes a novel hybrid graph transformer architecture, termed Hi-MGT, for the toxicity identification. An innovative aggregation strategy, referred to as GNN-GT combination, enables Hi-MGT to simultaneously and comprehensively aggregate local and global structural information of molecules, thus elucidating more informative toxicity information hidden in molecule graphs. The results show that the state-of-the-art model outperforms current baseline CML and DL models on a diverse range of toxicity endpoints and is even comparable to large-scale pretrained GNNs with geometry enhancement. Additionally, the impact of hyperparameters on model performance is investigated, and a systematic ablation study is conducted to demonstrate the effectiveness of the GNN-GT combination. Moreover, this study provides valuable insights into the learning process on molecules and proposes a novel similarity-based method for toxic site detection, which could potentially facilitate toxicity identification and analysis. Overall, the Hi-MGT model represents a significant advancement in the development of alternative non-animal testing approaches for toxicity identification, with promising implications for enhancing human safety in the use of chemical compounds.

New Approach Method for drug discovery and development aimed at regulatory acceptance

Recently, the importance of safety assessment based on mechanism of action for drug discovery has been emphasized, and international organizations are increasingly requesting safety assessment using non-animal test methods (Hereafter referred to as alternative method) that are not based on animal experiments using human-derived cells or tissues. However, it is clear that the variety of phenomena captured in animal studies cannot be covered by a stand-alone alternative method, as has been developed in the past, and there are some cases that are not intended to assess human toxicity based on comparison with the data of animal experiments. Expectations are therefore growing for the use of the New Approach Method (NAM) in drug discovery. In this article, we summarize the current status of alternative methods for reproductive toxicity testing and the regulatory acceptance of safety assessments by the Microphysiological system (MPS) as examples regarding NAM.

Big Question to Developing Solutions: A Decade of Progress in the Development of Aquatic New Approach Methodologies from 2012 to 2022.

In 2012, 20 key questions related to hazard and exposure assessment and environmental and health risks of pharmaceuticals and personal care products in the natural environment were identified. A decade later, this article examines the current level of knowledge around one of the lowest-ranking questions at that time, number 19: "Can nonanimal testing methods be developed that will provide equivalent or better hazard data compared with current in vivo methods?" The inclusion of alternative methods that replace, reduce, or refine animal testing within the regulatory context of risk and hazard assessment of chemicals generally faces many hurdles, although this varies both by organism (human-centric vs. other), sector, and geographical region or country. Focusing on the past 10 years, only works that might reasonably be considered to contribute to advancements in the field of aquatic environmental risk assessment are highlighted. Particular attention is paid to methods of contemporary interest and importance, representing progress in (1) the development of methods which provide equivalent or better data compared with current in vivo methods such as bioaccumulation, (2) weight of evidence, or (3) -omic-based applications. Evolution and convergence of these risk assessment areas offer the basis for fundamental frameshifts in how data are collated and used for the protection of taxa across the breadth of the aquatic environment. Looking to the future, we are at a tipping point, with a need for a global and inclusive approach to establish consensus. Bringing together these methods (both new and old) for regulatory assessment and decision-making will require a concerted effort and orchestration. Environ Toxicol Chem 2024;43:559-574. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.