The Epi2SensA method is a method similar to the validated EpiSensA assay for assessing the skin sensitization potential of chemicals. The Epi2SensA protocol includes adaptation (changes to exposure conditions and the controls) for using an alternative reconstructed human epidermis (RhE) model, the EpiDerm™ model. The interlaboratory validation study evaluated the reliability and predictive capacity of Epi2SensA according to OECD Performance Standards. Four laboratories (Mattek, Now Part of Sartorius, Eurofins Munich, Burleson Research Technologies, Inc., and Food and Drug Safety Center) conducted blinded testing of 20 coded reference substances representing various chemical categories and sensitization potencies. Statistical analysis using modified acceptance criteria (a 60% cell viability threshold) and a modified prediction model (requiring at least two positive gene markers) demonstrated substantially improved performance compared to the original EpiSensA criteria. The between-laboratory reproducibility (BLR) was 85%, the average within-laboratory reproducibility (WLR) was 83.3%, and the average predictivity parameters were 88.1% for sensitivity, 88.9% for specificity, and 88.3% for accuracy. Epi2SensA achieved performance metrics comparable to the validated reference method (EpiSensA), supporting regulatory acceptance of the Epi2SensA assay using the EpiDerm™ model (Mattek Corporation, Now Part of Sartorius, Ashland, MA, USA) as an alternative RhE source for OECD TG 442D skin sensitization testing.

The term "New Approach Methodologies" (NAMs) comprises a plethora of approaches with the potential for testing the safety, efficacy, and biological effects of chemicals, pharmaceuticals, and other substances without relying exclusively on traditional animal testing methodologies. While it is debateable whether "new" is the appropriate terminology - some NAMs have been around for several decades, while others have been developed recently - many scholars have observed that the effective uptake of NAMs in regulatory toxicology remains a challenge. The interdisciplinary CHANGE project applies a systems thinking approach to this challenge by modelling the behaviour of the regulatory toxicology system shaped by system-level factors and their interconnections. This article reports findings from two three-day workshops held in Oslo in summer 2024 (Workshop 1 "Explore") and 2025 (Workshop 2 "Reflect") with experienced practitioners from regulatory agencies, industry, civil society organisations and academia. Through structured collaborative activities, including small- and large-group discussions, modelling exercises and reflection activities, considerable qualitative data was gathered. Most sessions were recorded, transcribed, and analysed using a multi-step thematic analysis explicitly guided by systems thinking principles, enabling the identification of systemic structures and feedback mechanisms and their visualization in the form of a systems model. Insights from Workshop 1 "Explore" were revisited and stress-tested in Workshop 2 "Reflect", deepening the robustness of the resulting systems model. This systems model shows structural, economic, social, cultural and technical factors that are interlinked through reinforcing and balancing feedback loops that promote or inhibit the effective use of NAMs. The systems model shows potential leverage points for system change, for instance enhancing the stability and reliability of the supply chain or ensuring that actors in the regulatory toxicology system have both educational and institutional support to effectively change established practices.

In this paper, we present an operational conceptualisation of external validity for in vitro studies, to support the development of the forthcoming INVITES-EX tool. We define external validity as consisting of a concept of applicability and a concept of generalisability, and explain how it applies to in vitro comparative effect size studies. We argue that external validity assessment is relevant at two stages of evidence synthesis, during study screening and during evidence integration. Because evidence integration is complex and likely best supported by guidance, we conclude that INVITES-EX should be targeted at assessing the applicability of individual studies and used when screening studies for inclusion in a systematic review or chemical assessment.

Abstract Cytochrome P450 (CYP) enzymes play a key role in the metabolism of both xenobiotics and endogenous compounds, and the activity of some CYP isoforms are susceptible to induction and/or inhibition by certain chemicals. As CYP induction and inhibition can significantly alter the in vivo fate of xenobiotics i.e., levels of parent chemicals and/or metabolites, and thus toxicity, CYP induction/inhibition data is needed for regulatory chemical toxicity hazard assessment. Utilizing available human in vivo pharmaceutical data, a successful validation was previously conducted on the in vitro HepaRG™ CYP induction test method for measurement of induction of three key human CYP enzymes CYP1A1/1A2, 2B6 and 3A4. However, further validation data was required to demonstrate applicability of the test method to also accurately detect CYP induction mediated by industrial and pesticidal chemicals. Here we report on the supplementary validation of the HepaRG™ CYP enzyme induction test method carried out in two laboratories under the auspices of the EU Horizon2020-funded project “GOLIATH”, to expand the chemical applicability domain beyond pharmaceutical chemicals. Successful transfer was demonstrated and reproducibility assessed for the original 10 selected proficiency pharmaceuticals, plus three reference inducers together with six additional non-pharmaceutical ‘augmentation chemicals’. The method and chemical selection were found to be reliable and relevant for the routine assessment of human CYP induction. For the augmentation chemicals being proposed as additional proficiency chemicals, the test method achieved a reasonable but not optimum reproducibility. Recommendations are proposed to improve the test method’s specificity, reflecting the inherent uncertainty around borderline CYP inducing chemicals. Plain language summary Cytochrome P450 (CYP) enzymes help break down drugs and other chemicals in the body. Their activity can be increased (induced) or decreased (inhibited), which can change how toxic a chemical is and when it is excreted. Because of this, CYP data is important for chemical safety assessments. A laboratory-based method using HepaRG cells was previously validated to measure induction of key CYP enzymes (CYP1A1/1A2, CYP2B6 and CYP3A4) using pharmaceutical chemicals. This study aimed to show that it also works well for industrial and pesticidal chemicals. In the EU funded GOLIATH project, two laboratories tested 10 pharmaceutical and 6 non-pharmaceutical chemicals. The method showed good reliability overall and strong reproducibility for pharmaceuticals. For non-pharmaceutical chemicals, results were acceptable but less consistent. The study concludes that the method is useful for routine testing, but improvements are needed to increase accuracy and better handle chemicals that show weak or borderline CYP induction effects.

This paper examines why robust causality assessment is central to toxicology yet increasingly difficult in networked biological systems, including Adverse Outcome Pathway (AOP) networks and multi-omics datasets. Classical causal frameworks (e.g., Koch/Dale reasoning and Bradford Hill-type considerations) remain useful for scoping, but they offer limited operational guidance for multifactorial, nonlinear, and feedback-dominated mechanisms. We therefore synthesize a modern toolbox for causality analysis in toxicology spanning directed acyclic graphs for explicit causal assumptions, quantitative and probabilistic approaches for integrating evidence across key events, and network/dynamical methods that help identify influential hubs and points of vulnerability in AOP networks. Building on evidence-based toxicology, we propose an operational workflow that links (i) structured scoping and model specification, (ii) protocolized evidence retrieval across in vivo, in vitro, in silico and human data streams, (iii) risk-of-bias appraisal and quantitative synthesis of effect sizes and dose–response, (iv) mechanistic integration and targeted perturbation assays, and (v) translation via dual-strand certainty rating (mechanistic vs difference-making evidence) and Evidence-to-Decision tables. We discuss how explainable AI can support scalable integration and transparency. A worked developmental neurotoxicity example illustrates how this pipeline can support regulatory recommendations while explicitly documenting uncertainty.

The transition from traditional animal-based approaches and assessments to New Approach Methodologies (NAMs) marks a scientific revolution in regulatory toxicology, with the potential of enhancing human and environmental protection. However, implementing the effective use of NAMs in regulatory toxicology has proven to be challenging, and so far, efforts to facilitate this change frequently focus on singular technical, psychological or economic inhibitors. This article takes a system-thinking approach to these challenges, a holistic framework for describing interactive relationships between the components of a system of interest. In this case, the regulatory toxicology system. We do so by analysing and interpreting a very large qualitative data set of experts' observations, collected in a 3-day interactive workshop and three follow-up online workshops with a heterogeneous sample of experts representing major actors from the global regulatory toxicology system. We identified leverage points (where a small change within a system can have a disproportionately large effect) in the six core aspects-infrastructure, processes, culture, technology, goals, and actors-in the regulatory toxicology system to facilitate the effective use of NAMs. Identified systematic leverage points include the need for a functioning incentive structure for effectively discovering, developing, validating and using NAMs within academia, regulation, and industry; and measures that prevent or mitigate unwanted effects of using NAMs that acknowledge clashes between scientific, regulatory, political and social processes. The results serve as a basis for follow-up activities that reflect on the actual effectiveness of these levers and that develop measures for the regulatory toxicology system.

Epidemiological studies have raised concerns about health risks from occupational exposure to inhalation anesthetics. This study aimed to systematically search, select and appraise the evidence from animal studies to derive occupational exposure levels (OELs) for isoflurane and sevoflurane. Twenty-four studies for isoflurane and 7 studies for sevoflurane were included, which reported on neurological and fertility outcomes. When ranking adverse outcomes by derived benchmark dose lower bounds (BDMLs), male fertility was considered as the most critical effect for both substances. Assessment factors cover uncertainties regarding interspecies and intraspecies differences. Using the BMDL as point of departure we derived 8-h time-weighted average (TWA) OELs of 0.9 mg/m3 (0.12 ppm) for isoflurane and of 0.8 mg/m3 (0.09 ppm) for sevoflurane. For peak exposures, 15-min TWA OELs of 30 mg/m3 (3.9 ppm) and 25 mg/m3 (3.0 ppm) were derived, respectively. These levels are expected to protect workers from health effects caused by these anesthetic gases.

The glucocorticoid receptor (GR) belongs to the family of steroid receptors (SRs). These receptors regulate a vast selection of cell-, tissue-, and organism biology, and are also targets of endocrine disrupting chemicals warranting design and validation of in vitro assays. Here we report a "blinded" ring trial of an in vitro cell-based GR transactivation assay with four involved laboratories. The laboratories set up the assay and tested 34 selected chemicals with remarkably good concordance. There was agreement between all laboratories for the classification of activity in 97 % of the cases, and three or more laboratories were always in agreement. The within laboratory concordance was very high (99.6 %) with only one of all 272 triplicates deviating. The assay was, thus, deemed easily transferable and reproducible within and between laboratories, since they would arrive at the same qualitative results. Furthermore, for the chemicals with solid data regarding GR activation or inhibition, the laboratories arrived at the expected conclusion in all cases. Overall, the transfer and validation were successful, and the method is under evaluation to become an OECD test guideline. The method is expected to become valuable in tiered approaches for assessing chemicals or environmental samples together with other similar methods.

Background Systematic reviews (SR), an evidence-synthesis tool, have been increasingly used in toxicology, for regulatory decision-making and allocation of research funding resources. A critical domain of methodological quality is publicly available in the SR protocol that describes the review methods prior to the review. Earlier work showed that only a small number of published SRs in environmental health had identifiable protocols, but did not evaluate their completeness of reporting. Thus, there is a critical need to assess the number of protocols published in peer-reviewed literature related to environmental/occupational toxicology and how much those SR protocols adhere to current reporting standards such as PRISMA-P.Objective The overarching objective of this review is to assess the reporting quality of SR protocols in environmental and occupational toxicology, published in peer-reviewed journals.Methods and analysis This protocol has been reported following PRISMA-P and PRISMA-S checklist and study selection, data extraction, and reporting quality assessment were piloted by independent reviewers. Four electronic databases (PubMed, Scopus, Web of Science, EMBASE) were selected and will be searched for SR protocols using pre-defined strings. Both title/abstract and full-text screening will follow detailed eligibility criteria based on the population concept context (PCC) framework. We will include peer-reviewed, self-identified SR protocols that aim to assess the adverse effects of environmental and occupational exposures. We will perform data extraction using the form that includes general bibliographic information, exposure, adverse effect information, evidence streams, and guidelines/checklists used in protocol reporting or development. A reporting assessment form consisting of 15 PRISMA-P-based items, of which eleven with identical binary responses (reported vs not reported) address critical elements of SR protocol. Assessments of the eleven quality items will provide data for the analysis of the reporting quality of SR protocols. We employ reporting quality as it provides a practical and suitable approach to evaluating the inclusion of essential SR elements, under the assumption that these elements would be reported if they had been appropriately planned rather than overlooked. The screening and data extraction will be conducted by two independent reviewers. Disagreements will be discussed if needed with the support of a third reviewer. The data analysis will describe the total number of SR protocols and trends over time, and absolute frequencies and/or proportion of bibliographic information items. It will summarize the reporting quality of all included protocols by a histogram and rank the quality criteria by their level of adherence across the included protocols.

Metabolism disrupting chemicals (MDCs) are a class of endocrine disrupting substances that promote metabolic changes leading to metabolic disorders in humans. Central to assessing their adverse effects is the need to better understand their modes of action (MoA). Cytochrome P450 (CYP) enzymes play a major role in xenobiotic metabolism, but also catalyse many endogenous metabolic reactions. Therefore, modulation of CYP functionality may impact homeostasis, contributing to adverse outcomes. At the functional level, alteration of the activity of human CYPs by MDCs largely remains unexplored. In this study we investigated the capability of six candidate MDCs, bisphenol A (BPA), perfluorooctanoic acid (PFOA), tributyltin (TBT), dichlorodiphenyldichloroethylene (p,p'-DDE), triclosan (TCS) and triphenylphosphate (TPP) to induce CYP1A2, CYP2B6 and CYP3A4 activities in the human hepatic HepaRG cell line. The CYP induction test method previously validated for pharmaceuticals was optimised and selected MDCs were tested in the context of the European Horizon 2020 GOLIATH project. Induction was revealed using a cocktail of CYP-selective probe substrates, followed by probe metabolite quantification by mass spectrometry. All MDCs except TCS induced CYP activities. PFOA, TBT, p,p'-DDE and TPP induced CYP1A2, TPP being the most potent inducer. BPA, PFOA, TBT and TPP induced CYP2B6, PFOA being the most potent inducer. BPA, PFOA, TBT, p,p'-DDE and TPP all induced CYP3A4, p,p'-DDE and BPA being the most potent inducers. These results highlight the capability of candidate MDCs to induce key CYP activities in a human relevant hepatic model, paving the way for a better understanding of MDCs mechanisms of action.

Organising workshops that successfully address complex research problems is a challenge, especially when the research involves interest-holders with diverse roles and expertise and potentially conflicting values and viewpoints. In this article, we describe and reflect on the approach we took to organising the CHANGE workshop, held in Oslo in June 2024. CHANGE is a complex 3-year project involving the collecting, analysing, and developing cross-sector consensus on a challenging topic. The approaches on which we reflect include fundamental aspects of interest-holder engagement, workshop design, methodological approach, and inclusive participation. Based on our reflections, we present a series of recommendations for consideration by anyone in the general research community using workshops as part of a research process.

Background Systematic reviews and other types of evidence syntheses use rigorous methods to identify and synthesise all of the relevant evidence to answer a question. A robust search strategy is crucial to conducting a high quality evidence synthesis, as an inadequate search may miss relevant evidence and produce biased findings. Methodological recommendations endorse searching for ‘grey literature’, i.e., reports published outside of traditional commercial publishing. However, there is currently a lack of clarity about what is considered to be the relevant types of grey literature and how to deal with it.Objectives This survey will aim to answer three questions: (1) How do those conducting evidence syntheses on environmental health and toxicology (EHT) topics understand ‘grey literature’; (2) Where do they search for grey literature?; (3) How do they deal with included grey literature evidence?Methods Participants will be individuals who conduct systematic reviews and evidence syntheses in EHT, without restrictions on: previous experience in conducting evidence synthesis, age, gender or geographic location. Survey will be disseminated via relevant professional organisations, systematic review and evidence synthesis organisations, and social media. It will consist of a mix of demographic and content questions. Descriptive statistics will be reported as frequencies and percentages; free-text responses will be analysed thematically.Ethics The survey was exempted from a requirement to undergo ethics review. The first page of the survey will provide the respondents with information about the project and ethics approval; respondents will provide consent by clicking on a button to advance to the survey.

Context In vitro toxicity studies are increasingly being included as evidence in systematic reviews and chemical risk assessments. INVITES-IN, a tool for assessing the internal validity of in vitro studies, is under development in a process consisting of four consecutive studies. Study One in the creation of INVITES-IN was the development of an “item bank” database of 405 concepts (“items”) of potential relevance for assessing the internal validity of in vitro toxicity studies. The items were gathered from both focus group discussions and a purposive literature sample. In this paper we present the second study in the creation of INVITES-IN, i.e. the methods and results for identifying items for consideration when assessing the potential for bias in an in vitro study.Method A two-round digital Delphi survey, followed by online Delphi panel discussions guided by a moderator, was performed. The Delphi participants were experienced with both in vitro models and systematic review methods.Results Fifteen experts completed both Delphi rounds, and thirteen participated in a guided Delphi panel discussion. Of the 405 items in the bank, the experts agreed that 372 should be considered when assessing the potential for bias in an in vitro study. Items gathered from both the literature sample and the focus group discussions (Study One) were considered to be important for the assessment of the potential for bias in an in vitro study; 83–100% of the items collected from the literature sample were identified to be important and 91% (127) of the new items discovered in the focus group discussions of Study One were identified to be important.Discussion The 372 retained items will be interpreted into a manageable set of study appraisal criteria and a supporting guidance that will constitute the INVITES-IN study appraisal tool. In terms of lessons for tool development, the high retention of items included in tools designed for assessment of human and animal studies to in vitro studies suggests that many validity concepts are generally applicable across multiple study designs. Therefore, tool development processes should benefit from drawing on assessment tools outside the immediate domain of interest. Tool development would also likely benefit from supplementing literature reviews with focus group discussions, as our results demonstrate that the use of focus group discussions with domain experts was a pragmatic and valuable approach to increasing coverage of items in a tool development process.Conclusion In conclusion, this study demonstrates the value of using rigorous methods to ensure a comprehensive dataset as the starting point for creation of an assessment tool, though the direct application of Delphi methods to item banks may be an unnecessary step in tool development.

Background While some per- and polyfluoroalkyl substances (PFAS) are immunosuppressants, whether they have an adverse effect on infectious disease morbidity is unclear. We conducted a systematic review and meta-analysis of epidemiologic data on the association between an incremental increase in serum concentration of any of 12 PFAS and the risk or rate of infectious disease (ID). Methods From 25 reports representing 18 unique study populations, we conducted meta-analyses stratified on exposure type (log-transformed or absolute scale) and outcome type (risk or rate). To synthesize data that could not be combined with meta-analysis due to different exposure or outcome types, we additionally conducted vote counting and calculated combined p-values. Results A small positive association between PFAS exposure and ID risk or rate was more frequently reported than not, though in the synthesized data statistical significance was present only in a few instances. The meta-analyses and combined p-value analyses had many similar findings. In the combined p-value analyses, statistically significant positive associations were noted between Perfluorononanoic acid and lower respiratory tract infection (LRTI) event rates, Perfluorooctanesulfonamide and LRTI event rates and LRTI risk and rates combined, Perfluorooctanoic acid and Perfluorodecanoic acid with all ID risk and rates combined, and Perfluoroundecanoic acid with all ID risk. Conclusion We identified moderate evidence of positive associations that were of variable size but usually small; the certainty of evidence was, however, generally low or very low and diminished by the possible influences of multiple testing and covariance among results not accounted for in the analyses. Prospero registration CRD42024551990.

Risk of bias is a critical factor influencing the reliability and validity of toxicological studies, impacting evidence synthesis and decision-making in regulatory and public health contexts. The traditional approaches for assessing risk of bias are often subjective and time-consuming. Recent advancements in artificial intelligence (AI) offer promising solutions for automating and enhancing bias detection and evaluation. This article reviews key types of biases-such as selection, performance, detection, attrition, and reporting biases-in in vivo, in vitro, and in silico studies. It further discusses specialized tools, including the SYRCLE and OHAT frameworks, designed to address such biases. The integration of AI-based tools into risk of bias assessments can significantly improve the efficiency, consistency, and accuracy of evaluations. However, AI models are themselves susceptible to algorithmic and data biases, necessitating robust validation and transparency in their development. The article highlights the need for standardized, AI-enabled risk of bias assessment methodologies, training, and policy implementation to mitigate biases in AI-driven analyses. The strategies for leveraging AI to screen studies, detect anomalies, and support systematic reviews are explored. By adopting these advanced methodologies, toxicologists and regulators can enhance the quality and reliability of toxicological evidence, promoting evidence-based practices and ensuring more informed decision-making. The way forward includes fostering interdisciplinary collaboration, developing bias-resilient AI models, and creating a research culture that actively addresses bias through transparent and rigorous practices.

Context Study reliability impacts toxicology assessments and the confidence associated with them. Klimisch et al. (1997) first formalised reliability evaluation by standardising study reliability. Then, in 2009 ToxRTool was developed to improve the transparency and objectivity of Klimisch et al.’s (1997) reliability assessment under the EU chemical regulation REACH. Objective Given the observed trend showing higher ToxRTool use since 2020, we intend to further explore how ToxRTool is used and in what context(s). This protocol outlines our plan to to discuss current usage patterns by summarising ToxRTool applications in peer-reviewed scientific literature.Planned methods The JARS-Qual (Levitt et al. 2018) checklist guides this protocol. Since this project assesses toxicological studies, we use study characteristics of interest (e.g., data type, exposure, field of study) and bibliographic information. The search and confirmation of a picklist, code book, and categorization groups has been completed. The search occurred in November 2023 where five databases (PubMed, Embase, Scopus, Web of Science, and Google Scholar) were assessed without any language restrictions for publications from 2009 to 2024. After duplication removal, a total of 530 publications were identified and uploaded to Covidence (2025). Of the 530 publications, a randomly selected sample set were used to confirm the eligibility criteria, extraction steps, and decision-making process. The remaining publications have not been assessed nor has any data been extracted.Discussion ToxRTool helps reviewers evaluate study reliability, so an understanding of how ToxRTool is used or has been modified can inform toxicologists and evidence-based practitioners about current usage patterns. The results could also clarify any incorrect interpretations of ToxRTool use and illustrate useful applications of ToxRTool.

Identification of Endocrine-Disrupting Chemicals (EDCs) in a regulatory context requires a high level of evidence. However, lines of evidence (e.g. human, in vivo, in vitro or in silico) are heterogeneous and incomplete for quantifying evidence of the adverse effects and mechanisms involved. To date, for the regulatory appraisal of metabolism-disrupting chemicals (MDCs), no harmonised guidance to assess the weight of evidence has been developed at the EU or international level. To explore how to develop this, we applied a formal Expert Knowledge Elicitation (EKE) approach within the European GOLIATH project. EKE captures expert judgment in a quantitative manner and provides an estimate of uncertainty of the final opinion. As a proof of principle, we selected one suspected MDC -triphenyl phosphate (TPP) - based on its related adverse endpoints (obesity/adipogenicity) relevant to metabolic disruption and a putative Molecular Initiating Event (MIE): activation of peroxisome proliferator activated receptor gamma (PPARγ). We conducted a systematic literature review and assessed the quality of the lines of evidence with two independent groups of experts within GOLIATH, with the objective of categorising the metabolic disruption properties of TPP, by applying an EKE approach. Having followed the entire process separately, both groups arrived at the same conclusion, designating TPP as a “suspected MDC” with an overall quantitative agreement exceeding 85%, indicating robust reproducibility. The EKE method provides to be an important way to bring together scientists with diverse expertise and is recommended for future work in this area.

The focus on implementation of systematic review (SR) principles in chemical risk assessments (CRAs) is growing as it has the potential to advance the rigour and transparency of the CRAs. However, the SR and CRA communities use their own specific terminologies. Understanding the meaning of core SR and CRA terms and where they overlap is critical for application of SR methods and principles in CRAs. Moreover, it will increase the possibility for cross-sectorial collaboration, avoid misunderstandings, and improve communication among risk assessors, researchers, and policy makers. We present a process for the cross-mapping of core CRA terms and core SR terms. Core terms for study appraisal, evidence synthesis and integration used in the SR and CRA communities will be included. The outcome will be an overview of how core SR terms map onto core CRA terms and vice versa, and a description of the relationship and conceptual overlap between the terms. The cross-mapping is divided in three phases, where in the first phase the core SR and CRA terms will be identified. In the second phase, existing SR and CRA definitions will be mapped. In the third phase, descriptions of the relationship and conceptual overlap between the terms will be derived. The third phase will include weekly one-hour online meetings for SR and CRA experts.