In vitro toxicology assays are widely used in the context of toxicology to study in vivo outcomes and to improve mechanistic understanding of toxic responses. For this purpose, a better characterization of actual exposure and the fate of chemicals within the cellular test system is required. The present study aimed to develop a novel model, INSIGHT (In Silico Guide for Harmonized in vitro Testing), that integrates physiological and physicochemical parameters to better describe chemical fate in vitro and to guide assay design. The newly developed model, integrating the dynamic features of the Virtual Cell Based Assay model with the partitioning framework of the Virtual In Vitro Distribution model, was calibrated using both a large literature dataset and original experimental data, comprising a total of 42 chemicals and 7 commonly used cell lines: HepaRG, HepG2, 3T3 Balb/c, PC12, MCF-7, RTgill-W1, and HEK293. These cell lines were selected for their diverse tissue and species origins, metabolic capacities, and potential for functional transport mechanisms, known to influence chemical kinetics. The INSIGHT model demonstrated flexibility and robustness across a range of cell lines when the parameters driving their metabolic activity or functional transport were informed. The present study underscores the pivotal function of logPow determination and the necessity for accurate calibration of partition coefficients, permeability, and metabolic processes to account for variability across cell lines and tested chemicals. This approach supports and facilitates enhanced experimental design and advancing quantitative in vitro-to-in vivo extrapolation (qIVIVE) in toxicology and strengthens next generation risk assessment (NGRA) workflows. Plain language summaryIn vitro cell-based tests are increasingly being used as an alternative to traditional animal experiments for evaluating chemical safety. However, the actual exposure of cells to tested chemicals during these experiments is often uncertain and difficult to determine, which can limit the interpretation of results. In the present study, we introduce INSIGHT, a physiologically based model describing how chemicals are distributed, transported into cells, and metabolized over time in in vitro systems. By integrating physicochemical properties with the biological characteristics of different cell types, INSIGHT improves understanding of cellular exposure and supports better experimental design. The model was evaluated using data from multiple chemicals and commonly used cell lines, demonstrating consistent performance across diverse experimental conditions. INSIGHT contributes to new approach methodologies by improving the assessment of relevant exposure and supporting the replacement, reduction and refinement (3Rs) of animal testing in chemical safety assessments.

In vitro toxicology assays are widely used in the context of toxicology to study in vivo outcomes and to improve mechanistic understanding of toxic responses. For this purpose, a better characterization of actual exposure and the fate of chemicals within the cellular test system is required. The present study aimed to develop a novel model, INSIGHT (In Silico Guide for Harmonized in vitro Testing), that integrates physiological and physicochemical parameters to better describe chemical fate in vitro and to guide assay design. The newly developed model, integrating the dynamic features of the Virtual Cell Based Assay model with the partitioning framework of the Virtual In Vitro Distribution model, was calibrated using both a large literature dataset and original experimental data, comprising a total of 42 chemicals and 7 commonly used cell lines: HepaRG, HepG2, 3T3 Balb/c, PC12, MCF-7, RTgill-W1, and HEK293. These cell lines were selected for their diverse tissue and species origins, metabolic capacities, and potential for functional transport mechanisms, known to influence chemical kinetics. The INSIGHT model demonstrated flexibility and robustness across a range of cell lines when the parameters driving their metabolic activity or functional transport were informed. The present study underscores the pivotal function of logPow determination and the necessity for accurate calibration of partition coefficients, permeability, and metabolic processes to account for variability across cell lines and tested chemicals. This approach supports and facilitates enhanced experimental design and advancing quantitative in vitro-to-in vivo extrapolation (qIVIVE) in toxicology and strengthens next generation risk assessment (NGRA) workflows. Plain language summaryIn vitro cell-based tests are increasingly being used as an alternative to traditional animal experiments for evaluating chemical safety. However, the actual exposure of cells to tested chemicals during these experiments is often uncertain and difficult to determine, which can limit the interpretation of results. In the present study, we introduce INSIGHT, a physiologically based model describing how chemicals are distributed, transported into cells, and metabolized over time in in vitro systems. By integrating physicochemical properties with the biological characteristics of different cell types, INSIGHT improves understanding of cellular exposure and supports better experimental design. The model was evaluated using data from multiple chemicals and commonly used cell lines, demonstrating consistent performance across diverse experimental conditions. INSIGHT contributes to new approach methodologies by improving the assessment of relevant exposure and supporting the replacement, reduction and refinement (3Rs) of animal testing in chemical safety assessments.

Recognizing the significant impact that NAMs can have in biomedical research and drug development, many key funding bodies in India have also taken concrete steps to propel such technologies.In 2019, the Indian Council of Medical Research (ICMR) developed a white paper on alternatives to animal use (Swaminathan et al., 2025).Very recently, two key research groups in India working on such non-animal human-relevant technologies have been recognized as ICMR -Collaborating Centres of Excellence 6 .Biotechnology Industrial Research Assistance Council (BIRAC), Department of Biotechnology (DBT) released a call for proposals for developing in vitro and in vivo preclinical models for drug discovery in 2023 7 .In this workshop it was discussed how we can fast-track these efforts via dedicated investments and strategic partnerships to develop precise, affordable, and indigenous human-relevant technologies.The Biomedical Research for the 21 st Century (BioMed21) Collaboration Workshop was jointly organized by Humane World for Animals India and BIRAC, DBT, Government of India (GoI), along with the Division of Strategic Alliances, Office of Principal Scientific Advisor (PSA), GoI as the outreach partner to bring together national and international stakeholders from academia, industry, and key funding bodies. Workshop inaugurationAlokparna Sengupta (Managing Director, Humane World for Animals India) welcomed the participants and stated how the organization is promoting a shift towards advanced human biologybased technologies in preclinical safety and biomedical research via a combination of science, policy, education, and training.The workshop was inaugurated by Dr Jitendra Kumar (Managing Director, BIRAC, DBT), who emphasized the relevance of the workshop and recognized the area of human-based technologies as one of the emerging areas in biomedical research and drug development.He commented that the low success rate of the current drug development pipeline called for better model systems, but more efforts towards awareness are needed.1 https://cdsco.gov.in/opencms/resources/UploadCDSCOWeb/2022/new_DC_rules/NDCTR_G.S.R.%20175(E)%20dt_09.03.2023_Use%20of%20induced%20Pluripo-tent%20Stem%20Cells%20additional%20testing%20methods.pdf 2 https://www.fda.gov/news-events/press-announcements/fda-announces-plan-phase-out-animal-testing-requirement-monoclonal-antibodies-and-other-drugs 3 www.nih.gov/news-events

Advanced molecular technologies allow for the generation of reproducible, sequence-defined recombinant antibodies.As in all areas of science, time and resource constraints can slow a widescale transition; however, the rapid expansion of recombinant antibodies in commercial catalogs makes major progress achievable today.Here, practical opportunities to increase the use of sequenced antibodies are highlighted, aligning with a call from the scientific community for more reproducibility and less reliance on animals in research (Ayoubi et al., 2023;Bradbury and Plckthun, 2015).A review of CiteAb's compilation of the top 100 cited antibodies 1 , published in October 2025, reveals that numerous antibodies listed could be replaced with existing catalog recombinant options, as detailed in Table 1.Ten of the top 25 antibodies in CiteAb's 2024 list are polyclonal secondary antibodies.The composition of a polyclonal antibody varies depending on the individual immune response of the animal used for production, and this inherent variability has long led scientists to call for their replacement with more reproducible antibodies.Manufacturer catalogs offer a growing number of sequence-defined alternatives that are either entirely animal-free or, while using animals during development, eliminate animal use during production.These include individual monoclonal antibodies that can replace polyclonal antibodies, as well as multiclonal cocktails composed of defined monoclonals.Further, two of the top 25 antibodies are polyclonal primary antibodies, both of which have catalog recombinant options, as shown in Table 1.Monoclonal antibodies that have recombinant replacements are also frequently cited.Four of the top 25 antibodies in CiteAb's list are monoclonal antibodies produced via hybridomas, all of which have existing catalog recombinant options listed in Table 1, and five of the top 100 are monoclonals produced via the ascites method.This method inflicts such considerable suffering that scientific committees in Europe and the U.S. have discouraged its use for animal welfare reasons (NRC, 1999; EC, 2020) in addition to issues with reproducibility.More than 20 years ago, the European Commission's EU Reference Laboratory for alternatives to animal testing's (EURL ECVAM's) Scientific Advisory Committee concluded that alternative procedures were equal to or better than the ascites method.In 2020, the organization reaffirmed that the method should no longer be acceptable for use under any circumstances 2 .Sequence-defined alternatives for each of these five ascites antibodies, which do not involve the ongoing use of animals, are listed within Table 1.For consumers who have contracts with or have preferred antibody vendors, there are an increasing number of companies offering recombinant antibodies for the same targets as their traditional counterparts.As an example presented within Table 1, Abcam has numerous animal-production-free recombinant antibodies that can be used in place of their polyclonal primary antibodies.Such options effectively remove a potential logistical barrier and streamline the transition.The growing availability of recombinant antibodies allows scientists to select reagents that, with proper characterization, can enhance research quality while advancing animal welfare.The information presented here serves as a guide for scientists to identify sequence-defined antibodies ready for immediate use.Beyond these examples, myriad additional catalog recombinant antibodies are readily accessible through commercial catalogs and in the Recombinant Antibodies & Mimetics Database 3 -offering scientists a practical opportunity to incorporate more reliable reagents in their research today.

Reproducibility of cell culture experiments between laboratories needs to be improved by ensuring more complete reporting of methodology in scientific papers. The minimum reporting standards sug-gested here cover various cell culture methods including monolayers, stem cells, organoids, and microphysiological systems (MPS). The standards build on existing guidance like Good Cell Culture Practice (GCCP 2.0) and OECD test reporting guidelines on how to quality-assure in vitro work, focusing on transparency and completeness of reporting. Key elements to be reported include full details of cell source and identity, cell quality control and characterization, materials and reagents used, culture conditions and protocols, experimental design, data analysis, data availability, and legal and ethical aspects. For complex models, additional details need to be provided such as cell ratios, microenvironment conditions, functional characterization, etc. The guidance for Good In Vitro Reporting Standards (GIVReSt) is part of a broader initiative of evidence-based toxicology encompassing the improvement of the quality of in vitro studies for safety assessments and regulatory decisions. In summary, GIVReSt addresses incomplete reporting as a major factor affecting repro-ducibility of cell culture experiments by providing clear standards around transparency and rigor in reporting. The integration of agentic artificial intelligence (AI) is envisioned to streamline compliance checking, providing real-time feedback and accelerating scientific discovery by making high-quality evidence more accessible. This should lead to more reliable cell culture research overall.

Gut microbiota play a central role in human health, notably through the production of metabo­lites, including short-chain fatty acids, secondary bile acids, vitamins or neurotransmitters. Beyond contributing to gut health, these microbial metabolites significantly impact multiple organ systems by activating key signaling pathways along the gut-organ axes, including the gut-liver, gut-brain, and gut-bone axes. Chemicals ingested through food such as food additives, extensively used to enhance the texture, preservation and appearance of foods, may interact with our gut microbiota, altering metabolite production, and this can have consequences for our health. However, gut microbial metabolism is currently overlooked in toxicology. While efforts are underway to develop standardized human-based new approach methodologies to assess compound-microbiome inter­actions, anchoring those assays within the adverse outcome pathway (AOP) framework would offer a structured way to connect changes in gut microbial metabolism to adverse health outcomes. Using human-based models enhances the relevance of the results while supporting the reduction of animal-based testing in toxicology research.

Prediction of hepatotoxicity in humans remains an unresolved challenge. Recently, an in vitro/in silico method was established to predict blood concentrations of test compounds with an increased risk of causing human hepatotoxicity. In the present study, we addressed the question whether gene expression data can improve the quality of hepatotoxicity prediction compared to cytotoxicity analysis alone. A particular challenge is that high-dimensional gene expression data must be sum­marized into variables that allow the determination of the lowest test compound concentration that causes altered gene expression. To address this challenge, we analyzed 60 hepatotoxic and non-hepatotoxic substances in a concentration-dependent manner for cytotoxicity and expression of 3,524 probes previously reported to be influenced by hepatotoxicants. The toxicity separation index (TSI) was applied to quantify how well specific summary variables of gene expression can differen­tiate between the set of hepatotoxic and non-hepatotoxic substances. The best TSI was obtained when the lowest concentration of a test compound that led to differential expression of two genes when compared to vehicle controls was considered positive. Furthermore, the best gene expression-based summary variable was superior to cytotoxicity-based variables alone, and the combination of the best summary variables of gene expression and cytotoxicity data further improved the TSI compared to each category alone. In conclusion, the method used to derive summary variables of gene expression is critical, and the best summary variables improve the prediction of hepatotoxic substances in relation to oral doses and blood concentrations in humans.

Annex G of ISO 10993-11:2017 lists 24 substances that are allegedly material-mediated pyrogens (MMPs) in medical devices and recommends using the rabbit pyrogen test (RPT) for MMP evaluation. We aimed to establish whether medical devices contain MMPs; determine if MMPs are pyrogens or uncouplers of oxidative phosphorylation (UOPs); complete a survey of RPT failures; and develop a UOP screening list. A literature search was conducted to identify MMPs associated with medical devices. Concurrently, data mining was conducted to search for MMPs in medical device extractables databases and collect RPT data from industry and contract research labs. The literature search produced the conclusions: (1) Annex G’s endogenous biological compounds are pyrogenic; (2) Annex G’s drugs and UOP substances are not pyrogenic but thermogenic; (3) numerous other UOPs were identified; and (4) nearly all UOPs are poorly soluble in saline (the RPT’s injection solvent). The data mining found two Annex G MMPs in medical device extracts that were not pyrogenic and confirmed that the RPT failure rate was low, with investigated failures caused by endotoxin or test incompatibilities, not by Annex G MMPs. Since no pyrogenic Annex G MMPs or saline soluble UOPs were found in medical device extracts, the RPT is unnecessary. If UOPs are detected during chemical characterization studies, an ISO 10993-17:2023 toxicological risk assessment can be used to determine their hazard potential. Residual biological contamination can be evaluated with the human monocyte activation test, which detects all biological pyrogens. The term “MMP” is inaccurate and obsolete. Plain language summary Pyrogens cause fevers in mammals. Medical devices contaminated with bacterial components or chemicals generate a material-mediated pyrogenic (MMP) response. The rabbit pyrogen test (RPT) is used to detect MMPs even though its accuracy is questionable. An international standard lists 24 chemicals that are alleged to be medical device MMPs. To evaluate this belief, a search of the scientific literature was conducted to identify MMPs associated with medical devices along with a data mining effort that searched for MMPs found by chemistry laboratories that test medical devices. Failure rates for the RPT were also studied. Only two of the 24 chemicals were found in medical devices, and they were not pyrogens, plus the RPT failures were not due to MMPs. It was concluded that MMPs do not exist and therefore the RPT could be replaced with a human blood cell test that detects all types of biological pyrogens.