The gut microbiome converts the prenylated polyphenol isoxanthohumol (iXN), a natural constituent of hops found in beer, into 8-prenylnaringenin (8-PN), a potent phytoestrogen associated with endocrine-disrupting effects. Following oral exposure, interindividual differences in microbiome composition may lead to variable systemic 8-PN concentrations and consequently to differences in susceptibility to toxicity. To characterize the contribution of gut microbiota to health effects of hop polyphenols, a human physiologically based kinetic (PBK) model that includes microbial 8-PN formation was developed. Ex vivo fecal fermentation coupled to LC–MS/MS revealed substantial interindividual variation in biotransformation capacity. Derived kinetic parameters were incorporated into the PBK model, which was subsequently used to predict systemic 8-PN exposure while accounting for interindividual variability. Model simulations indicated that high iXN metabolizers experience approximately two-fold more internal 8-PN exposure than low metabolizers. Estrogenicity of the predicted uterine 8-PN concentrations was assessed via alkaline phosphatase induction in Ishikawa cells. Even in high metabolizers, systemic 8-PN concentrations appeared to remain below levels of concern regarding endocrine disruption. These findings highlight the importance of accounting for interindividual variability in gut microbial biotransformation when predicting xenobiotic toxicokinetics and illustrate the applicability of microbiome-competent PBK modeling for predicting the systemic fate of gut microbial metabolites.

Per- and polyfluoroalkyl substances (PFAS) are widely used chemicals known for their persistence, bioaccumulation, and adverse health effects, particularly on the immune system. Epidemiological studies link PFAS exposure to immunosuppression, with increased infection susceptibility and reduced vaccine efficacy. In this paper, we describe the workflow we used to establish an integrated testing strategy (ITS) combining in vitro and in silico methods to model PFAS inhibition of antibody production and to define a tolerable daily intake. This strategy was based on data generated within an EFSA-sponsored project. Using human peripheral blood mononuclear cells, the effects of PFAS on antibody production were assessed. Mathematical models were then applied to determine PFAS free concentrations in vitro, while Physiologically Based Kinetics (PBK) modeling enabled quantitative in vitro to in vivo extrapolation (QIVIVE) to translate in vitro effects into external doses. In addition, the Universal Immune System Simulator was used to predict immune-related outcomes and threshold doses for sensitive populations. Following this strategy, we were able to demonstrate that the oral equivalent effect doses derived through QIVIVE were similar to, or lower than, the tolerable weekly intake established by EFSA for PFAS, indicating that our approach is conservative. We demonstrate the possibility of using alternative methods for studying PFAS toxicity, offering insights into their dynamics and kinetics without animal testing. The strategy provides a promising framework for assessing other chemicals, advancing toxicology toward more human-relevant and ethical practices.

Per- and polyfluoroalkyl substances (PFASs) are man-made organofluoride chemicals widely present in the environment, with exposure associated to various adverse health effects, including immunotoxicity. Recently, we showed that PFASs can directly impair antibody production, leading to decreased immunoglobulin (Ig) M and IgG release in human peripheral blood mononuclear cells (PBMCs) obtained from both male and female healthy donors. However, the underlying molecular mechanisms remain largely unknown. In this study, we aimed to address this gap by performing RNA sequencing to identify pathways and genes potentially involved in the observed immunotoxic effects. PBMCs were exposed to selected PFASs for 24 h, and to assess effects on antibody secretion, a subset was subsequently stimulated with CpG oligodeoxynucleotide ODN2006 and rhIL-2 for an additional six days. Transcriptomic analysis indicated activation of the glucocorticoid receptor (GR) and associated signaling pathways, supported by the upregulation of several GR-target genes and the prediction of glucocorticoids or the GR agonist dexamethasone as upstream regulators in Ingenuity Pathway Analysis. Moreover, the inhibitory effects of PFASs on antibody secretion were shown to be reversable by the GR antagonist Mifepristone, supporting the involvement of the GR in PFAS-mediated suppression of antibody secretion. Overall, this research advances our understanding of PFAS-induced immunotoxicity and identifies potential biomarkers for evaluating PFAS exposure and its associated health effects.

The current testing strategy for the assessment of developmental immunotoxicity (DIT) in European chemicals regulations has well recognised limitations. In response to these limitations, the Partnership for the Assessment of Risks from Chemicals (PARC) initiated a 4-year DIT project in May 2025. This project comprises 14 partner institutions and will tackle two primary objectives: a) to enhance the DIT knowledgebase and refine the understanding of critical phases of immune system development, and b) to facilitate the transition towards the use of New Approach Methodologies (NAMs) in DIT risk assessment. The first objective will be approached by reviews of existing literature and the continued advancement of a physiological map of human immune system development. The reviews and the physiological map will in turn serve as foundational tools to support NAMs and Adverse Outcome Pathway (AOP) development. Addressing the second objective, the project has a short-term aim to promote a testing strategy that leverages current immunotoxicity assays and their modifications to inform regulatory decision processes such as screening, prioritisation and read-across analyses. In the longer term, novel NAMs, encompassing developmental processes, will be developed and assessed for their regulatory applicability. While the primary focus of this PARC project is on the enhancement of human DIT risk assessment, it also aims to contribute to the advancement of ecotoxicological evaluation of immunotoxicity.

To create a regulatory infrastructure for the effective use of NAMs, the CHANGE project aims to organise three workshops to Collaborate and Harmonise the Assessment of Next Generation Evidence. To better ensure the success of the CHANGE approach, project organisers invited a group of participants to provide feedback on the first workshop held in Oslo on 18-20 June 2024. This report represents the participants' perspective on the CHANGE working methodology and serves as a companion piece to the CHANGE organisers' publication "Improving how we use workshops when solving complex research problems: Reflections from the CHANGE Project", which provides a detailed description of the methodology, outputs, and conclusions from the workshop. The report includes feedback from most participants in response to the workshop evaluation as well as personal experiences from the authors. The workshop successfully facilitated stimulating engagement with a diversity of perspectives, though representation could be further broadened across sectors and geographies. Additionally, future workshops could refine the explanation of novel approaches to participants, as well as improve how information gathering is structured and formatted for feedback. Overall, participants were enthusiastic about CHANGE and believe the approach holds great promise in shaping future effective use of NAMs for chemical safety assessments. The report concludes with recommendations for follow-up workshops in 2025 and 2026, aiming to contribute to a regulatory infrastructure open to the acceptance and effective use of NAMs and to the use of similar workshops to address other emerging science policy issues.

The gut microbiome converts the prenylated polyphenol isoxanthohumol, a natural constituent of hops found in beer, to 8-prenylnaringenin (8-PN), a potent phytoestrogen, raising concerns about potential endocrine-disruption. Interindividual differences in microbiome composition may result in varying internal exposures to 8-PN and susceptibility to toxicity. To improve the understanding of 8-PN toxicokinetics, a human physiologically based kinetic (PBK) model was extended to include gut microbial 8-PN formation. Respective parameters were obtained from ex vivo fermentations using pooled and individual stool samples to predict average internal exposure while accounting for interindividual differences. This revealed twofold higher internal 8-PN exposure in high metabolizers compared to low metabolizers. Further, we measured estrogenicity of predicted uterus concentrations of 8-PN using alkaline phosphatase assays and found that even in high metabolizers, systemic 8-PN concentrations remain below estrogenicity thresholds. This study broadly demonstrates the applicability of microbiome-competent PBK modeling for quantifying health impacts of gut microbial metabolites.

The increasing variety and quantity of new chemical substances have raised concerns about their potential immunotoxic effects, making it essential to assess their impact on human health. One key concern is the reduction of antibody production, as seen with per- and poly-fluoroalkyl substances (PFASs), commonly known as "forever chemicals." Both in vivo and epidemiological data show that PFASs have immunosuppressive effects, leading to reduced antibody responses, particularly following vaccination. In animal studies, the T cell-dependent (TD) antibody response is the gold standard for assessing chemical effects on immune function. This study utilized two in vitro approaches to investigate the effects of chemicals on antibody production using human peripheral blood mononuclear cells. Initial tests used unstimulated, negative (vehicle), and positive (rapamycin) controls to confirm the robustness of the models. Subsequently, four long-chain PFASs (PFOA, PFOS, PFNA, and PFHxS) were tested. Keyhole limpet hemocyanin (KLH) was used to mimic the TD response, while a TLR9 agonist and IL-2 activated B cells for T cell-independent (TI) immunoglobulin production. The results demonstrated the ability to reproduce TD and TI responses in vitro with robust, reproducible outcomes across a cohort of 20 human donors. The data, consistent with existing literature, showed a significant reduction in anti-KLH IgM production, especially for PFOA in male donors. Similar trends were observed for all PFASs in suppressing total TI IgG and IgM production. These methods closely replicated in vivo conditions, offering a potential alternative to animal models in immunotoxicity assessments.

Hop extracts containing prenylated polyphenols such as 8-prenylnaringenin (8-PN) and its precursor isoxanthohumol (iXN) are popular among women seeking natural alternatives to hormone therapy for postmenopausal symptoms. Due to structural similarities with estrogens, these compounds act as estrogen receptor agonists. Especially 8-PN, described as the most potent phytoestrogen known to date, poses a potential risk for endocrine disruption. Therefore, its use as a hormone replacement raises concerns for human health. However, a significant challenge in assessing the potential endocrine-disruptive effects of hop polyphenols is the lack of data on their toxicokinetics. Particularly, information on in vivo concentrations in target tissues is lacking. To address this gap, we developed a physiologically based kinetic (PBK) model tailored to female physiology. The model was used to predict the levels of hop polyphenols in human blood and target tissues under realistic exposure scenarios. The predictions suggest that iXN and 8-PN concentrations in target tissues reach the low nanomolar range after dietary supplementation. This study enhances our understanding of internal concentrations of iXN and 8-PN after dietary consumption and is of direct applicability for respective risk assessment.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Perfluorinated substances (PFAS) are a class of synthetic chemicals widely used in industry, to which people and ecosystems are exposed. Epidemiological studies have shown that PFAS can cause immunosuppression, increased risk of infections and decreased response to vaccination, with the underlying mechanism(s) of action still remaining elusive. The aim of this project was to fill some of the data gaps identified in the 2020 EFSA Opinion, using new approach methodologies (NAMs). In particular, we aimed to get information on the mode of action for the immunosuppression effects observed in epidemiological studies (i.e., reduction in the vaccination efficacy and possible increase in the susceptibility to infectious disease), and to address the immunotoxicity of PFAS other than PFOS and PFOA (PFNA and PFHxS), including the assessment of a possible common mode of action and to provide insight into the relative potencies of the tested PFAS. To reach these goals, an integrated testing strategy (ITS) consisting of in vitro and in silico methods was developed. The effects of PFAS were investigated using target immune human cell-based in vitr o models, suitable to assess the relevant immunotoxic parameters observed in epidemiological studies (i.e. decreased antibody production). Results obtained fully support the evidence from human epidemiological studies. Furthermore, mathematical fate and distribution models were used to identify nominal concentration of PFAS in the in vitro cell system and physiologically based kinetic (PBK) models were used to perform quantitative in vitro to in vivo extrapolation. The ‘Universal Immune System Simulator’ was used to complete the ITS and investigate the reduced response to vaccination also on vulnerable populations. The use of these selected NAMs may provide a tool to support, by providing mechanistic information, regulatory risk assessment and to study the immunotoxic potential of other PFAS. The participation of immunotoxicologists, molecular biologists, risk assessors, and computational experts within the Consortium, together with EFSA's engagement, ensured the successful performance of this project and delivery of a NAMs-based strategy that allows generating mechanistic information on PFAS immunotoxicity and support risk assessment.

There is a clear need to develop new approach methodologies (NAMs) that combine in vitro and in silico testing to reduce and replace animal use in chemical risk assessment. Physiologically based kinetic (PBK) models are gaining popularity as NAMs in toxico/pharmacokinetics, but their coverage of complex metabolic pathways occurring in the gut are incomplete. Chemical modification of xenobiotics by the gut microbiome plays a critical role in the host response, for example, by prolonging exposure to harmful metabolites, but there is not a comprehensive approach to quantify this impact on human health. There are examples of PBK models that have implemented gut microbial biotransformation of xenobiotics with the gut as a dedicated metabolic compartment. However, the integration of microbial metabolism and parameterization of PBK models is not standardized and has only been applied to a few chemical transformations. A challenge in this area is the measurement of microbial metabolic kinetics, for which different fermentation approaches are used. Without a standardized method to measure gut microbial metabolism ex vivo/in vitro, the kinetic constants obtained will lead to conflicting conclusions drawn from model predictions. Nevertheless, there are specific cases where PBK models accurately predict systemic concentrations of gut microbial metabolites, offering potential solutions to the challenges outlined above. This review focuses on models that integrate gut microbial bioconversions and use ex vivo/in vitro methods to quantify metabolic constants that accurately represent in vivo conditions.

Humans can be exposed to per- and polyfluoroalkyl substances (PFASs) via many exposure routes, including diet, which may lead to several adverse health effects. So far, little is known about PFAS transport across the human intestinal barrier. In the current study, we aimed to assess the transport of 5 PFASs (PFOS, PFOA, PFNA, PFHxS and HFPO-DA) in a human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cell (IEC) model. This model was extensively characterized and compared with the widely applied human colonic adenocarcinoma cell line Caco-2 and a human primary IEC-based model, described to most closely resemble in vivo tissue. The hiPSC-derived IEC layers demonstrated polarized monolayers with tight junctions and a mucus layer. The monolayers consisted of enterocytes, stem cells, goblet cells, enteroendocrine cells, and Paneth cells that are also present in native tissue. Transcriptomics analysis revealed distinct differences in gene expression profiles, where the hiPSC-derived IECs showed the highest expression of intestinal tissue-specific genes relative to the primary IEC-based model and the Caco-2 cells clustered closer to the primary IEC-based model than the hiPSC-derived IECs. The order of PFAS transport was largely similar between the models and the apparent permeability (Papp) values of PFAS in apical to basolateral direction in the hiPSC-derived IEC model were in the following order: PFHxS > PFOA > HFPO-DA > PFNA > PFOS. In conclusion, the hiPSC-derived IEC model highly resembles human intestinal physiology and is therefore a promising novel in vitro model to study transport of chemicals across the intestinal barrier for risk assessment of chemicals.