Deliverable D5.4 1st closing data gaps report WP5 – Task 5.1

D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Partnership for the Assessment of Risks from Chemicals Deliverable D5.4 1st closing data gaps report WP5 – Task 5.1 This partnership has received funding from the European Union ’s Horizon Europe research and innovation programme under Grant Agreement No 101057014. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Technical reference Work package WP5 – Hazard Assessment Task T5.1 – Toxicity testing addressing data gaps of concern Dissemination level 1 PU = Public Lead Beneficiary/ Responsible AE ANSES, FR Contributing Participants UNIVIE (AT), BfR (DE), NIPH (NO), INSA (PT), ANSES (FR), UGent (BE), BPI (EL), ISS (IT), INRAE (FR), CNRS (FR), UMIL (IT) EEA, EFSA, ECHA Responsible author(s) Kiara Aiello Holden / BfR / [email protected] Anne-Cathrin Behr / BfR / anne-cathrin.b[email protected] Doris Marko / UNIVIE / [email protected] Jana Asselman / UGent / Jana.Assel[email protected]e Katerina Kyriakopoulou / BPI / [email protected] Co-authors Hubert Dirven / NIPH / [email protected] Igor Snapkow / NIPH / [email protected] Maria João Silva / INSA / [email protected] Henriqueta Louro / INSA / [email protected] Sabrina Tait / ISS / sabrin[email protected] Emanuela Testai / ISS / emanue[email protected] Franca Maria Buratti / ISS / franca.bur[email protected] Catherine Viguie / INRAE / [email protected] Daniel Zalko / INRAE / [email protected] Sakina Mahouty-Kodja / CNRS / [email protected] Emanuela Corsini / UMIL / [email protected] Regina Puts / BfR / regina[email protected] Internal Reviewers2 Name of the Internal Reviewer(s) / short name of institutions/email addresses Gilles Rivière / ANSES / [email protected] Thalia de Castelbajac / ANSES / -- Philip Marx-Stoelting / BfR / [email protected] Celia Garcia Arenas / BfR / Celia.Garcia-Arena[email protected]d.de External Reviewers3 Name of the External Reviewer(s) / short name of institutions/email addresses Hans Steinkellner / EFSA / [email protected] Maria Anastassiadou / EFSA / [email protected] Vessela Vitcheva / ECHA / [email protected] Pascal Sanders/Anses/ pascal.sander[email protected] D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 1 PU = Public 2 WP/task co-leader(s) from the same WP 3 Independent Reviewers that should not be part of the task related to the AD/D or part of projects related to the Task Document history “Funded by the European Union. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.” Due date of deliverable 30 April 2025 Actual submission date 25/06/2025 Version Date Reviewer name/Institutions Short description of changes 1 19/12/2024 After agreement with PARC WP5 CT and input from PARC T5.1 Leaders, drafted by Kiara Aiello, BfR High-level draft version 1 (v1) 2 19/12/2024 Anne-Cathrin Behr, BfR draft version 1 (v1) 3 31/01/2024 Authors of D5.4 First compiled version reviewed by PARC T5.1 Leaders 4 20/03/2024 Authors of D5.4 and Internal reviewers Review and work on deliverable 5 05/05/2025 External reviewers Draft version with comments provided by reviewers 6 24/06/2025 Authors and WP5 co-leaders Final version with comments from reviewers taken into account D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Abstract This deliverable summarizes the preliminary results of the PARC WP5 Task 5.1, aiming to close key data gaps for substances of emerging concern. Focus is placed on two major substance groups: natural toxins and bisphenol A (BPA) alternatives, addressing both human and environmental health. For human health, prioritized mycotoxins (enniatins including beauvericin and Alternaria toxins) were tested across genotoxicity, endocrine disruption, developmental neurotoxicity, and immunotoxicity endpoints, employing both OECD test guidelines and new approach methodologies (NAMs). In a parallel project, hazard testing of BPA alternatives examined metabolism, bioactivation, and toxicological profiles using as well OECD test guidelines and in vitro systems aligned with regulatory relevance. On the environmental side, aquatic organisms such as Daphnia magna, Lymnaea stagnalis, and Chlorella vulgaris were used to assess the ecotoxicity of selected natural toxins and BPA alternatives under standard OECD and ISO guidelines. Both single and mixture exposures were tested, revealing speciesand compoundspecific effects and emphasizing the relevance of temperature and combined stressors in ecological risk assessment. Altogether, this first data gap report contributes to strengthening risk assessment capabilities across the EU by supporting better prioritization and regulation of under-studied substances. The data are being shared and discussed with regulatory agencies (EFSA, ECHA, EEA), and further testing is ongoing to complete hazard characterizations. Key Words Natural toxins, BPA alternatives, genotoxicity, endocrine disruptive effects, immunotoxicity, developmental neurotoxicity (DNT), ecotoxicology, in vitro, risk assessment, new approach methodologies (NAMs) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Table of contents Document history ___________________________________________________________ 3 Abstract ___________________________________________________________________ 4 Key Words _________________________________________________________________ 4 Table of contents ___________________________________________________________ 5 Authors and Acknowledgements _______________________________________________ 6 Acronyms _________________________________________________________________ 6 Introduction _______________________________________________________________ 9 Part I: Natural toxins: Human Health & Environment _______________________________ 9 1. Introduction ____________________________________________________________ 9 2. Human Health effects of Natural toxins _____________________________________ 10 2.1. Substances ______________________________________________________________________________________________________ 11 2.2. Endpoints and performed and planned studies ______________________________________________________________ 12 2.3. Results __________________________________________________________________________________________________________ 14 3. Environmental effects of natural aquatic toxins _______________________________ 21 3.1. Substances ______________________________________________________________________________________________________ 21 3.2. Target species, endpoints and methods ______________________________________________________________________ 22 3.3. Results __________________________________________________________________________________________________________ 24 4. Conclusions _____________________________________________________________ 28 Part II: BPA Alternatives: Human Health & Environment ____________________________ 30 1. Introduction ___________________________________________________________ 30 2. Substances ____________________________________________________________ 30 3. Human Health effects of BPA alternatives ___________________________________ 31 3.1 Methods and results ____________________________________________________________________________________________ 31 3.1.1 Metabolic Fate and Bioactivation Studies ___________________________________________________________________ 32 3.1.2 Endocrine Disruption _________________________________________________________________________________________ 33 3.1.3 Immunotoxicity _______________________________________________________________________________________________ 34 3.1.4 Developmental Neurotoxicity ________________________________________________________________________________ 39 • Neurosphere Assay by [IUF (DE)] _________________________________________________________________________ 40 3.1.5 Carcinogenicity _______________________________________________________________________________________________ 41 4.1 Testing methods ________________________________________________________________________________________________ 44 4.2 Results per organism ___________________________________________________________________________________________ 49 4. Conclusions ___________________________________________________________ 60 Scientific achievements _____________________________________________________ 63 D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Interactions with regulators __________________________________________________ 68 References _______________________________________________________________ 68 Annex ___________________________________________________________________ 72 A.1 Methods: Toxins human health ____________________________________________ 74 A.2 Methods: Toxins environment _____________________________________________ 76 A.3 Methods: BPA alternatives human health ____________________________________ 76 A.3 Methods: BPA alternatives environment _____________________________________ 85 Authors and Acknowledgements We would like to sincerely thank all experts contributing to this report and to the broader work within the PARC WP5.1 Task. The full list of contributors is available in Annex (Section 0). We are especially grateful to the internal reviewers who provided detailed feedback on the Immunotoxicity and Developmental Neurotoxicity sections, helping to ensure the accuracy and clarity of the content presented. Internal reviewers from PARC WP5 Task 5.2, per endpoint: Immunotoxicity Etienne Blanc / INSERM / [email protected] Nicola Smith / NIPH / [email protected] Igor Snapkow / NIPH / [email protected] Developmental Neurotoxicity Tamara Tal / UFZ / tamara[email protected] Acronyms ALT – Altenuene AME – Alternariol Monomethyl Ether AOH – Alternariol AOP – Adverse Outcome Pathway AR SSTA – Androgen Receptor Steroidogenesis and Transcriptional Activation Assay ATX-I – Altertoxin I BEA – Beauvericin BMC30 – Benchmark Concentration for 30% response BPA – Bisphenol A D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 BPAF – Bisphenol AF BPAP – Bisphenol AP BPB – Bisphenol B BPE – Bisphenol E BPF – Bisphenol F BPP – Bisphenol P BPPH – Bisphenol P Hydroquinone BPZ – Bisphenol Z BPS – Bisphenol S BPS-MAE – Bisphenol S-MAE BPS-MPE – Bisphenol S-MPECaco-2 – Human Colorectal Adenocarcinoma Cells CYN – Cylindrospermopsin DA – Domoic Acid DHT – Dihydrotestosterone DMSO – Dimethyl Sulfoxide DNT – Developmental Neurotoxicity DON – Deoxynivalenol E-Screen Assay – Estrogenic Activity Assay in MCF-7 Cells EC50 – Effective Concentration for 50% effect ENNs – Enniatins ER SSTA – Estrogen Receptor Steroidogenesis and Transcriptional Activation Assay ETS – Electron Transport System γH2Ax Assay – Phosphorylation Assay for DNA Damage Response GR – Glucocorticoid Receptor HABs – Harmful Algal Blooms HCEC-1CT – Human Colonic Epithelial Cells (non-tumorigenic) HepG2 – Human Liver Cancer Cell Line hiPSC(s) – Human Induced Pluripotent Stem Cell(s) H-NMR – Proton Nuclear Magnetic Resonance HPLC-UV – High-Performance Liquid Chromatography with Ultraviolet Detection HPRT Assay – Hypoxanthine-Guanine Phosphoribosyltransferase Assay IC50 – Half-Maximal Inhibitory Concentration (a measure of a substance’s potency) IL-1β, IL-2, IL-6, IL-8, IL-10 – Interleukins (cytokines) JHASA – Juvenile Hormone Activity Screening Assay LC50 – Lethal Concentration for 50% effect LDH – Lactate Dehydrogenase (used in cytotoxicity assays) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 LOAEL – Lowest Observed Adverse Effect Level LPS – Lipopolysaccharide MAP2, ELAVL2, GRIK4 – Neuronal markers MC-LR – Microcystin-LR MCT8 – Monocarboxylate Transporter 8 MCT8 transporter – Monocarboxylate transporter 8 NAMs – New Approach Methodologies NF-κB – Nuclear Factor Kappa B NIS – Sodium/Iodide Symporter NIS transporter – Sodium iodide symporter NPC(s) – Neural Progenitor Cell(s) PBPK – Physiologically Based Pharmacokinetic PSD95, gephyrin – Synaptic plasticity markers QSAR – Quantitative Structure-Activity Relationship RT-qPCR / qRT-PCR – Quantitative Reverse Transcription Polymerase Chain Reaction RTgill-W1 – Fish Cell Line Acute Toxicity using the RTgill-W1 cell line SAR Analysis – Structure-Activity Relationship Analysis SoM Scoring – Site of Metabolism Scoring SOP – Standard Operating Procedure STX – Saxitoxin STTX: Stemphyltoxin III T – Testosterone T3 – Triiodothyronine (thyroid hormone) TBA – Terbuthylazine TCR – T-cell Receptor TDAR – T Cell–Dependent Antibody Response TeA – Tenuazonic Acid TEN – Tentoxin TDI – Tolerable Daily Intake TNF-α – Tumor Necrosis Factor Alpha TH – Thyroid Hormone YTX – Yessotoxin ZEN – Zearalenone D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Introduction PARC task 5.1 Closing data gaps of concern aims to investigate and close existing data gaps through toxicity testing. Following a prioritization exercise and further consultation with partners involved in hazard and risk assessment in national and European agencies, the following groups of substances have been selected for the first round of testing: natural toxins as well as alternatives to bisphenol A (BPA alternatives) [Marx-Stoelting et al., 2023]. Under T5.1, several projects have been initiated to fill regulatory gaps in hazard assessment, following consultations with EU agencies (Table 1). Table 1: Outline of the projects approved under PARC WP5T5.1 Data Gaps Project Status P51.1.a Toxins Hazard identification and hazard characterisation of the mycotoxins enniatins including beauvericin and Alternaria toxins in order to close data gaps and improve risk assessment for human health 05/2022 – 04/2026 Preliminary data was obtained and further studies are ongoing P5.1.1.b BPA HumTox Hazard assessment of bisphenol A alternatives to close data gaps of concern for human health and improve their risk assessment 05/ 2022 – 10/2026 Preliminary data was obtained and further studies are ongoing P5.1.1.c TG+ EnnB1 OECD test guideline-conform animal study with additional endpoint omics-enhanced in vivo study on Enniatin B1 05/2024 – 04/2027 Studies will be initiated by CRO in May 2025 Additional analysis will be performed by selected PARC partners P5.1.1.d PlasticLeach Hazard characterization of leachable chemicals present in plastics 05/2025 – 01/2029 P5.1.2.a Natural toxins aqua Toxicity assessment of naturally occurring toxins on aquatic organisms 05/2022 - 04/2025 First data gaps are filled and project will be finished P5.1.2.b BPA alternatives Adverse effects of individual compounds and mixtures of BPA alternatives on organisms from different taxa 05/2022 – 04/2026 Preliminary data was obtained and further studies are ongoing This report aims to provide an overview of the preliminary results from PARC WP5 Task 5.1 on data gaps proving information on the first results obtained to close the prioritized data gaps (human health and environment health), the scientific achievements (publications, oral presentations etc.) and the interactions developed with appropriate regulators (e.g. EU Agencies). Part I of this deliverable focuses on natural toxins. We will present data from project P5.1.1.a, which examines human toxicity, and project P5.1.2.a, which investigates environmental effects. Part II of the deliverable follows a similar structure, presenting data on BPA alternatives. Specifically, data from project P5.1.1.b, focusing on human health, and project P5.1.2.b, which focuses at environmental toxicity, will be shared. Part I: Natural toxins: Human Health & Environment 1. Introduction Natural toxins have no manufacturer or supplier that is responsible for providing hazard data. Due to climate changes, the human exposure to natural toxins will likely increase (Gobler, 2020) and regulatory agencies have asked for hazard data to improve the risk assessment. This will ultimately result in recommendations on which D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Substance without S9 with S9 Result summary Enniatins ENN A - - - ENN A1 - - - ENN B - - - ENN B1 - - - BEA ongoing ongoing Alternaria toxins AOH ongoing ongoing AME pending pending ATX-I ongoing ongoing ALT ongoing ongoing TeA ongoing ongoing TEN ongoing ongoing Green with a minus (-): negative results. red with a plus (+); positive results ENN A, A1, B, and B1 did not induce γH2AX activation at non-cytotoxic concentrations. However, at a toxic concentration of 10 µM, where cell viability (DAPI) was approximately 60–80%, ENN B, A, and B1 triggered γH2AX activation (see Table 8). No induction of γH2AX was observed for TEN, AME, ALT, and BEA. In contrast, ATX-I and TeA induced γH2AX activation even at non-cytotoxic concentrations [BfR (DE)]. Table 8: Status of γH2Ax data in HepaRG cells after 24h of incubation [BfR (DE)] Substance IC50 (µM) γH2Ax Result summary Highest test concentration (µM) Cell survival at highest test concentration (DAPI) relative to control Enniatins ENN A 60,54 (+), equivocal 10 75% ENN A1 18,31 - 10 99% ENN B 10,93 (+), equivocal 10 55% ENN B1 41,94 (+), equivocal 10 72% BEA 13,34 - 2 95% Alternaria toxins AOH pending pending pending pending AME > 100 - 100 100% ATX-I 83,61 + 3 92% ALT > 100 - 100 96% TeA > 1000 (+), equivocal 1000 95% TEN > 100 - 100 91% Green with a minus (-): negative results. red with a plus (+); positive results Results were interpreted as positive if fold change > 1,5 on two or more adjacent concentrations Endocrine effects D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 In the ER SSTA assay (Table 9), ENNs were tested at concentrations ranging from 3 nM to 1 µM, with cytotoxicity remaining below 20% of the control. No agonist or antagonist effects were observed for any of the four ENNs. AME was tested at concentrations between 100 nM and 30 µM, TeA between 30 nM and 10 µM, and BEA between 3 nM and 1 µM. No antagonist effects were detected for these three mycotoxins. However, AME exhibited an agonist effect with an EC50 of 4.8–8.8 µM, whereas TeA acid and BEA showed no agonist effects. TEN and ALT were tested at concentrations from 100 nM to 30 µM, and neither exhibited agonist nor antagonist effects. ATX-I was tested within a range of 30 nM to 10 µM. No agonist effect was observed, but an antagonist effect was detected, with a LOAEL of 1 µM [INRS (FR)]. Table 9: Status of the results of the ER SSTA in hERα-HeLa9903 cells [INRS (FR)] Substance Dose range (µM) Results Agonist assay Antagonist assay Enniatins ENN A 0 - 1 µM - - ENN A1 0 - 1 µM - - ENN B 0 - 1 µM - - ENN B1 0 - 1 µM - - BEA 0 - 1 µM - - Alternaria toxins AOH pending pending pending AME 0 - 30 µM + TP50: 4,8 - 8,8 µM - ATX-I 0 - 10 µM - + LOAEL 1 µM ALT 0 - 30 µM - - TeA 0 - 10 µM - - TEN 0 - 30 µM - - Green with a minus (-): negative results. red with a plus (+); positive results Regarding the AR SSTA assay, a sigmoidal concentration-response curve and an over 6.4-fold induction of AR activity using DHT was achieved. However, results with Mestanolone, so far, do not fit well in the sigmoidal curve (maximum response and thus plateau was usually achieved already at a concentration of 1.0 x 10−10 M). Standardisation with positive control Mestanolone and the reference compounds for the antagonist assay are currently ongoing. Experimentation with the test chemicals (Enniatins and Alternaria toxins) diluted in DMSO will follow [BPI (GR)]. The steroidogenesis assay (OECD TG 456) was performed to assess the endocrine potential of the mycotoxins. T According to the TG 456, cytotoxicity of ENNA, A1, B, B1 and BEA was evaluated as a preliminary step to exclude concentrations reducing vitality > 20%. The IC50 values are showed in Table 10. In two out of three biological replicates, ENNB and B1 showed an increase of E2 of 60% and 10%, respectively. The hormone assessment of the other mycotoxins are in progress [ISS (IT), BfR (DE)]. Table 10: Preliminary results of the Steroidogenesis Assay [ISS (IT) and BfR (DE)] Substance IC50 (µM) Steroidogenesis Max concentration tested (µM) E2 T Enn iati ns ENN A 1,06 ± 0,08 ongoing ongoing D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 ENN A1 3,48 ± 0,50 ongoing ongoing ENN B > 10 1 ↑ 60% increase (1) ongoing ENN B1 7,14 ± 0,30 1 ↑ 10% increase (1) ongoing BEA 3,08 ± 0,50 ongoing ongoing Alternaria toxins AOH pending pending pending pending AME ongoing ongoing ongoing ongoing ATX-I ongoing ongoing ongoing ongoing ALT ongoing ongoing ongoing ongoing TeA ongoing ongoing ongoing ongoing TEN ongoing ongoing ongoing ongoing (1) Preliminary results of two out of three biological replicas; statistical analysis was not performed An E-screen assay was performed to evaluate the endocrine potential of the Alternaria toxins. The preliminary results are summarized in Table 11. Table 11: Preliminary results of the E-Screen assay [BPI (GR)] Reference compound Results 17-𝜷-estradiol (positive control) Estradiol treatment (10-10 M - 10-8 M) has resulted in a statistically significant fold increase in cell proliferation. The optimal concentration was set at 10-9 M, which led to an increase in the range of 2-2,5 compared to control. This is in accordance with scientific literature using MCF-7 cells from ATCC and estradiol treatment (fold change of 1,5-3). The cell system is considered responsive to ER-mediated proliferation agonists. Fulvestrant (ICI 182,780) (inhibition control) Treatment of cells with the pure antagonist ICI 182,780 led to a statistically significant decrease in cell proliferation compared to control. Co-incubation of estradiol with ICI 182,780 (10-7 M ICI 182,780 and 10-9 M 17-𝛽-estradiol) also significantly decreased the magnitude of E2-induced cell proliferation as expected. Mycotoxins Alternaria toxins AOH Ongoing. A range of concentrations per compound will be tested (alone or in combination with ICI 182,780) to assess agonists and antagonists of the ER-mediated proliferation. We are currently working with Tenuazonic acid. AME ATX-I ALT TeA TEN Immunotoxic effects In the NF-κB reporter gene assay, AOH and ATX-I suppressed the LPS-induced activation of the NF-kB pathway in the absence of S9 (Table 12). TeA did not interact with the pathway (up to 250 µM). Suppression of the pathway by AME was observed only at cytotoxic concentrations, thus no clear indication of a real interaction with the pathway could be concluded. Also, suppression of the pathway by ENNA, ENNA1, ENNB, D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 ENNB1 and BEA was observed only at cytotoxic concentrations, thus no indication of a clear interaction with the pathway was observed [UNIVIE (AT)]. Table 12: Status of the results of the NF-κB reporter gene assay and CellTiter Blue assay in the presence of LPS stimulation in the absence of S9 [UNIVIE (AT)] Substance Cell viability (%) * Luciferase activity (%) * Summary results Lowesst active conc. Highest active conc. Active concentration range (µM) Lowest active conc. Highest active conc. Enniatins ENN A ≈ 60* ≈ 50* 2,5 - 5,0 ≈ 70* ≈ 40* No clear immunosuppressive effects (concurrent cytotoxicity) ENN A1 ≈ 80 ≈ 70* 2,5 - 5,0 ≈ 80* ≈ 50* ENN B ≈ 70* ≈ 65* 2,5 - 5,0 ≈ 80 ≈ 75 ENN B1 ≈ 65* ≈ 60* 2,5 - 5,0 ≈ 70* ≈ 60* BEA ≈ 60 5 ≈ 80* Alternaria toxins AOH ≈ 99 ≈ 85# 1 - 20 ≈ 80 ≈ 18 + AME ≈ 76# ≈ 77# 5 - 20 ≈ 84 ≈ 62 No clear immunosuppressive effects (concurrent cytotoxicity) ATX-I ≈ 90 ≈ 98 1 - 20 ≈ 95 ≈ 56 + ALT ongoing ongoing ongoing ongoing ongoing TeA - - - - - - up to 250 µM TEN ongoing ongoing ongoing ongoing ongoing * Results are expressed in percentage compared to the control. Treatment conditions: 20h, with LPS stimulation # Significantly different compared to the control (p<0,05) § Experiments in the presence of S9 fractions are going Regarding the TLR reporter gene assay, results are summarized in Table 13. AOH and AME show some reduction of LPS-induced TLR-NFκB signal, while ATX-I increased the TLR-NFκB signal in the absence of LPS. ALT, TeA and TEN did not significantly impact the TLR-NFκB signal alone or in combination with LPS [STAMI (NO)]. Table 13: Status of the results from TLR reporter gene assay [STAMI (NO)] Substance Highest test concentration (µM) * TLR2 TLR4 Induce TLRNFk-β pathway Inhibit LPS activated TLRNFk-β pathway Induce TLRNFk-β pathway Inhibit LPS activated TLRNFk-β pathway Alternaria toxins AOH 30 - + - + AME 10 - + - + ATX-I 20 + - + - ALT 10 - - - - TeA 100 - - - - TEN 10 - - - - Green with a minus (-): negative results. red with a plus (+); positive results * No cytotoxicity was observed at the dose ranges tested (alamarBlue) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Additional assays and in silico analysis The in silico analysis, utilizing Site of Metabolism Scoring, indicated that ENNs and BEA are primarily metabolised through the Phase I CYP450 system (Table 14). While the Meteor software predicted only hydroxylations and demethylations, additional theoretical metabolites are described in the literature. The Structure-Activity Relationship (SAR) assessment suggested that BEA has a higher toxic potential compared to the ENNs [IMR (NO)]. Table 14: In silico predictions for biotransformation and toxicity based on Site of Metabolism Scoring and Structure-Activity Relationship (SAR) analysis [IMR (NO)] Substance Biotransformation Predicted biotransformation reactions that can produce metabolites Toxicity Enniatins ENN A Phase 1, CYP450 Oxidative N-demethylation and hydroxylation of terminal methyl Potential hepatoxic compounds after hydrolysis and ring-opening ENN A1 Phase 1, CYP450 Oxidative N-demethylation and hydroxylation of terminal methyl - ENN B Phase 1, CYP450 Oxidative N-demethylation - ENN B1 Phase 1, CYP450 Oxidative N-demethylation and hydroxylation of terminal methyl - BEA Phase 1, CYP450 Oxidative N-demethylation and para hydroxylation at monosubstituted benzene compounds Several metabolites with alert for hepatoxicity since the benzene rings can be transformed to alkylphenls. Alkylphenols can cause chromosome aberration. The predicted intrinsic clearance on human, rat and mice hepatocytes were summarized in Table 15 [ANSES (FR)]. Table 15: Intrinsic Clearance on human and rat and mice hepatocytes [ANSES (FR)] Substance Parameters Unit Human Rat Mice Enniatins ENN A Half-life H ongoing ongoing n.d. Constant of elimination (Ke) /h ongoing ongoing n.d. ENN A1 Half-life H ongoing ongoing n.d. Constant of elimination (Ke) /h ongoing ongoing n.d. ENN B Half-life H 1,02 0,48 0,29 Constant of elimination (Ke) /h 0,68 1,43 2,41 ENN B1 Half-life H 1,07 0,55 n.d. Constant of elimination (Ke) /h 0,65 1,27 n.d. Alternaria toxins TeA Half-life H ongoing ongoing n.d. Constant of elimination (Ke) /h ongoing ongoing n.d. TEN Half-life H ongoing ongoing n.d. Constant of elimination (Ke) /h ongoing ongoing n.d. n.d. not determined D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 3. Environmental effects of natural aquatic toxins This project contributes to the effect assessment of natural toxins in aquatic ecosystems using standard OECD testing procedures that can be directly linked to risk assessment approaches. Particularly, the project also provides comprehensive information on the ecotoxicity of natural toxins in mixtures using standard OECD tests and therefore contributes to a more realistic risk assessment of natural toxins and their impact on achieving good environmental status (i.e., water framework directive and marine framework directive). 3.1. Substances Procurement of substances: Substances were either commercially acquired or collected through a harmful algae producer obtained from a certified culture collection (Table 16). The producer was Microcystis aeruginosa (PCC 7806). In experiments where this producer was used, it was cultured by our partner, UGent (BE), to ensure consistency across all partners working with the same organism and minimize batch to batch variation. For experiments involving Chloarella vulgaris, producer strains that could grow in the same culture medium were selected: Microcystis aeruginosa (LEGE 91094) and Aphanizomenon ovalisporum (LEGE X-001). As each partner tested different toxins in various test systems, a centralized purchase of these strains was not necessary. Partner UAVR (PT) also added additional reference chemicals to assess the effects of binary mixtures. The chemical substances used in this study included cadmium chloride anhydrous (CdCl2, 99% purity, FLUKA, Sigma-Aldrich), and TBA (C9H16ClN5, molecular weight: 229,7 Daltons), tested as a commercial formulation (SAPEC, containing 500 g/L active ingredient TBA). Table 16: List of natural aquatic toxins investigated in P5.1.2.a, visual structures are obtained from PubChem Substance Abbreviation CAS no. Molecular weight (g/mol) Structure Visual structure Cyanobacterial toxins Domoic acid DA 14277-97-5 311,3 C15H21NO6 Yessotoxin YTX 112514-54-2 114344,0 C55H82O21S2 Saxitoxin STX 35523-89-8 299,3 C10H17N7O4 Microcystin-LR MC-LR 101043-37-2 995,2 C49H74N10O12 D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Cylindrospermopsin CYL 143545-90-8 415,4 C15H21N5O7S Mycotoxins Zearalenone ZEN 17924-92-4 318,4 C18H22O5 Deoxynivalenol DON 51481-10-8 296,3 C15H20O6 Enniatin A1 ENN A1 4530-21-6 667,9 C34H59N3O9 Enniatin B1 ENN B1 19914-20-6 653,8 C35H61N3O9 3.2. Target species, endpoints and methods In this section a summary of the organisms, different endpoints and methods used is presented. More detailed description of the methods can be found in the Annex of this deliverable. Following the REACH regulation, model algae and invertebrate species are used to assess effects of natural toxins on life history endpoints of these organisms. The selected model species include Chlorella vulgaris LEGE Z-001, Lymnea stagnalis, Daphnia magna, Nitocra spinipes and Acartia tonsa. These are standard model species across freshwater, estuarine and marine environments in current REACH regulations and environmental risk assessment frameworks. Table 17 summarizes the test guidelines followed by the different partners on the target species selected. Table 17: Test guidelines followed on the selected model species Test guideline Organism OECD 211: Daphnia magna reproduction test Daphnia magna ISO14669: Determination of acute lethal toxicity to marine copepods Nitocra spinipes Acartia tonsa ISO18220: Larval development test with the harpacticoid copepod Nitocra spinipes Nitocra spinipes OECD 243: Lymnaea stagnalis Reproduction Test Lymnea stagnalis OECD 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test Chlorella vulgaris Table 18 presents an overview of the different tests performed to assess single toxins. Acute and chronic toxicity experiments were executed and the following endpoints were evaluated: mortality, swimming speed, development and reproduction. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Table 18: Overview of all conducted tests with single toxins Substance Organism Lifestage Exposure Time Endpoint DA N. spinipes Adult Acute Mortality Naupli Acute Mortality Chronic Development Chronic Reproduction YTX N. spinipes Adult Acute Mortality Naupli Acute Mortality Chronic Development Chronic Reproduction STX N. spinipes Naupli Chronic Development MC-LR N. spinipes Naupli Chronic Reproduction Adult Acute Swimming speed Acute Mortality A. tonsa Adult Acute Swimming speed Acute Mortality L. stagnalis Naupli Chronic Reproduction ZEN L. stagnalis Naupli Chronic Reproduction Additional mixtures studies are currently ongoing and Table 19 collects the mixtures that have been tested. Chronic exposure experiments with Lymnaea stagnalis evaluated effects of microcystin, 0.5 to 50 µg/L, Zearalenon, 0.030 to 0.50 mg/L, while mixture studies on Deoxynivalenol (Don), Zearalenone (Zen), Enniatin A1 (EnnA1) and Enniatin B1 (EnnB1) based on environmental relevant levels is currently ongoing for this organism. The lowest mixture was 0.1, 0.05, 0.03 and 0.01 µg/L of Don, Zen, EnnA1 and EnnB1, respectively. The remaining tested concentrations consisted of the same relative ratios of the four mycotoxins but concentrated 10, and 100 times compared to the lower mixture. Besides L. stagnalis, the combined toxicity of cyanotoxins and anthropogenic contaminants on the growth of Chlorella vulgaris, focusing on both single and binary mixture exposures, has been also analysed. The test substances included cadmium chloride (CdCl₂), terbuthylazine (TBA), microcystin-LR (MC-LR), and cylindrospermopsin (CYN), due to their environmental relevance and potential interactive effects. A fixed ray experimental design was used for all experiments. For MC-LR combination with TBA and Cd, the experimental design consisted of single exposures to 7 concentrations of MC-LR and 5 concentrations of TBA or Cd and 25 combinations of MC-LR and TBA or MC-LR and Cd. The nominal concentrations for MC-LR single exposures ranged from 0.5 to 80 mg.L-1 and for combined exposures, from 0.5 to 20 mg.L-1. TBA and Cd nominal concentrations ranged from 0.03 to 0.48 and 0.05 to 5 mg.L-1 in single and combined exposures. For CYN combination with TBA and Cd, the experimental design consisted of single exposures to 4 concentrations of CYN and 5 concentrations of TBA and Cd and 25 combinations of substances for each case. The nominal concentrations for CYN single exposures ranged from 10 to 80 mg.L-1 and for combined exposures, from 0.5 to 20 mg.L-1. TBA and Cd nominal concentrations ranged from 0.03 to 0.48 and 0.05 to 5 mg.L-1 in single and combined exposures. Interactions between chemicals in binary mixtures were analyzed to determine whether they exhibited synergistic, additive, or antagonistic effects. For experiments with copepods, we used bloom- D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 relevant cell densities of M. aeruginosa and A. minutum in our mixture experiments. As these two species differ in cell size, we standardized biomass based on carbon content. The average carbon content per cell was 1.71 ± 0.48 pg cell-1 for M. aeruginosa and 469.70 ± 30.59 pg cell-1 for A. minutum. Accordingly, four cell densities of M. aeruginosa were selected, ranging from 0.25 to 2 × 106 cells mL-1 with a two-fold dilution between levels. To achieve an equivalent biomass (carbon content) ratio, four corresponding cell densities of A. minutum were chosen, ranging from 1000 to 8000 cells mL-1, also using a two-fold dilution series. Microcystis aeruginosa blooms have been reported to occur in estuaries by previous studies, in densities ranging from 0.029 x 106 to 2.7 x 106 cells mL1 (Bormans et al., 2020; Taş et al., 2006). Alexandrium minutum blooms have been observed at densities between 104 to 106 cells L-1 (Bravo et al., 2008; Cosgrove et al., 2014). Nitocra spinipes is an estuarine copepod species with a broad salinity tolerance (Svetlichny and Hubareva, 2014; Wulff, 1972). For Daphnia, a previous acute toxicity test conducted by the University of Birmingham reported a 72 h EC50 with immobility at 9.5 × 106 cells mL-1. To facilitate a two-fold dilution series, we selected 1 × 107 cells mL-1 as the highest exposure concentration of M. aeruginosa, resulting in four exposure levels. The same cell densities of A. minutum (1000 to 8000 cells mL-1) were maintained for consistency. Table 19: Summary of mixtures tested Organism Test guideline Mixture Status Lymnea stagnalis OECD 243 MC-LR + ZEN done MC-LR + DON ongoing MC-LR + ENN A1 ongoing MC-LR + ENN B1 ongoing Chlorella vulgaris OECD 201 MC-LR + TBA ongoing MC-LR + CdCl₂ ongoing CYN + TBA ongoing CYN + CdCl₂ ongoing Daphnia magna OECD 211 Saxitoxin producer + microcystin LR producer ongoing Nitocra spinipes ISO14669/ ISO18220 Saxitoxin producer + microcystin LR producer ongoing 3.3. Results Results from UGent and UAVR are a summary from results of published work (see scientific achievements). All results are based on nominal concentrations. Firstly, we determined acute effect concentrations for N. spinipes (Table 20). Secondly, we observed significant impacts of yessotoxin and saxitoxin on the brood size of N. spinipes (Table 21). Lastly, elevated temperatures markedly intensify the reproductive toxicity of algal toxins including domoic acid, yessotoxin and saxitoxin (Table 21). Notably, the concentrations of the four algal toxins in natural seawater, in the absence of blooms, are currently below the effective concentrations identified in this study, suggesting a low risk of exposure for marine copepods. Furthermore, exposure to MC-LR producing cyanobacteria resulted in decreased swimming speed, increased inactivity, and higher mortality in A. tonsa, compared to the nontoxic algae (Table 22, 23). No effects were observed for N. Spinipes (Table 22, 23). For L. stagnalis, all results are presented as nominal concentrations. No effects at tested concentrations were observed for MC-LR and ZEN (Table 24). Table 20: Acute toxicity tests with N. spinipes to determine the 50% Lethal concentration [UGent (BE)] from Liu et al. 2024 Substance Temperature Adults Nauplii Concentration Range LC50 Concentration Range LC50 D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 DA 15°C 0 - 1000 µg L-1 25,97 ± 11,96 µg L-1 0 - 600 µg L-1 97,24 ± 6,45 µg L-1 * YTX 15°C 0 - 150 µg L-1 ND 0 - 120 µg L-1 ND DA 20°C 0 - 1000 µg L-1 17,15 ± 3,34 µg L-1 0 - 600 µg L-1 81,99 ± 3,89 µg L-1 YTX 20°C 0 - 150 µg L-1 ND 0 - 120 µg L-1 ND DA 25°C 0 - 1000 µg L-1 8,79 ± 1,93 µg L-1 0 - 600 µg L-1 57,26 ± 6,82 µg L-1 * YTX 25°C 0 - 150 µg L-1 ND 0 - 120 µg L-1 ND * Tests were conducted at 18/22°C instead of 15/25°C due to mortality of the Nauplii at 15/25°C. ND: LC50 could not be determined, as even in the highest concentrations tested mortality did not reach 50% Table 21: Chronic toxicity tests with N. spinipes to determine effects on developmental and reproductive toxicity [UGent (BE)] from Liu et al. 2024 Substance Temperature Developmental toxicity Reproductive Toxicity Concentration Range LOEC Concentration Range LOEC DA 18°C 0 - 10 µg L-1 No Effects 0 - 10 µg L-1 No Effects YTX 18°C 0 - 5 µg L-1 No Effects 0 - 5 µg L-1 No Effects STX 18°C 0 - 20 µg L-1 5 µg L-1 0 - 20 µg L-1 10 µg L-1 MC-LR 18°C 0 - 200 µg L-1 100 µg L-1 0 - 200 µg L-1 No Effects DA 20°C 0 - 10 µg L-1 No Effects 0 - 10 µg L-1 No Effects YTX 20°C 0 - 5 µg L-1 No Effects 0 - 5 µg L-1 5 µg L-1 STX 20°C 0 - 20 µg L-1 No Effects 0 - 20 µg L-1 10 µg L-1 MC-LR 20°C 0 - 200 µg L-1 No Effects 0 - 200 µg L-1 No Effects DA 22°C 0 - 10 µg L-1 No Effects 0 - 10 µg L-1 No Effects YTX 22°C 0 - 5 µg L-1 No Effects 0 - 5 µg L-1 5 µg L-1 STX 22°C 0 - 20 µg L-1 No Effects 0 - 20 µg L-1 10 µg L-1 MC-LR 22°C 0 - 200 µg L-1 No Effects 0 - 200 µg L-1 200 µg L-1 The table reports the lowest observed effect concentrations Table 22: Acute toxicity tests with N. spinipes and Acartia tonsa to determine effects of MC-LR producer on swimming behaviour and mortality under a combination of increased temperature and salinity at a fixed ratio of MC-LR producer strain [UGent (BE)] from Liu et al. 2025. Variable N. spinipes A. Tonsa % of Inactivity Average speed % of Inactivity Average speed Temperature x MC-LR producer 20°C No Effects No Effects + + 24°C No Effects No Effects + + 26°C No Effects No Effects + + 28°C No Effects No Effects + + Salinity x 8 ppt No Effects No Effects + + D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 - Carcinogenicity Next, a brief introduction regarding the endpoints and assays performed, followed by the results obtained so far (or in progress/expected) are presented. Further methodological details can be found in Annex A.3. 3.1.1 Metabolic Fate and Bioactivation Studies The elucidation of metabolic pathways and bioactivation routes is critical for a sound assessment of hazard and risk of substances. Understanding how a substance is metabolized within an organism helps determine its toxicological impact, including the formation of reactive metabolites that could pose a risk to health. Such knowledge is fundamental for making informed decisions regarding safety standards, regulatory measures, and risk management, as recognized by OECD and regulatory agencies. These challenges are being addressed in this task, and most of the ongoing activities are described below. Ongoing pilot biotransformation studies by [INRAE (FR)] include performing mass balance analyses in HepaRG cells, followed by radio-HPLC profiling, and using radiolabeled molecules (³H or ¹⁴C) to identify key metabolites and quantify the biotransformation of parent compounds (primary and secondary metabolites). To date, all prioritized molecules, except BPS-MAE, have been obtained as radiolabeled standards, with ongoing purification and stability testing. These molecules were either purchased, synthesized or made available by [INRAE (FR)]. To complement these efforts, [ISS (IT)] has developed a diode-HPLC array method (DAD) to detect and quantify the bioavailability of BPA alternatives in in vitro assays: BPZ, BPE, BPS-MAE, BPP, BPAP, and TCBPA. The metabolism, using isoform specific enzymes and liver microsomes, and biokinetics studies will provide crucial data for Physiologically Based Pharmacokinetic (PBPK) modelling. The data generated - including kinetic parameters, i.e., Km, Vmax, hepatic clearance, - feed directly into quantitative structure-activity relationships (QSARs) and physiologically based exposure models. The metabolism study has been initiated with BPZ testing various concentrations (5-10-20-40 µM) and incubation times (0-10-20-30-60 min). No significant plastic adsorption on polypropylene safe-lock tubes, used for centrifugation, was observed. However, reliable metabolic data could not be obtained due to BPZ instability in the incubation buffer and low solubility in MeOH - a solvent required for incubation with recombinant enzymes and Human Liver Microsomes (DMSO was avoided as it may interfere with enzymatic activity). The next step will be to evaluate BPE, which shows better (higher) solubility in water matrix, to obtain metabolic kinetic data. The ongoing work of this endpoint performed by both partners is summarized in Table 27. Table 27: Summary of the metabolism study from radio-HPLC profiling by [INRAE (FR)] and diode-HPLC array method (DAD) by [ISS (IT)], both in HepaRG cells. Blue, completed. Light blue, ongoing, Yellow planned for PARC Year 4 –until PARC M54 (May 2025 – October 2026) Substance Viability test in HepaRG Radio-labeled - Availability of standards - Purity & stability confirmation over time Radio-labeled Mass balance study in HepaRG cells Radio-labeled Radio-HPLC profiling (quantification of metabolites) Radio-labeled Structural identification of major metabolites Radio-labeled Results summary Further determine kinetic constants (Km, Vm) for major metabolites Isoform specific metabolism study at 5-1020-40 µM, and incubation times 0-1020-30-60 min [ISS (IT)] BPA Completed Ongoing May 2026 BPZ May 2026 Stability problems in MeOH, DMSO can interfere with enzymatic activity D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 No reliable data obtained BPE May 2026 Ongoing BPS-MAE Radio-labeled molecule availability remains uncertain Pergafast 201 May 2026 BPP May 2026 BPAP May 2026 TCBPA May 2026 TBBPA May 2026 3.1.2 Endocrine Disruption The endocrine system plays a crucial role in development, regulating growth, metabolism, reproduction, and overall homeostasis in the body. Because it relies on precise communication between organs, any interference can have widespread effects on the entire organism, making endocrine disruption a key concern for hazard and risk assessment. In this context, in vitro studies are being conducted to assess the effects of prioritized BPA alternatives on steroidogenesis, using the OECD TG 456, and thyroid hormone regulation, are being performed. - Steroidogenesis Assessment by [ISS (IT)]: The Human H295R Steroidogenesis Assay (OECD TG 456) was used to evaluate the ability of BPA alternatives to alter synthesis of the key steroid hormones—17β-estradiol (E2) and testosterone (T)—in human adrenal cells. Preliminary results from two out of three biological replicates indicate a dose-dependent induction of E2 and inhibition of T levels. Further hormone level assessments are in progress. Cytotoxicity assessment in human adrenal cells has been completed for all eight prioritized BPA alternatives, using BPA as a reference compound, obtaining the following relative cytotoxicity ranking: BPP > BPZ > BPS-MAE > TBBPA> BPAP > BPA, BPE, TCBPA. All the described data is summarized in Table 28 below. Table 28: Preliminary results of Human H295R Steroidogenesis Assay (OECD TG 456), conducted by [ISS (IT)]. Blue, completed. Light blue, ongoing, Substance Cytotoxicity IC50 [µM] Steroidogenesis assay Max concentration tested [µM] E2 T BPA > 100 50 ↑ 33% induction ↓ 90% inhibition BPZ 37,76 ± 4,57 10 ↑ 22% induction ↓ 60% inhibition BPE > 100 50 ↑ 37% induction ↓ 90% inhibition BPS-MAE 57,13 ± 4,29 Ongoing Pergafast 201 BPP 26,43 ± 3,17 BPAP 63,37 ± 7,02 D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 TCBPA > 100 TBBPA 61,88 ± 2,42 - Thyroid Hormone Transport Disruption: T3 by MCT8 and I uptake by NIS [ISCIII (ES)]: The potential of BPA alternatives to interfere with thyroid hormone transport is being assessed through in vitro assays currently under consideration by EURL ECVAM / NETVAL (validation process ongoing, see Table 29 for preliminary results). These assays evaluate: • Cytotoxicity assessment of MDCK1 cells (via morphological alterations): completed for all BPA alternatives (Table 29). • MCT8 is a highly selective transporter of thyroid hormones, facilitating their entry into various tissues, including the brain, kidney, liver, and heart, thereby regulating neurological health and systemic metabolic balance. Triiodothyronine (T3) transport via MCT8 transporter in MDCK-hMCT8 cells (measuring T3 accumulation). T3 transport inhibition was tested in at least two replicates for all compounds. • Iodide (I-) uptake via the NIS transporter, a key step in thyroid hormone synthesis. Itransport inhibition was evaluated for two out of 13 substances. Table 29. Preliminary results of thyroid regulation disruption assays. Blue, completed. Light blue, ongoing. Substance Morphological Cytotoxicity (1h incubation) start at [µM] Max concentration tested [µM] T3 transport by MCT8 IC50 range [µM] Itransport by NIS IC50 range [µM] BPA > 250 1000 200-300 200-400 BPZ > 250 1000 600-700 Ongoing BPE No 1000 300-400 BPS-MAE > 250 1000 No Inhibitor Pergafast 201 > 100 1000 800-100 BPP > 250 1000 > 1000 BPAP > 250 1000 100-200 TCBPA No 1000 200-300 TBBPA No 1000 800-1000 No Inhibitor BPH > 250 1000 No Inhibitor BPS > 250 1000 700-800 BAGDE > 250 1000 700-800 Trans-CBDO > 250 1000 No Inhibitor Positive Control Silychristin (Potent inhibitor) 1000 0,5-2,0 N/A Positive Control ClO4- (Potent Inhibitor) N/A 1-5 3.1.3 Immunotoxicity BPA and its analogues have the potential to disrupt or modulate immune function, potentially increasing susceptibility to infections and altering inflammatory responses. Therefore, understanding how BPA analogues affect immune regulation is relevant for assessing their full impact on human health. Scientifically valid in vitro and in silico methods in line with the OECD Detailed Review Paper No. 360 on In Vitro Test Addressing D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Immunotoxicity with a Focus on Immunosuppression were used (https://www.oecd.org/en/publications/detailed-review-paper-on-in-vitro-test-addressing-immunotoxicitywith-a-focus-on-immunosuppression_667965bc-en.html). One of the methods used, based on Jurkat T cells and IL2 production, is similar to the recently accepted IL-2 Luc Assay (OECD TG444A). (OECD (2023), Test No. 444A: In Vitro Immunotoxicity: IL-2 Luc Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/27b10ba3-en.) - Immune Cell Targets and Function [UMIL (IT)]: The effect of BPA analogues on immune function is being investigated using human peripheral blood mononuclear cells (PBMCs) obtained from healthy male and female donors. Initial cytotoxicity screening (LDH assay) defined non-toxic concentrations for further analysis (Tables 30 and 31). The study focuses on: • Antibody production and Natural Killer cell (NK-cell) activity: BPA and its analogues were tested for their ability to alter B cell differentiation and antibody production (crucial for identifying, neutralizing, and marking pathogens for destruction) as well as natural killer (NK) cell lytic activity, involved in innate immunity defense the first-line response to infections and tumors. While the results show at non-cytotoxic concentrations (cell viability > 80%) a reduction in the release of both IgG and IgM for all tested substances, with differences based on sex and the specific immunoglobulin examined, no changes in NK-cell lytic activity were detected. • T-cell proliferation and differentiation: Ongoing analyses are examining BPA alternatives' on T-cell activation markers and cytokine production, which are central to the activation of adaptive immune responses and critical for infection resistance and immune regulation. Table 30: Summary of data from in vitro assay in PBMCs of healthy donors by UMIL (IT). Blue, completed. Light blue, ongoing. Substance NK-cell lytic activity PBMC 24h exposure + co-culture with K562 for 4h T-cell proliferation and differentiation PBMC 24h exposure + 4 days with anti-CD3 and anti CD28 Male donors Female donors BPA No significant changes compared to controls ongoing ongoing BPZ ongoing ongoing BPE ongoing ongoing BPS-MAE ongoing ongoing BPP ongoing ongoing BPAP ongoing ongoing TCBPA ongoing ongoing Table 31: Summary of data from in-vitro assays in PBMCs of healthy donors by UMIL (IT). Any value shown in the graph represents the mean ± SD, with n = 5 males and n = 5 females. Only statistically significant results are reported. Substance Determination of cell viability of 80% (CV80) PBMC 24h exposure CV80 [µM] T-cell independent antibody production PBMC 24h exposure + 6 days of stimulation with ODN2006 and IL-2 Effect on IgG and IgM release IC50 [µM] Male donors Female donors IgG IgM No genders difference D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 BPA 50,84 54,37 ↓ 50 µM female ↓ 50 µM male 38,29 ± 0,17 BPZ 13,81 17,82 ↓ 15 µM female ↓ 0,15 and 15 µM male 14,50 ± 0,12 1,5 µM male vs female BPE 51,87 59,33 ↓ 50 µM female ↓ 50 µM female 41,74 ± 0,40 BPS-MAE 35,80 60,65 ↓ 35 µM male ↓ 60 µM female ↓ 0,35, 3,5 and 35 µM male ↓ 60 µM female 33,22 ± 0,71 BPP 13,13 20,44 ↓ 15 µM female ↓ 1,5 and 15 µM male ↓ 15 µM female 9,89 ± 2,06 0,15 and 1,5 µM male vs female 15 µM male vs female BPAP 12,90 15,74 ↓ 15 µM female ↓ 15 µM male and female 12,42 ± 0,77 0,15 µM male vs female 15 µM male vs female TCBPA 17,59 30,23 ↓ 25 µM female ↓ 25 µM male 23,41 ± 2,25 2,5 and 25 µM male vs female - Immunometabolism and Inflammation Pathways [MUI (AT)]: • Tryptophan metabolism: Given critical role of tryptophan in immune regulation, BPA analogues' effects on inflammation-induced tryptophan breakdown are being investigated in human PBMCs and monocytederived cell lines by measuring the concentrations of tryptophan and its catabolite kynurenine. Experiments are still ongoing. • Metabolic markers: Proliferation assays and gene expression profiling provide insight into immunometabolic disruptions induced by BPA alternatives. Metabolic activity/viability was assessed by using resazurin as a probe. Primers were designed for relevant metabolic enzymes. Experiments for primer validation and cell treatments for RNA collection have been initiated. - Immunomodulation and Targeting the Glucocorticoid Receptor [UL FFA]: The effect of selected BPA analogues on the metabolic activity and the release of cytokines from in vitro models of human macrophages, T cells and B cells is being evaluated. Preliminary results point to BPP, BPAP, and BPZ as the most cytotoxic, with BPP showing IC50 values five times lower than BPA (Table 31). In silico screening (Endocrine Disruptome): a computational analysis helped to identify potential interactions of BPA alternatives with 12 hormone nuclear receptors, including the glucocorticoid system, by ranking BPA analogues based on their likelihood to bind to the receptors. BPS-MAE, PF201, BPZ, BPAP, and BPP exhibited higher binding potential (to all 12 nuclear receptors) than BPA, while BPE and TCBPA were less active. These results are gathered in a manuscript currently under revision. Glucocorticoid receptor (GR) signalling: to determine immunomodulatory potential; relevant for stress & hormone modulation. Experimental evaluations using macrophages (THP-1 derived), T cells (Jurkat cells), and B cells (lymphoblastoid cell lines, LCLs) assess how BPA analogues influence cytokine release and GR activation: • Macrophages (THP-1 derived): IL-1β was the most affected cytokine, showing biphasic modulation by BPP (stimulation at nanomolar doses, suppression at micromolar doses). IL-6 release was increased by BPSMAE, BPZ, TCBPA, and PF201, while TNF-α remained unchanged and only 10 micromolar TCBPA stimulates the release of IL-8. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 • T-cell responses (Jurkat cells): BPA analogues generally suppressed IL-2 release in low micromolar range (a classic marker for T cell activation), except for TCBPA, which stimulated IL-2 at low micromolar concentrations but suppressed it at higher doses. • B cells (Lymphoblastoid, LCLs): IL-2, IL-6, and IL-10 were suppressed by most BPA analogues at 10 µM, while TCBPA again stood out with different behaviour by IL-6 is increased by 10 uM TCBPA went rest of cytokines are mostly downregulated. Comparably to the results in THP-1 derived macrophages, TNFα was the least affected cytokine. The data described is summarized in a manuscript, which has been submitted to Ecotoxicology and Environmental Safety journal and is currently under review. Next steps - qRT-PCR protocol for glucocorticoid receptor modulation in THP-1 derived macrophages is being established. - Developing a stable GR-expressing T cell line (Jurkat cells) that will enhance mechanistic studies. Table 32: Summary of data from in-vitro cytotoxicity assays in macrophages (THP-1 derived), T cells (Jurkat cells) and LCLs viaresazurin method for24h exposure, n=3 by UL FFA. Blue, completed. Light blue, ongoing. Substance Jurkat T cells Cytotoxicity IC50 [µM] THP-1 derived macrophages Cytotoxicity IC50 [µM] Lymphoblastoid cell lines (LCLs) Cytotoxicity IC50 [µM] BPA 99,4 ± 5 122,5 ± 3,2 182,8 ± 16,1 BPZ 38,1 ± 1 44,8 ± 2,1 65,9 ± 4,9 BPE 208,2 ± 6 208,5 ± 4,6 220,2 ± 11,2 BPS-MAE 141,4 ± 7 145,2 ± 8,0 226,8 ± 34,5 Pergafast 201 148,1 ± 7,5 98,2 ± 5,6 178,8 ± 0,7 BPP 11,1 ± 0,1 18,8 ± 0,5 28,6 ± 1,5 BPAP 26,5 ± 0,9 36,9 ± 0,7 62,4 ± 8,5 TCBPA 41,5 ± 3 73,1 ± 3,4 89,8 ± 4,6 TBBPA - Calcium Homeostasis and Mechanistic Insights [ISS (IT)]: Calcium signalling is critical to immune cell activation. To investigate the pathways involved in immunotoxicity, ongoing studies are evaluating BPA analogues’ effects on intracellular calcium homeostasis in T cells (Jurkat cells). Short and long-term effects of the selected BPA analogues on both resting intracellular calcium concentration ([Ca2+]ᵢ) and regulated calcium responses are being evaluated. The findings will help highlight the potential of these substances to trigger and/or disrupt key calcium-mediated cellular events, which are central to T-cell activation. Preliminary results indicate (Table 33): BPP, BPAP, and BPZ significantly increase basal [Ca²⁺]ᵢ, while inhibiting the responses following T-cell receptor (TCR) activation. Effects are dose-dependent, occurring at lower concentrations than those affecting cell viability. Serum proteins (e.g., albumin) reduce BPZ potency, suggesting that the effect is linked to the analog's free fraction, and that binding interactions might affect toxicity. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Table 33: Summary of preliminary results from intracellular calcium homeostasis assays in Jurkat T cell line [ISS (IT)] Blue, completed. Light blue, ongoing. Blank row Not performed and not planned. Substance Acute effects (Whitin 5 min exposure) Long-term effects (24h) Max concentration tested (µM) Basal [Ca2+]i [Ca2+]i response to TCR activation Max concentration tested (µM) Basal [Ca2+]i [Ca2+]i response to TCR activation BPA 100 ongoing ongoing - planned planned BPZ 100 ↑ ↓ 20 ongoing ongoing BPE 100 ongoing ongoing - planned planned BPS-MAE - planned planned - planned planned Pergafast 201 BPP 10 ↑ ↓ - planned planned BPAP 100 ↑ ↓ - planned planned TCBPA 50 ongoing ongoing - planned planned TBBPA Conclusions on immunotoxicity Table 34 presents the key findings from tests assessing the immunotoxicity of BPA alternatives on human health. Table 34: Summary of Immunotoxicity Findings of BPA Alternatives Immune Function Key Finding Interpretation Regulatory Relevance T-Independent Antibody Production (B Cells – PBMCs, LCLs) BPA analogues reduced IgM and IgG production, with doseand sex-dependent effects (IgG suppression in females, IgM suppression in males). B-cell dysregulation may impair adaptive immunity weaking immune protection. Reduced antibody production could affect vaccination response and infection resistance, highlighting a risk of immunosuppression. NK-Cell Cytotoxicity (PBMCs – NK Cells) No significant alterations in NK-cell lytic activity across tested substances. NK-cell function appears resilient to BPA analogue exposure, meaning innate immune surveillance is not directly impaired. While no immediate concern, long-term effects and interactions with other immune stressors require further study. T-Cell Proliferation & Differentiation (PBMCs – CD4+/CD8+ T Cells, Jurkat Cells) BPA alternatives suppressed IL-2 release, except for TCBPA, which showed biphasic effects (stimulated at low doses, suppressed at high doses). IL-2 suppression suggests impaired T cell activation, which could lead to immune suppression. The biphasic response of TCBPA suggests that dosedependent effects influence immune function differently. IL-2 suppression raises concerns about T cell anergy, potentially affecting pathogen defense and autoimmune balance. Further evaluation is needed for low-dose effects. Glucocorticoid Receptor (GR) Signaling & Cytokine Modulation - BPP, BPAP, and BPZ were the most cytotoxic. - IL-1β showed biphasic modulation by BPP (stimulation at GR signaling disruptions may lead to immune suppression or excessive inflammation. BPP’s biphasic effect on IL1β suggests a shift GR disruption is a major regulatory concern due to its role in stress response and immune homeostasis. IL-6 elevation suggests potential chronic inflammatory risks, D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 (Macrophages, T Cells, LCLs) nanomolar doses, suppression at micromolar doses). - IL-6 increased with BPS-MAE, BPZ, TCBPA, and PF201. - TNF-α was largely unchanged. between inflammation activation and suppression. IL-6 elevation indicates a risk of chronic inflammation or autoimmunity. supporting the need for further evaluation of exposure limits. Calcium Homeostasis in Immune Cells (T Cells – Jurkat) - BPP, BPAP, and BPZ increased basal calcium levels but inhibited T-cell receptor (TCR) activation responses. - The presence of albumin reduced BPZinduced calcium effects, suggesting protein binding affects toxicity. Disruptions in calcium signaling can impair T cell activation, leading to weakened immune responses or dysregulated cell signaling. Mechanistic evidence of immunotoxicity – calcium homeostasis disruption is a recognized marker of immune dysfunction. These findings support AOP development for immune dysregulation. 3.1.4 Developmental Neurotoxicity The developing nervous system is particularly sensitive to chemical exposures, making developmental neurotoxicity (DNT) a significant concern for human health. To evaluate the potential neurotoxic effects of BPA alternatives, two complementary in vitro strategies are being applied, aiming to improve mechanistic understanding of how BPA alternatives disrupt neurodevelopmental processes These include hippocampal differentiation and neuronal proliferation, differentiation, and migration, neurite outgrowth, and mitochondrial function. - Hippocampal neurons in vitro screening by [CNRS (FR)]: This is an early-stage assay, under development to complement the established DNT in vitro battery (IVB), which includes assays for all the processes listed above. The DNT-IVB has gone through an initial evaluation by the OECD (OCDE, 2023). The DNT-IVB lacks endpoints for hippocampal development and function. By developing a method for differentiating hippocampal neurons from hiPSCs as a new complementary approach within the DNT-IVB framework, this new test system will enable targeted assessment of substances affecting hippocampal development and function. The hippocampus is a key brain region involved in learning and memory and a critical driver of cognitive abilities. Differentiation Process: Two hiPSC clones were successfully differentiated into hippocampal neural progenitors (hNPCs) and matured into neurons over 43 days. Key markers confirmed successful differentiation: - Neuronal Progenitor Cells marker: Nestin - Hippocampal progenitor markers: PROX1, ZBT20 - Neuronal markers: MAP2, ELAVL2, GRIK4 Preliminary in vitro screening revealed, that exposure of hNPCs to BPA, BPAP, BPE, BPZ at seven concentrations ranging from 0,001 to 100 µM (14-day exposure) resulted in concentration-dependent decrease in cell viability, The decrease was statistically significant at 100 µM for BPA, BPAP, BPZ, and from 10 µM for BPE. Subsequent experiments will study: - Cellular morphology: β-III tubulin immunocytochemistry (neurites per neuron, branch length), neuronal network formation. - Synaptic Plasticity markers: Western blot for synapsin 1, PSD95, gephyrin, synaptogenesis D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 - Gene expression and hormonal pathways: receptors for sex steroids and thyroid hormones (TH) and DNA methylation extracted from molecular analysis Table 35. Test system enabling targeted assessment of substances affecting hippocampal development and function Substance Cell viability at 0.001, 0.01, 0.1, 1 μM, 14-day incubation Neuronal morphology β-III tubulin Pre and Post synaptic markers Gene expresion analysis neurites x neuron Length Branches x neuron synapsin1 PSD95 gephyrin BPA 100 µM ongoing ongoing ongoing planned planned planned planned BPZ 100 µM ongoing Ongoing ongoing “ “ “ “ BPE 10 µM “ “ “ “ “ “ “ BPS-MAE planned Pergafast 201 BPP planned BPAP 100 µM “ “ “ “ “ “ “ TCBPA planned TBBPA • Neurosphere Assay by [IUF (DE)] This is an established high-content, high-throughput model, assessing seven key neurodevelopmental events as part of the DNT-IVB approach. This assay [Koch et al. 2022] is part of the DNT in vitro test battery (DNT-IVB) (DNTIVB; Blum et al. 2023; OECD377 2023) and evaluates seven key neurodevelopmental events using human fetal neural progenitor cells (hNPCs). Endpoints Assessed: Proliferation, differentiation, migration, neurite outgrowth, and mitochondrial function by using the following assays: - Neural progenitor proliferation (NPC1). - Radial glia, neuronal, oligodendrocyte migration (NPC2a/b/c). - Neuronal differentiation (NPC3), neurite outgrowth (NPC4), oligodendrocyte differentiation (NPC5). Experimental design and preliminary findings are summarized below, Table 36: NPC1 Assay (72h): Neurospheres exposed to 7 concentrations (20–0,027 µM). NPC2-5 Assays (72h): Neurospheres exposed to 7 concentrations (20–0,027 µM). plated on ECM for 5-day exposure. Radial glia migration (NPC2a): Inhibited by BPZ, BPAP, BPS-MAE at low micromolar concentrations. Neuronal differentiation (NPC3): Impaired at submicromolar doses for BPP and BPAP. Oligodendrocyte differentiation (NPC5): Strong disruption by BPAP (BMC30: 0.155 µM). The final classification as specific, unspecific, borderline hit is pending and will be performed after final classification (few evaluations pending). This will be added either in the final report of the BPA project (M54, October 2026) or in the 2nd Data Gaps report (M60, May 2027). Table 36. Summary of benchmark concentrations (BMCs) across all endpoints of the Neurosphere assay D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Substance BPA BPAP BPE BPP BPZ BPS-MAE TCBPA TBBPA* Cytotoxicity (NPC1) No Hit No Hit No Hit No Hit No Hit No Hit No Hit No Hit Mitochondrial activity (NPC1) No Hit No Hit No Hit No Hit No Hit No Hit No Hit No Hit NPC proliferation 72h (NPC1) No Hit No Hit No Hit No Hit No Hit No Hit No Hit No Hit Cytotoxicity 72h (NPC2-5) No Hit BMC10: 7.30µM No Hit BMC10: 3.03µM BMC10: 8.65µM No Hit BMC10: 2.43µM BMC20: 1.75µM Radial glia migration 72h (NPC2a) No Hit BMC10: 1.72µM No Hit BMC10: 3.77µM BMC10: 0.896µM BMC10: <0.027µM BMC10: 1.16µM BMC20: 1.75µM Cytotoxicity 120h (NPC2-5) No Hit BMC10: 6.11µM No Hit BMC10: 2.40µM BMC10: 6.82µM No Hit BMC10: 2.80µM BMC20: 0.63µM Mitochondrial activity 120h (NPC2-5) No Hit BMC30: 4.97µM No Hit BMC30: 3.14µM BMC30: 7.97µM No Hit BMC30: 1.94µM BMC20: 1.38µM Radial glia migration 120h (NPC2a) No Hit BMC10: 3.81µM No Hit BMC10: 1.87µM BMC10: 3.99µM No Hit BMC10: 2.89µM BMC20: 1.93µM Neuronal migration 120h (NPC2b) No Hit BMC30: 8.22µM No Hit BMC30: >2.22µM BMC30: 6.69µM No Hit BMC30: 6.16µM BMC20: 2.6µM Oligodendrocyte migration 120h (NPC2bc) No Hit BMC30: 8.56µM No Hit BMC30: >2.22µM BMC30: 15.8µM No Hit BMC30: 4.10µM BMC20: 2.23µM Neuronal differentiation 120h (NPC3) No Hit No Hit No Hit BMC30: 0.691µM BMC30: >6.66µM No Hit BMC30: 6.64µM BMC20: 2.18µM Subneurite length per nucleus 120h (NPC4b) No Hit No hit (N=1) No Hit (N=2) BMC30: >2.22µM BMC30: >6.66µM No Hit (N=2) BMC30: >6.66µM (N=1) BMC20: 2.31µM Mean neurite area without nuclei 120h (NPC4a) No Hit BMC30: 8.70µM (N=1) No Hit (N=2) BMC30: 2.27µM No Hit No Hit (N=2) BMC30: >6.66µM (N=1) BMC20: 2.49µM Oligodendrocyte differentiation 120h (NPC5) No Hit BMC30: 0.155µM No Hit BMC30: >2.22µM BMC30: >6.66µM No Hit BMC30: >6.66µM BMC20: 0.55µM The endpoint analysis was performed as a percentage of the solvent control, and different benchmark response values were used as described in Blum et al 2022 and indicated as subscript character. BMCs were calculated using the CRStats software (available at https://github.com/ArifDoenmez/CRStats), e.g., BMC10 is defined as the concentration, that reduces or increases the respective response to 90% or 110% of the solvent control. No hit indicates that the respective BMR was not reached. * TBBPA results were obtained in Klose et al 2021 using different BMRs. Classification of results in specific, unspecific and borderline hits is still pending. 3.1.5 Carcinogenicity Carcinogenicity refers to a chemical’s potential to cause cancer through genotoxic or non-genotoxic mechanisms. Given its critical implication for public health, regulatory agencies emphasize rigorous assessment of this risk (EFSA Scientific Committee; Scientific Opinion on genotoxicity testing strategies applicable to food and feed safety assessment. 2011). To ensure safety of prioritized BPA alternatives, in vitro assays are being conducted to evaluate both genotoxic and non-genotoxic carcinogenicity. The three experimental approaches planned to assess genotoxic and genotoxic carcinogenicity were developed as follows: - Genotoxicity Carcinogenicity Testing: D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 Group 1 Group 2 Group 3 Tests BPA BPZ BPE BPSMAE Pergafast 201 BPP BPAP TCBPA BPPH BPSMPE BPB BPF BPS BPAF known as Selenastrum capricornutum) (OECD TG 201) Lemna sp. Growth Inhibition Test (OECD TG 221) P X X - - X - - - - - P p - Soil organisms Effects on soil micoorganisms: Carbon transformation test (OECD TG 217 and ISO 11274) - O O O O O O - O - - - O - Effects on soil microorganisms: Nitrogen transformation test (OECD TG 216) - P P P P P P - P - - - P - Earthworm reproduction test (OECD 222) - O O - - - - - - - - - - - Amphibians In vitro assays with A6 epitheliallike cells (ECACC 89072613) of Xenopus laevis X X X X - - - - - - - - - X Behaviour toxicity assay with larvae of Xenopus laevis X X X - - - - - - - - - X Frog embryo teratogenicity assay (ASTM E1439-12) X X X X - - - - - - - - - X In vitro / high throughput assays Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay (OECD TG249) X X X X - X X X X X X X X Microtox® Acute Toxicity Test - X X X-X X X X - X X-X - X-X - X-X YES assay, in vitro screen with Saccharomyces cerevisiae X X X - - - X - - - - - X X “x” indicates that the assay was performed for the corresponding substance, in few cases, more than one partner performed the same assay with the same substance. O: on going P: planned D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 49 4.2 Results per organism - Aquatic invertebrates Three partners performed acute (immobilization) toxicity test on Daphnia magna (OECD TG 202) [BPI (EL), INERIS (FR)] and SU (SE)] and the results are presented below. There are four other partners [IISPV (ES), IEP-NRI (PL), UG-PL (PL) and UAVR (PL)] with ongoing experiments and/or experiments that will be carried out with mixtures and presented in the next deliverable. The results by BPI (EL) are presented in Table 40 as nominal and measured concentrations: Table 40. Daphnia magna acute immobilization test: The EC50 values (48h) both nominal and measured for each bisphenol alternative tested. Nominal and measured values are considered the same when they present a ±20% difference. Bisphenols from Group 3 tested EC50 range 48hrs/mg/L Nominal Measured BPB BPF BPS BPAF Red colour: values 1-10 mg/L, Green colour: values >100 mg/L Partner SU (SE) performed acute and reproduction toxicity assessment on Daphnia magna (OECD TG 202 and OECD TG 211 respectively). The results are presented in Table 41 as nominal concentrations: Table 41. Daphnia magna acute immobilization and reproduction tests: LC50 values (24 & 48h) and NOEC values (21d & 7d), respectively, for each bisphenol alternative tested. Endpoint Daphnia, survival Daphnia, reproduction Daphnia, growth LC50, 24h LC50, 48h NOEC, 21d LOEC, 21d NOEC, 7d LOEC, 7d BPA BPZ BPE BPS-MAE Not tested -- -- -- -- -- BPB Not tested -- -- -- -- -- BPF BPS BPAF Red colour: values 0.015-1 mg/L, Orange colour: values 1-10 mg/L and Green colour: values 10-100 mg/L Therefore, BPAF is the most toxic bisphenol for Daphnia magna (for both survival and reproduction), following by BPZ, while BPA, BPE and BPF are quite less toxic but in the same order of magnitude. BPS seems to be the less toxic bisphenol for Daphnia magna. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 50 Partner SLU (SE) performed reproductive toxicity assessment using the freshwater mollusk Greatpond snail (Lymnaea stagnalis) (OECD TG 243). The results are presented in Table 42 as nominal concentrations: Table 42. Lymnaea stagnalis Reproduction Test: The effects of the bisphenol alternatives tested, on fecundity and mortality Bisphenols tested BPA No effects up to 0.50 mg/L (nominal) reduced fecundity at 1.6 mg/L (nominal) BPZ No effects up to 0.59 mg/L (nominal) increased mortality at 1.9 mg/L (nominal) BPS-MAE No effects up to 0.63 mg/L (nominal) increased fecundity at 2.0 and 6.0 mg/L. (nominal) Partner SDU (DK) also performed reproductive toxicity assessment using the freshwater mollusk Great pond snail (Lymnaea stagnalis) and the results are summarised in Tables 43 and 44: Tables 43. Lymnaea stagnalis Reproduction Test results using BPE in different exposure concentrations A) BPE Embryo exposure test Exposure (µg/L) Growth Reduction (%) Rotation Speed Reduction Heartbeat Rate (bpm) Hatching Rate (%) Malformations Observed Control (0) 0 No 83.70 No 5,000 21.5 No 39.71 Yes (& slower dev.) 10,000 36.1 Yes (Day 4) 0 (Day 6) Yes (& slower dev.) A) Embryo exposure test results on growth reduction, rotation speed, heartbeat rate, hatching rate and malformations. The Hatching rate is depicted in ranges: Red color: values 0-10%, and Green color: values 50-100%. B) BPE Reproduction test Exposure (µg/L) (TWA concentr.*) Growth & Weight Gain Mortality Rate (%) Laid Egg Masses Control (0) Yes No change 4.02 Yes No change 11.80 Yes No change 47.36 Yes No change 187.32 Yes No change 872.59 Yes No change B) Reproduction test results on growth and weight gain, mortality rate and laid egg masses. The mortality rates are depicted in ranges: Green color: values 0-10% Orange color: values 10-50% *time-weighted average (TWA) concentrations C) BPE Transgenerational study Exposure (µg/L) F1 Embryo Development in Clear Water F1 Embryo Development in BPE Exposure Growth & Hatching Success Control (0) No significant change No significant change Normal 5,000+ No significant change Significant reduction Lower than controls C) Transgenerational study results on F1 embryo development and growth and hatching success D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 51 Tables 44. Lymnaea stagnalis Reproduction Test results using BPAP in different exposure concentrations A) BPAP Embryo exposure test Exposure (µg/L) Growth Reduction (%) (Day 9) Heartbeat Reduction Development Stage Reached Hatching Rate (%) (Day 18) Mortality Rate (%) Control (0) 0 No Normal 0 500 26.8 Yes Normal NS* 1,000 65.6 Yes Did not reach veliger stage 100 A) Embryo exposure test results on growth reduction, heartbeat, developmental stage, hatching rate and mortality. The Hatching rate is depicted in ranges: Green color for 50-100% Orange color for 20-50% Red color for 0-20% B) BPAP Reproduction test Exposure (µg/L) (TWE concentr.*) Egg Mass Reduction (%) BPAP 28dEC50 (µg/L) Feeding Behaviour (g/snail over 3 days) Growth & Weight Gain Survival Rate (%) BPAP 28dLC50 (µg/L) Control (0) 0 1.12 NS 16.28 59.43 NS NS 36.47 NS** NS NS 86.44 NS NS Null 217.34 NS NS Null 451.82 NS 0.013 Null B) Reproduction test results on egg mass reduction, EC50, feeding behaviour, growth and weight, survival and LC50. The Survival rate is depicted in ranges: Green color for 60-100% Orange color for 20-60% EC50 and LC50 values are depicted in ranges: Dark red color for LC50/EC50 values 10-100 mg/L and Light red color for LC50/EC50 values >100 mg/*NS: Not stated *time-weighted average (TWA) concentrations **NS: Not stated Partner Cefas (UK) performed 10-day acute sediment toxicity bioassay with the marine polychaete Arenicola marina and the results are presented in Table 45: Table 45. Arenicola marina 10-day acute sediment toxicity bioassay: EC50 casts and mortality concentrations (both nominal and verified) for all bisphenol alternatives tested. The EC50 values are depicted in ranges: Chemical EC50 Casts Nominal Conc. (mg/kg) EC50 Casts Verified Conc. (mg/kg) EC50 Mortality Nominal Conc. (mg/kg) EC50 Mortality Verified Conc. (mg/kg) BPA BPZ BPAP BPAF Red colour: values 0.5-2, D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 52 Orange colour: values 2-5 and Green colour: values 5-10 Surviving organisms were used for the following biomarkers analysis: Cellular Energy Allocation (CEA) BPA and BPAP did not significantly impact the electron transport system (ETS) in A. marina. BPZ significantly increased ETS at 1mg/kg compared to the control group (p=0.001). Exposure to 3mg/kg sediment gave a similar ETS increase although this was not statistically significantly compared to the control samples. There was a 53 and 58% decrease in ETS at 1 and 3 mg/kg BPAF respectively. The organisms exposed to BPA and the analogues had less carbohydrates than the control organisms. No significant difference was found at 1mg/kg in BPAP or BPZ (low treatment). At 3mg/kg (high treatment) of both BPAP and BPZ significantly decreased the levels of carbohydrates found in the test organisms. At the low concentration for BPA (3mg/kg) there was no significant difference relevant to the controls but did display a significant decrease in carbohydrates at the high concentration(10mg/kg) BPA (p=0.04). The organisms exposed to BPAF showed significant decrease in carbohydrate availability at both 1 (low) and 3 (high) mg/kg . Overall, none of the chemicals tested showed any significant impact to protein or lipid content in A. marina compared to the control organisms. Glutathione S-transferase (GST) Surviving A. marina in the BPA and BPZ spiked sediment had an increased production of GST on average compared to the individuals in the control sediment. However, this increase was not significantly different from the controls. BPAF did cause significant increase in GST production compared to the controls. BPAP also affected GST production, with a significant increase at 1mg/kg (low) and 3 mg/kg relative to the control. Partner Cefas (UK) also performed the 42-day chronic toxicity test using the freshwater amphipod Hyalella azteca, spiking sediment with BPAF and the results are presented in Table 46: Table 46. The 42-day chronic toxicity test on Hyalella azteca exposed to different BPAF concentrations: Verified concentrations and % survival are presented Nominal Conc. of BPAF (mg/kg) Verified Conc. (mg/kg) Survival (%) 0 0 0,03 <LOQ 0,1 0,03 0,3 0,18 1 0,7 3 2,58 Green colour: Survival 83-100% The results indicate that BPAF caused no significant effect on Hyalella Azteca survival. In addition, it did not result on any statistically significant effect on the number of nauplii per surviving female and final dry weight per individual. The partner UAVR (PT) performed the Regeneration assay - freshwater planarian Girardia tigrine. The results are summarised in Table 47: Table 47. The Regeneration assay on G. tigrina after several bisphenol alternatives exposure: The blastema, photoreceptor and auricle regeneration are presented along with the mortality rate Bisphenols tested Concentration (mg/L) Blastema Regeneration Photoreceptor Regeneration Auricle Regeneration Mortality (%) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 53 BPA 0,04 Not reported Not reported 1 Only 2 planarians regenerated 2,5 5 BPZ 0,16 0,4 1 Regenerated initially, then died Not evaluated Not evaluated 2,5 Not evaluated Not evaluated BPS-MAE 2,5 BPAF 0,4 1 Not evaluated Not evaluated 2,5 Not evaluated Not evaluated The mortality rate is depicted in ranges: Green colour: 0-40% and Red colour: 40-100%. The regeneration rate is depicted in ranges: Green colour: increased regeneration, Light green: not affected, Yellow: slightly delayed, Orange: reduced or delayed, slightly 0-40% and Red colour: 40-100% The partner INERIS (FR) was in charge of assessing the biological effects of several bisphenols in order to fill data gaps regarding their potential endocrine activities in Daphnia magna using recently adopted OECD test guidelines for endocrine activity, i.e. TG 253 - Short-term Juvenile Hormone Activity Screening Assay using Daphnia magna (JHASA). As a first step, the toxicity of the selected bisphenols was assessed in Daphnia using OECD TG 202 and in zebrafish embryo using a refined zebrafish embryo toxicity (FET) assay (OECD TG 236 performed on tg(cyp19a1b:GFP) embryos). This first step allowed to define non-toxic concentrations of bisphenols to test in TG 253 and 250. The summarized results are the following: BPA, BPB, BPF and BPS have been evaluated in OECD TG 202 and TG 253. The toxicity of BPA in TG 202 was 14 mg/L. The toxicity of bisphenol substitutes ranged from 8 mg/L (BPB) to 81 mg/L (BPS). In TG 253, BPA and BPB were inactive. BPF and BPS induced no change in the sex-ratio of juveniles (no male production). BPS induced the production of aborted eggs while BPF inhibited the molting of females and the production of the second brood. - Zebrafish The partner NIB (SI), BPI (EL) and other partners as indicated below performed fish acute toxicity tests according to the OECD TG 236. Lethal effects, calculated LC50 values for the tested bisphenols are presented in Table 48. Table 48. The Fish Embryo Acute Toxicity (FET) Test: The nominal LC50 values after 96hrs of several bisphenol alternatives exposure is presented D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 54 Bisphenols tested LC50 96hrs/mg/L (SE) Nominal BPA BPAP BPPH BPB BPF BPS BPAF The nominal EC50 values are presented in ranges: Red colour: values 1-10 mg/L; Orange colour: values 11-100 mg/L Green colour: values >100 mg/L The partner UAVR (PT) tested specific bisphenols as indicated in the table in Annex A.4 with OECD TG 236 (FET test). Preliminary results of the FET indicate that most alternatives tested were more toxic than BPA, with lower lethal concentrations (LC50) values and lower effect concentrations for malformations (both EC10 and EC50). Although the high concentrations tested may not be environmentally realistic due to the compounds’ low solubility, these findings highlight the critical need for continued efforts to identify truly safer BPA alternatives. The effects of BPA and alternative compounds were also evaluated through a complementary behavioural approach and results are still being analysed. The partner INERIS (FR) was in charge of assessing the biological effects of several bisphenols in order to fill data gaps regarding their potential endocrine activities in zebrafish embryo models using recently adopted OECD test guidelines for endocrine activity, i.e. TG N°250 - Detection of Endocrine Active Substances, acting through estrogen receptors, using transgenic tg(cyp19a1b:GFP) Zebrafish embrYos (EASZY assay). The summarized results are the following: BPA and 14 bisphenols substitutes were tested using the refined OCDE TG N°236 and the TG N°250. The list of bisphenol substitutes tested is: BPA, BPB, BPE, BPC, BPC-Cl, BPF, BPS, BPS-MAE, BPS-MPE, BPZ, TCBPA, BPAF, BPAP, 4-4’ODP 9 BPA substitutes were more toxic than BPA in the refined FET assay. The LC50 for BPA was 14 mg/L and the toxicity of the substitutes range from 0.8 mg/L (TCBPA) to >100 mg/L (BPS) At non-toxic concentrations, 14 bisphenols were assessed for their potential estrogenic activities in the EASZY assay. BPA and 13 substitutes tested elicited an estrogenic activity in the EASZY while TCBPA was inactive. Among the active substances, nine elicited stronger estrogenic activity than BPA. The EC50 for BPA was 0.4 mg/L while for bisphenols substitutes EC50 ranged from 0.0047 mg/L to 34 mg/L. The toxicity and potential estrogenic activity of BPP was not evaluated yet. As regards partner INERIS (FR) it was initially planned to assess the endocrine and reproductive effect of one bisphenol substitute in the OECD TG N°229 but it was not possible to perform this experiment due to “human factors” beyond partener’s control. In place of performing the TGN°229, they assessed the metabolic endocrine activity of some bisphenol substitutes in a novel zebrafish embryo assay, called EMERGE (Effect of metabolic endocrine disruptors in the Gut of zebrafish embryos) which allow to evaluate the effect of chemicals on the intestinal expression of the cyp3a65 gene. The EMERGE assay is not endorsed by OECD and was developed within the Horizon EU project OBERON. In EMERGE, BPA and several bisphenols were able to induce the expression of the cyp3a65 gene in the developing intestine revealing their potential metabolic endocrine disrupting effect D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 55 The partner SDU (SE) was involved in the investigation of the effects of Bisphenol A (BPA)-alternatives like BPE, BPAP, and BPS-MAE and associated mixtures in the model animals, zebrafish (Danio rerio). They investigate possible adversity for endpoints such as heart rate, growth (larvae length), hatching rate, inflation of the posterior swimbladder chamber, eye development (eye size), development of retina structures such as retinal pigmentary layer (RPE), and inner plexiform layer (IPL), development of the brain (brain size) and associated swimming performance in a light/dark transition test (LDTT). They exposed zebrafish larvae to BPAP, BPE, and BPS-MAE and the summarized results are indicated in Table 49: Table 49. Zebrafish exposure to BPE, BPS-MAE and BPAP: LOEC values for several endpoints are presented Endpoint BPE LOEC BPS-MAE LOEC BPAP LOEC Larvae length decreased (mm) 5 days Heart rate decreased 48 hpf (bpm) N/A Heart rate decreased 96 hpf (bpm) N/A Heart rate increased 48 hpf (bpm) N/A N/A Heart rate increased 96 hpf (bpm) N/A N/A Hatching rate (%) a Swimbladder inflation a a a Swimming performance changed (dark) Swimming performance changed (light) Eye size (diameter µm^2) a Retinal pigmentary layer (RPE) a a Inner plexiform layer (IPL) a a Brain size 5 days a a a The LOEC values are presented in ranges: Green: >1500 µg/L Yellow: 500-1500 µg/L Orange: 51-500 µg/L Red: 5-50 μg/L (a; experiments in progress, N/A not applicable) - Alga / Aquatic plants The partner BPI (EL) performed alga growth Inhibition tests and the results are shown in Table 50. Table 50. The Alga growth inhibition test with BPF and BPS: The EC50 values, measured after 72 hours are presented. The nominal values are compared to the measured. Bisphenols tested EC50 72hrs/mg/L (SE) Nominal Measured BPF BPS The EC50 values are presented in ranges: Red colour: values 10-50 mg/L Green colour: values 50-120 mg/L D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 56 The partner SU (SE) performed Alga Inhibition tests with the following results as presented in Table 51 in nominal concentrations: Table 51. The Alga growth inhibition test with several bisphenol alternatives: The nominal EC50 values, measured after 72 hours are presented Bisphenols tested Nominal EC50 values BPA BPF BPS BPAF The EC50 values are presented in ranges: Red colour: values 1-10 mg/L Green colour: values 10-30 mg/L The more toxic bisphenol was BPAF, following by BPF and BPA. As regards BPS a difference between the results derived by two partners was observed. This difference may be caused by different solvents used by each lab and the fact that the endpoints by SU was based on nominal and not measured concentration. Nevertheless, further discussion should be carried out in order to interpretate these results The partner IEP-NRI performed growth inhibition test on Lemna and the results are shown in Table 52. Table 52. The L. stagnalis inhibition test using bisphenol alternatives Bisphenols tested EC50 mg/L Nominal frond number (f.n) frond area (f.a) BPZ BPE BPP The nominal EC50 values are presented in ranges: Red colour: values 1-10 mg/L and Green colour: values >10 mg/L - Soil organisms The partner BPI (EL) performs earthworm reproduction tests to assess the reproduction toxicity of BPZ and BPE on the earthworm Eisenia foetida (OECD TG 222). The LD50 values for BPZ is > 250 mg kg/dry soil, while the tests with BPE and the reproduction phase of the test with both bisphenols are ongoing. The partner IISPV (ES) investigated the effects of bisphenols on soil microorganisms (OECD TG 217 and TG 216), but the tests are still ongoing. - Amphibians The partner UAVR (PT) investigated the possible effects of bisphenols in in vitro and in vivo amphibian models and the results are presented in Table 53: Table 53. in vitro (A6 cell line) and in vivo amphibian exposure to several bisphenol alternatives: 72hLC50 values (for in vitro protocols) and 96h LC50 values along with 24h EC50 values (in vivo protocols) are presented Bisphenols tested In vitro 72hLC50 mg/L (A6 cell line) In vivo 96hLC50 mg/L 24hEC50 mg/L for malformations D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 57 BPA Not reported BPS-MAE BPZ Not reported BPE BPAF (24h preliminary) BPS NA (no activity ) BPS-4-allyl ether NA (no activity ) The LC50 and EC50 values are presented in ranges: Red colour: values 1-10 mg/L Orange colour: values 10-50 mg/L In vitro assays results suggest BPA to be more toxic to A6 cell line than the other tested bisphenols. For BPAF, preliminary assays using the A6 cell line indicate that this cell-based model is slightly more sensitive to BPAF than to BPA. In vivo definitive assays show that X. laevis embryos are more sensitive to BPAF than to BPA (available published data and preliminary assays). Preliminary data for BPZ showed a similar toxicity trend of higher toxicity than BPA. - In vitro / high throughput assays The partner BPI (EL), IEP-NRI (PL) and IISPV (ES) performed microtox tests with the following results for 5, 15 and 30 min of exposure. There was not a remarkable difference in LC50 comparing the different times and the results derived by the three partners are presented in Table 54. Table 54. The Microtox® Acute Toxicity Test using several bisphenol alternatives: The 15 min EC50 values. The nominal values are compared to the measured. Nominal and measured values are considered the same when they present ±20% difference. A) BPI (EL) results B) IEP-NRI (PL) results and C) IISPV (ES) results A) Bisphenols tested EC50 15min - mg/L Nominal* Measured BPAF BPF BPS-MPE BPAP BPP BPE BPZ *Nominal and measured values are considered the same when the present a maximum ±20% difference B) Bisphenols tested EC50 30 min mg/L Nominal BPAF BPF BPS-MAE BPS-MPE BPPH D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 64 Yvonne Kohl, Maria João Silva, Hubert Dirven, Jessica Dietrich, Doris Marko A Short-Term Exposure to Saxitoxin Triggers a Multitude of Deleterious Effects in Daphnia magna at Environmentally Relevant Levels Albano Pinto1,2, Inês P. E. Macário1,2, Sérgio M. Marques1,2, Maria João Botelho3,4, Jana Asselman5, Patrícia Pereira1,2, Joana L. Pereira1,2 Peer reviewed publications Zebra-K, a kinematic analysis automated platform for assessing the sensitivity, habituation and prepulse inhibition of acoustic startle in adult zebrafish Main author: Demetrio Raldúa Co-authors: Marija Stevanović, Niki Tagkalidou, Cristiana Roberta Multisanti, Sergi Pujol, Ouwais Aljabasini, Eva Prats, Caterina Faggio, Josep Maria Porta, Peer reviewed publications Hazard characterisation of the mycotoxins enniatins and beauvericin to identify data gaps and improve risk assessment for human health Anne-Cathrin Behr, Christiane Kruse Fæste, Amaya Azqueta, Ana M. Tavares, Anastasia Spyropoulou, Anita Solhaug, Ariane Vettorazzi, Ann-Karin Olsen, Birgit Mertens, Bojana Zegura, Camille Streel, , Dieynaba Ndiaye, Eliana Spilioti, Estelle Dubreil, Franca Maria Buratti, Francesco Crudo, Gunnar Sundstøl Eriksen, Igor Snapkow, João Paulo Teixeira, Josef D. Rasinger, Julie Sanders, Kyriaki Machera, Lada Ivanova, Laurent Gaté, Ludovic Le Hegarat, Matjaz Novak, Nicola M. Smith, Sabrina Tait, Sónia Fraga, Sonja Hager, Albert Braeuning, Henriqueta Louro, Maria João Silva, Hubert Dirven, Jessica Dietrich Peer reviewed publications Unrevealing immunotoxic effects of bisphenol A substitutes on human macrophages, T and B lymphocytes using in vitro models Nina Franko, Tijana Markovič, Pia Žižek, Anja Kodila, Irena Mlinarič Raščan, Marija Sollner Dolenc Peer reviewed publications Motor and non-motor effects of acute MPTP in adult zebrafish: insights into Parkinson’s disease Main author: Demetrio Raldúa Co-authors:Marija Stevanović, Niki Tagkalidou, Irene Romero-Alfano, Gustavo Axel ElizaldeVelázquez, Selene Elizabeth Herrera-Vázquez, Eva Prats, Cristian Gómez-Canela, Leobardo Manuel Gómez-Oliván Peer reviewed publications Temperature dependent sensitivity of the harpacticoid copepod Nitokra spinipes to marine algal toxins Wenxin Liu, Ilias Semmouri, Colin Janssen, Jana Asselman Peer reviewed publication (uploadingdata to zenodot) Temperature and salinity affect growth and toxin content of cyanobacterium Microcystis aeruginosa (PCC 7806) in estuarine environments Wenxin Liu, Ilias Semmouri, Colin Janssen, Jana Asselman Peer reviewed publication (under revision) Unlocking nature's arsenal: discovery of the Alternaria mycotoxins alterperylenol and altertoxin I as novel immunosuppressive and antiestrogenic compounds in vitro Francesco Crudo, Vanessa Partsch, Dennis Braga, Ruzica Blažević, Judith M. Rollinger, Elisabeth Varga, Doris Marko Peer reviewed presentation (data available on demand) Effects of binary mixtures of cyanotoxins and xenobiotics on the growth rate of the freshwater algae Chlorella vulgaris Carlos Pinheiro, Joana Azevedo, Alexandre Campos, Vítor Vasconcelos, Susana Loureiro Attendance in international and national events (conference, congress, workshop, etc) Closing data gaps on natural toxins effect on human health: Immunotoxic effects of Alternaria toxins using reporter cell lines and a 3D lung exposure model. Solveig Krapf, Anne Straumfors, Steen Mollerup Attendance in international and national events (conference, congress, workshop, etc) Towards next generation risk assessment for Environmental Health: how PARC Work Package 5 is contributing Celia Garcia Arenas, Kiara Aiello Holden, Thalia de Castelbajac, Ondrej Adamovsky, Katerina Kyriakopoulou, Jana Asselman, Gilles Riviere, Philip Marx-Stölting Attendance in international and national events (conference, congress, workshop, etc) Innate immune responses of Alternaria toxins in vitro: Receptor activation, inflammation induction, and signal transduction Booshra Ahmed, Solveig Krapf, Anne Straumfors, Paal Graff, Steen Mollerup. Attendance in international and national events D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 65 (conference, congress, workshop, etc) Closing data gaps on natural toxins effect on human health: Immunotoxic effects of Alternaria toxins using a co-culture lung exposure model Solveig Krapf, Anne Straumfors, Steen Mollerup National Institute of Occupational Health (STAMI) Gydas vei 8, 0333 Oslo, Norway" Attendance in international and national events (conference, congress, workshop, etc) Immunomodulating effects of Alternaria toxins activation on the toll like receptor – NFkB/AP-1 signalling pathway Solveig Krapf, Anne Straumfors, Steen Mollerup. Attendance in international and national events (conference, congress, workshop, etc): poster Investigating the genotoxic effects of the Alternaria toxin Tenuazonic Acid in human liver cells Beatriz Guerreiro, Célia Ventura, Henriqueta Louro, Maria João Silva Attendance in international and national events (conference, congress, workshop, etc): Poster METABOLISM OF ENNIATIN B IN PRIMARY MOUSE, RAT AND HUMAN HEPATOCYTES Estelle DUBREIL, Lada IVANOVA, Charlène GENDRE, Mariam MADJHOUB, Valérie FESSARD, Ludovic LE HEGARAT, Christiane K. FAESTE, Jérôme HENRI Attendance in international and national events (conference, congress, workshop, etc): Poster Steroidogenesis assay (OECD TG 456) to fill data gap on BPA alternatives and on the natural mycotoxins Enniatins and Beuvericin: preliminary results from two PARC projects Lucia Coppola, Gabriele Lori, Elena Bossù, Livia Manna, Daniele Sadutto, Sabrina Tait Attendance in international and national events (conference, congress, workshop, etc): Poster Contribution to the characterisation of Alternaria toxins’ genotoxicity in human liver cells B. Guerreiro, C. Ventura, H. Louro, M. J. Silva Attendance in international and national events (conference, congress, workshop, etc): Poster Influence of Temperature on Acute and Chronic Toxicity of Marine Algal Toxins — A Case Study with Copepod Nitokra spinipes Wenxin Liu, Ilias Semmouri, Colin Janssen, Jana Asselman Attendance in international and national events (conference, congress, workshop, etc): Poster Acute Toxicity of Harmful Algae on Marine Zooplankton in the Context of Climate Change Wenxin Liu1, Suzanne Bulckaert, Ilias Semmouri, Colin Janssen, Jana Asselman Attendance in international and national events (conference, congress, workshop, etc): Poster Genotoxicity assessment of enniatins and Alternaria toxins with the in vitro micronucleus assay and the SOS/umu test Streel Camille, Vettorazzi Ariane, Sanders Julie, Anthonissen Roel, Azqueta Amaya and Mertens Birgit Attendance in international and national events (conference, congress, workshop, etc): Poster Closing regulatory data gaps on the genotoxicity and non-genotoxic carcinogenesis of natural toxins and bisphenols Maria João Silva, Kiara Aiello-Holden, Doris Marko, Jessica Dietrich, Henriqueta Louro, Hubert Dirven Attendance in international and national events (conference, congress, workshop, etc) presentation Genotoxicity assessment and potency ranking of enniatins and Alternaria toxins with the in vitro micronucleus assay Streel Camille, Sanders Julie, Anthonissen Roel and Mertens Birgit Attendance in international and D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 66 national events (conference, congress, workshop, etc) INTERSPECIES VARIATIONS IN THE METABOLISM OF ALTENUENE AND TENTOXIN IN PRIMARY HEPATOCYTES Eszter Borsos, Charlène Gendre, Elisabeth Varga, Estelle Dubreil, Jérôme Henri, Ludovic Le Hegarat, Doris Marko Attendance in international and national events (conference, congress, workshop, etc) - poster Mycotoxin Metabolism Matters: Unlocking the Immunotoxicity of Enniatins and Beauvericin Dino Grgic, Ibrahim Elesh, Vanessa Partsch, Francesco Crudo, Sonja Hager, Doris Marko Attendance in international and national events (conference, congress, workshop, etc) - to be decided (poster or presentation) Assessing the effects of phycotoxin mixtures on marine zooplankton: insights from copepod responses at different life stages Ilias Semmouri, Luca Deroma, Colin R. Janssen, Jana Asselman Attendance in international and national events (conference, congress, workshop, etc) Table 58: Scientific outputs of T5.1. regarding BPA alternatives Title Co-authors Type of dissemination Unrevealing immunotoxic effects of bisphenol A substitutes on human macrophages, T and B lymphocytes using in vitro models Nina Franko, Tijana Markovič, Pia Žižek, Anja Kodila, Irena Mlinarič Raščan, Marija Sollner Dolenc Peer reviewed publications Temperature and salinity affect growth and toxin production of cyanobacterium Microcystis aeruginosa (PCC 7806) in estuarine environments Wenxin Liu, Ilias Semmouri, Colin Janssen, Jana Asselman Peer reviewed publications Endocrine disruption, adverse outcomes and occurrence of newly emerging BPA substitutes Nina Franko, Anja Kodila, Marija Sollner Dolenc Peer reviewed publications Endocrine disrupting toxicity of bisphenol A and its analogs: impact on the neuro-immune system Erica Buoso, Mirco Masi, Roberta Limosani, Chiara Oliviero, Martina Iulini, Francesca Passoni, Marco Racchi and Emanuela Corsini Peer reviewed publications Comprehensive Toxicity Scoring of 26 BPA Alternatives Based on Eight Cell-Based Bioassays and Abiotic CYP Oxidation Test V. Srebny, L. Henneberger, J.Huchthausen, M. König, S. Mälzer, and B. I. Escher Peer reviewed publications Which in vitro liver model suits best for predictive toxicological research: A comparison Yvonne Kohl, Gizem Erdogan, Sylvia Wagner Attendance in international and national events (conference, congress, workshop, etc): Poster Temperature and salinity affect growth and toxin production of estuarine cyanobacterium Microcystis aeruginosa Wenxin Liu, Ilias Semmouri, Colin Janssen, Jana Asselman Attendance in international and national events (conference, congress, workshop, etc) - presentation Studio in vitro del bisfenolo A e dei suoi analoghi nella regolazione della risposta immunitaria Francesca Carlotta Passoni, Martina Iulini, Valeria Bettinsoli, Valentina Galbiati, Marina Marinovich, Emanuela Corsini Attendance in international and national events (conference, congress, workshop, etc) Linking chemical disruption of molting processes to survival of crustaceans Knut Erik Tollefsen, Bjørn Henrik Hansen, Simon Schmid, You Song, Celine Vågå, Li Xie. Attendance in international and national events (conference, congress, workshop, etc) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 67 Is it safer? Evaluating the toxicity of Bisphenol A Alternatives in Zebrafish Embryos Maria S. Costa, Marta S. Monteiro, Susana Loureiro, Inês Domingues Attendance in international and national events (conference, congress, workshop, etc) In vitro effects of BPA and its analogues on antibody production Martina Iulini Attendance in international and national events (conference, congress, workshop, etc): Presentation In vitro effects of bisphenol A analogues on human B and T lymphocytes activation F. C. Passoni, A. Maddalon, V. Bettinsoli, V. Galbiati, and E. Corsini. Università degli Studi di Milano, Milan, Italy. Attendance in international and national events (conference, congress, workshop, etc) Impact of endocrine disruption on the number of hair cells in neuromasts of the lateral line organ of zebrafish larvae (Danio rerio) Ellen Vandeputte Evelyn Stinckens, Lucia Vergauwen, Dries Knapen Attendance in international and national events (conference, congress, workshop, etc) Impact of endocrine disruption on the number of hair cells in neuromasts of the lateral line organ of zebrafish larvae Ellen Vandeputte, Evelyn Stinckens, Lucia Vergauwen, Dries Knapen Attendance in international and national events (conference, congress, workshop, etc): poster Impact of endocrine disruption on the number of hair cells in neuromasts of the lateral line organ of zebrafish larvae Ellen Vandeputte, Evelyn Stinckens, Lucia Vergauwen, Dries Knapen Attendance in international and national events (conference, congress, workshop, etc) presentation IDO-1 activation is a key event, driving immunotoxicity Lucia Parrakova, Oriol Ruiz Catalan, Pablo Monfort-Lanzas, Dietmar Fuchs, Johanna M Gostner Attendance in international and national events (conference, congress, workshop, etc) ENDOCRINE DISRUPTION IN DAPHNIA MAGNA: FROM MOLECULAR TO POPULATION LEVELS MADALENA VIEIRA , MARIA PAVLAKI , SUSANA LOUREIRO Attendance in international and national events (conference, congress, workshop, etc) Effects of newly emerging bisphenols on Tcell activation Nina Franko, Marco Bertolli, Anja Kodila, Emanuela Corsini and Marija Sollner Dolenc Attendance in international and national events (conference, congress, workshop, etc) Effects of Bisphenols to Amphibians and Fish: An Integrative Approach Barreto M., Costa M.S., Vilarinho I., Marques B., Quintaneiro C., Monteiro M.S., Domingues I., Oliveira M. and Lopes I. Attendance in international and national events (conference, congress, workshop, etc) Ecotoxicological Evaluation of Bisphenol A and alternatives: A Comprehensive in silico Modelling Approach Liadys Mora Lagares and Marjan Vračko Attendance in international and national events (conference, congress, workshop, etc) Contribution to the hazard assessment of substances alternative to bisphenol A: genotoxic and carcinogenic effects in mammalian cells Pereira M., Tavares A., Louro, H., Silva M.J. Attendance in international and national events (conference, congress, workshop, etc): Poster Comparative Toxicity Assessment of Bisphenol A and Its Analogues (BPAF and BPZ) Using Zebrafish ( Danio rerio ) Embryos and Larvae as Model Organisms Maria S. Costa, Inês Vilarinho, Marta S. Monteiro, Isabel Lopes and Inês Domingues Attendance in international and national events (conference, congress, workshop, etc) - poster Closing regulatory data gaps on the genotoxicity and non-genotoxic carcinogenesis of natural toxins and bisphenols Maria João Silva, Kiara Aiello-Holden, Doris Marko, Jessica Dietrich, Henriqueta Louro, Hubert Dirven Attendance in international and national events (conference, congress, workshop, etc) presentation BPA to Z: Harnessing Bioanalytical Tools for the Development of Hazard-Free Chemicals "V. Srebny, L. Henneberger, J.Huchthausen, M. König, S. Mälzer, and B. I. Escher Department Cell Toxicology, Attendance in international and national events (conference, congress, workshop, etc): Presentation D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 68 Helmholtz Centre for Environmental Research – UFZ Assessing the effects of phycotoxin mixtures on marine zooplankton: insights from copepod responses at different life stages Wenxin Liu, Ilias Semmouri, Luca Deroma, Colin Janssen, Jana Asselman Attendance in international and national events (conference, congress, workshop, etc) - poster Advancing the understanding of the life-stage specific impact of thyroid hormone system disruption in the zebrafish embryo model Imke Van Dingenen, Lucia Vergauwen, Dries Knapen Attendance in international and national events (conference, congress, workshop, etc): poster Interactions with regulators Ongoing monthly meetings with the Task/Activity Leaders 5.1.1, Project Managers, Endpoint leaders and partners. Our project reviewers are mostly experts from EFSA & ECHA and to have a better overview and understanding of our work they are joining our monthly meetings. The participants are as follow: Regulatory Agencies: ECHA, EFSA, EEA are welcome to join. So far, only experts from EFSA/ECHA have joined our monthly meetings. Project Managers of P5.1.1.a Natural toxins have been invited to the EFSA focus group on Alternaria, to share preliminary results. A5.1.1 Activity Leaders [NIPH and INSA] References Aiello Holden K, Zalko D, De Castelbajac T, et al. Additional Deliverable AD5.1 List of prioritized BPA alternatives for WP5 Hazard Assessment activities Workshop Report WP5, 2023, https://www.euparc.eu/sites/default/files/2023-08/PARC_AD5.1.pdf Alonso-Jauregui M, Font M, González-Peñas E, López de Cerain A, Vettorazzi A. Prioritization of Mycotoxins Based on Their Genotoxic Potential with an In Silico-In Vitro Strategy. Toxins (Basel). 2021 Oct 19;13(10):734. doi: 10.3390/toxins13100734 ASTM E1439-12 - Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) English language, DOI: 10.1520/E1439-12 Behr, AC., Fæste, C.K., Azqueta, A. et al. Hazard characterization of the mycotoxins enniatins and beauvericin to identify data gaps and improve risk assessment for human health. Arch Toxicol (2025). https://doi.org/10.1007/s00204-025-03988-3 Blum, J., Masjosthusmann, S., Bartmann, K. et al. (2022). Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals. Chemosphere 311, 137035. doi:10.1016/j.chemosphere.2022.137035 Blum J, Masjosthusmann S, Bartmann K, Bendt F, Dolde X, Dönmez A, Förster N, Holzer AK, Hübenthal U, Keßel HE, Kilic S, Klose J, Pahl M, Stürzl LC, Mangas I, Terron A, Crofton KM, Scholze M, Mosig A, Leist M, Fritsche E. Establishment of a human cell-based in vitro battery to assess developmental neurotoxicity hazard of chemicals. Chemosphere. 2023 Jan;311(Pt 2):137035. doi: 10.1016/j.chemosphere.2022.137035. Epub 2022 Oct 31. PMID: 36328314. CRStats software available at https://github.com/ArifDoenmez/CRStats Dellafiora L, Warth B, Schmidt V, Del Favero G, Mikula H, Fröhlich J, Marko D. An integrated in silico/in vitro approach to assess the xenoestrogenic potential of Alternaria mycotoxins and D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 69 metabolites. Food Chem. 2018 May 15;248:253-261. doi: 10.1016/j.foodchem.2017.12.013. Epub 2017 Dec 7. PMID: 29329852. EFSA Scientific Committee; Scientific Opinion on genotoxicity testing strategies applicable to food and feed safety assessment. EFSA Journal 2011; 9(9):2379. [69 pp.] doi:10.2903/j.efsa.2011.2379. EFSA (2024), EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), Knutsen, H. K., Åkesson, A., Bampidis, V., Bodin, L., Chipman, J. K., Degen, G., Hernández-Jerez, A., Hofer, T., Hogstrand, C., Landi, S., Leblanc, J.- C., Machera, K., Ntzani, E., Rychen, G., Sand, S., Schwerdtle, T., Vejdovszky, K., Viviani, B., … Bignami, M. (2024). Genotoxicity of beauvericin. EFSA Journal, 22(10), e9031. https://doi.org/10.2903/j.efsa.2024.9031 Gobler, J. Christopher. Climate Change and Harmful Algal Blooms: Insights and perspective, Harmful Algae, Volume 91, 2020, 101731, ISSN 1568-9883, https://doi.org/10.1016/j.hal.2019.101731 nternational Organization for Standardization. (1999). Water quality — Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) (ISO Standard No. 14669). Retrieved from https://www.iso.org/standard/25162.html#lifecycle International Organization for Standardization. (2016). Water quality — Larval development test with the harpacticoid copepod Nitocra spinipes (ISO Standard No. 18220). Retrieved from https://www.iso.org/standard/61809.html International Organization for Standardization. (2019). Soil quality — Determination of the water-retention characteristic — Laboratory methods (ISO Standard No. 11274). Retrieved from https://www.iso.org/standard/68256.html Koch K, Bartmann K, Hartmann J, Kapr J, Klose J, Kuchovská E, Pahl M, Schlüppmann K, Zühr E and Fritsche E (2022) Scientific Validation of Human Neurosphere Assays for Developmental Neurotoxicity Evaluation. Front. Toxicol. 4:816370. doi: 10.3389/ftox.2022.816370 Kollarova J, Cenk E, Schmutz C, Marko D. The mycotoxin alternariol suppresses lipopolysaccharideinduced inflammation in THP-1 derived macrophages targeting the NF-κB signalling pathway. Arch Toxicol. 2018 Nov;92(11):3347-3358. doi: 10.1007/s00204-018-2299-4. Epub 2018 Sep 3. PMID: 30175388; PMCID: PMC6208963. Komatsu, H. et al. (2021), “Adverse Outcome Pathway on inhibition of calcineurin activity leading to impaired Tcell dependent antibody response”, OECD Series on Adverse Outcome Pathways, No. 18, OECD Publishing, Paris, https://doi.org/10.1787/3c988dde-en. Louro, H., Vettorazzi, A., López de Cerain, A., et al. Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health. Archives of Toxicology, 98(2), 425-469 (2024), https://doi.org/10.1007/s00204-023-03636-8 Luckert, C., Braeuning, A., Lampen, A., & Hessel-Pras, S. (2018). PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids. Chemico-Biological Interactions, 288, 38-48, https://doi.org/10.1016/j.cbi.2018.04.017 Marx-Stoelting, P., Rivière, G., Luijten, M. et al. A walk in the PARC: developing and implementing 21st century chemical risk assessment in Europe. Arch Toxicol 97, 893–908 (2023). https://doi.org/10.1007/s00204-02203435-7 Mhaouty-Kodja S, Zalko D, Tait S, et al. A critical review to identify data gaps and improve risk assessment of bisphenol A alternatives for human health. Crit Rev Toxicol. 2024 Nov;54(10):696-753. doi: 10.1080/10408444.2024.2388712 OECD (2000), Test No. 216: Soil Microorganisms: Nitrogen Transformation Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264070226-en OECD (2000), Test No. 217: Soil Microorganisms: Carbon Transformation Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264070240-en D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 70 OECD (2004), Test No. 202: Daphnia sp. Acute Immobilisation Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264069947-en OECD (2006), Test No. 221: Lemna sp. Growth Inhibition Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264016194-en OECD (2009), Test No. 231: Amphibian Metamorphosis Assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264076242-en OECD (2011), Test No. 201: Freshwater Alga and Cyanobacteria, Growth Inhibition Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264069923-en OECD (2012), Test No. 211: Daphnia magna Reproduction Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264185203-en OECD (2012), Test No. 229: Fish Short Term Reproduction Assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264185265-en OECD (2013), Test No. 236: Fish Embryo Acute Toxicity (FET) Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264203709-en OECD (2016), Test No. 222: Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei), OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264264496-en. OECD (2016), Test No. 243: Lymnaea stagnalis Reproduction Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264264335-en OECD (2017), Guidance Document On The In Vitro Bhas 42 Cell Transformation Assay. OECD Environment, Health and Safety Publications. Series on Testing & Assessment, No. 231. ENV/JM/MONO(2016)1. Available at: https://one.oecd.org/document/ENV/JM/MONO(2016)1/en/pdf OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264071247-en OECD (2021), Test No. 249: Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/c66d5190-en OECD (2021), Test No. 250: EASZY assay - Detection of Endocrine Active Substances, acting through estrogen receptors, using transgenic tg(cyp19a1b:GFP) Zebrafish embrYos, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/0a39b48b-en. OECD (2021), Test No. 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264265295-en. OECD (2022), Detailed Review Paper on In Vitro Test Addressing Immunotoxicity With a Focus on Immunosuppression, OECD Series on Testing and Assessment, No. 360, OECD Publishing, Paris, https://doi.org/10.1787/667965bc-en. OECD (2023), Initial Recommendations on Evaluation of Data from the Developmental Neurotoxicity (DNT) In-Vitro Testing Battery, OECD Series on Testing and Assessment, No. 377, OECD Publishing, Paris, https://doi.org/10.1787/91964ef3-en OECD (2023a), Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264861-en. OECD (2023b), Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264366-en. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 71 OECD (2023c), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264122642-en. OECD (2023d), Test No. 444A: In Vitro Immunotoxicity: IL-2 Luc Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/27b10ba3-en. OECD (2023), Test No. 487: In Vitro Mammalian Cell Micronucleus Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264264861-en. OECD (2024), Test No. 253: Short-term Juvenile Hormone Activity Screening Assay using Daphnia magna (JHASA), OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/03cb5c08-en. Ondrej Adamovsky, Ksenia J. Groh, Anna Białk-Bielińska, et al. Exploring BPA alternatives – Environmental levels and toxicity review. Environment International, Volume 189, 2024, https://doi.org/10.1016/j.envint.2024.108728. Pavicich MA, Nielsen KF, Patriarca A. Morphological and chemical characterization of Alternaria populations from apple fruit. Int J Food Microbiol. 2022 Oct 16;379:109842. doi: 10.1016/j.ijfoodmicro.2022.109842. Epub 2022 Jul 20. PMID: 35878437. Reifferscheid G, Heil J, Oda Y, Zahn RK. A microplate version of the SOS/umu-test for rapid detection of genotoxins and genotoxic potentials of environmental samples. Mutat Res. 1991 Dec;253(3):215-22. doi: 10.1016/01651161(91)90134-t. Sakai A, Sasaki K, Hayashi K, et al. An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity. Mutat Res. 2011 Jul;725:57-77. doi:10.1016/j.mrgentox.2011.07.006. Sasaki K, Bohnenberger S, Hayashi K, et al. Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay. Mutat Res. 2012 Feb;744:42-53. doi:10.1016/j.mrgentox.2012.01.009 Schmey T, Tominello-Ramirez CS, Brune C, Stam R. Alternaria diseases on potato and tomato. Mol Plant Pathol. 2024 Mar;25(3):e13435. doi: 10.1111/mpp.13435. PMID: 38476108; PMCID: PMC10933620. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 72 Annex A.0 Participants A.01 Participants: Toxins human health Project Manager Enniatins : BfR (Germany): Anne-Cathrin Behr. Jessica Dietrich from 2022 until2024 Project Manager Alternaria toxins: UNIVIE (Austria): Doris.Marko Partners List (besides management): ANSES (France): Ludovic Lehegarat INRS (France): Laurent Gate, Dieynaba Ndiaye Sciensano (Belgium): Julie Sanders, Camille Streel NIPH (Norway): Hubert Dirven, Igor Snapkow, Nicola Margareta Smith IMR (Norway): Josef Rasinger STAMI (Norway): Steen Mollerup, Solveig Krapf INSA (Portugal): Henriqueta Louro, Maria Joao Silva, NIB (Slovenia): Bojana Zegura, Matjaz Novak UNAV (Spain): A. Vettora BPI (Greece): E. Spilioti Fraunhofer-IBMT (Germany): Yvonne Kohl ISS (Italy): Sabrina Tait NVI (Norway): Gunnar Eriksen, Christiane Faste TUB (Germany): Roderich Suessmuth A.02 Participants: Toxins environment Project Manager UGent (Belgium): Jana Asselman. Researchers: Eveline Diopere Deputy Project Manager SLU (Sweden): Stefan Örn. Researcher: Gunnar Carlsson Partners List (besides management): UAVR (Portugal): Susana Loureiro UoB (United Kingdom): Pu Xia D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 73 A.03 Participants: BPA alternatives human health Project Manager BfR (Germany): Kiara Aiello Endpoint Leaders/Project Managers 1. Endocrine Disruptors: ISS (Italy): Sabrina Tait INRAE (FR): Catherine Viguié 2.Developmental Neurotoxicity CNRS (FR): Sakina Mhaouty-Kodja. Researcher: Lydie Naule 3. Immunotoxicity: UMIL (Italy): Emanuela Corsini. Researcher: Martina Lulini 4. Carcinogenicity: INSA (Portugal) Maria Joao Silva. Researcher: Ramos Carolina 5. Metabolism: INRAE (France): Daniel Zalko ISS (Italy): Buratti Franca. Emanuela Testai from 2022 until 2025. Partners List (besides management): ISS (Italy): Francesca Marcon (Carcinogenicity). Simonetta Palleschi (Immunotox) TTL (Finland) Jonna Weisell, Kukka Aimonen (Carcinogenicity) ULFFA (Slovenia) Marija Sollner Dolenc, Nina Franko (Immunotoxicity) MUI (Austria) Johanna Gostner ISCIII (Spain) Ana Isabel Cañas Portilla, Antonio De La Vieja Escolar (ED) INRS (France) Laurent Gate, Sophie Ndaw, Yves Guichard (Carcinogenicity) IBMT Fraunhofer (Germany) Sylvia Wagner, Yvonne Kohl Carcinogenicity) LIH (Luxembourg): Archibold Mposhi (joined Febrary 2024); Jonathan Turner. Nathalie Grova from 2022 until 15th March 2024 ) (DNT) NILU (Norway) Naouale El Yamani Carcinogenicity) A.04 Participants: BPA alternatives environment Project Manager BPI (Greece): Katerina Kyriakopoulou (for Data Gaps section of the porject) Partners List (besides management): MU (Czechia) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 80 was not clear if BSA could also affect BPZ bioavailability. No new peak, attributable to glucuronide formation, was detected when recombinant UGTs and human liver microsomes were used. Next step will be to use BPE as the test item, which shows higher solubility in water matrix, to obtain metabolic kinetic data. Endocrine effects Steroidogenesis assay The steroidogenesis assay was performed according to the OECD TG 456 (https://www.oecd.org/en/publications/test-no-456-h295r-steroidogenesis-assay_9789264122642-en.html), using the human adreno-carcinoma cell line (NCI-H295R cells from ATCC). Cells were maintained in DMEM/F12 medium supplemented with 1% ITS-Premix, 2.5% Nu-Serum, 1% Pen-Strep, 15 mM HEPES at 37°C, 5% CO2 in a humified atmosphere. At the end of 2023, the Corning company stopped producing the Nu-Serum causing a delay in the experimentation. We then substituted it with Nu-Serum IV, containing fetal bovine serum rather than newborn calf serum. To verify that the reproducibility of the assay was not affected, we compared the results of the quality control plates obtained using the two serums and including: non-treated and vehicle treated (DMSO 0,1%) cells, cells treated with the positive control forskolin (at 1 and 10 µM) and the negative control prochloraz (at 0.1 and 1 µM). The results were identical, assuring the robustness of the assay. All the chemicals (BPA, BPE, BPZ, BPP, BPAP, BPS-MAE, TCBPA, TBBPA, as well as forskolin and prochloraz) were dissolved in 2 ml DMSO in glass amber vials to obtain 100 mM stock concentrations, with no solubility problems. Seven 10-fold diluted concentrations were also prepared in DMSO in glass amber vials; all the solutions are stored at -20°C. To perform the assay, cells were used from passages 5 to 10; 150,000 cells/cm2 were plated in 24-wells plates in 1 ml complete medium. The next day, 1 µl DMSO (as vehicle control) or 1 µl 1000x concentrated stock solutions were added in the wells in triplicate. After 48 h incubation at 37°C, conditioned medium of each well was collected and stored at -80°C until analysis, whereas cell monolayers were assessed for vitality by performing the MTS assay. Concentrations decreasing vitality more than 20% were excluded from the hormone assessment; whenever possible, additional intermediate concentrations were added for a more precise assessment. For each compound, three independent experiments at different cell passages were performed. Cytotoxicity IC50 values were calculated with the drc package v3.0 in R 4.3.3. Commercial ELISA kits were used to assess E2 and testosterone levels. Samples were diluted 1:50 or left undiluted for E2 and testosterone assessment, respectively. The hormone interference test was performed according to the OECD TG 456 protocol; at the maximum concentration tested, the compounds interfered with hormone assessment by less than 20%, as required. Thyroid Hormone (T3) transport via the MCT8 Transporter Assay The T3 transport assays was conducted based on the OECD assay (under validation). MCT8 overexpressing cell line (MCDK1-MCT8) and control MDCK1 cells lines were used and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 1% Pen-Strep at 37 ºC, 5% CO2 in a humidified atmosphere. All the chemicals (BPA, BPZ, BPE, BPS-MAE, PERGAFAST 201, BPP, BPAP, TCBPA, TBBPA, BPH, BPS, BAGDE, TransCBDO, and positive control Silychristin) were dissolved in DMSO at stock concentration of 100 mM and stored at – 20 ºC until use. To perform the assay, 50,000 cells/well were seeded in 96-wells plates in 0.25 ml complete medium. After 24 hours, the medium was replaced with an uptake buffer containing 5 µM of T3 and various concentration of test substances (ranging from 0.01 – 1000 µM). The cells were incubated 30 min at 37°C, then washed twice with PBS. Iodinecontaining molecules were digested using ammonium persulfate to release the iodide, which was quantified using the colorimetric Sandell-Kolthoff reaction. Each substance was tested in triplicate and repeated in at least three independent experiments. IC50 values were calculated with a curve-fitting algorithm in GraphPad Prism. All substances were tested simultaneously using MDCK1-MCT8 and control MDCK1 cells, alongside the potent inhibitor Silychristin as a positive control for the assay. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 81 Iodide (I-) transport via the NIS symporter Assay The Itransport assay was conducted based on the OECD assay (under validation). Human NIS (Sodium/Iodide symorter) overexpressing cell line (MCDK1-hNIS) and control MDCK1 cells lines were used. Both cell lines were maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 1% Pen-Strep at 37 ºC, 5% CO2 in a humidified atmosphere. All the chemicals (BPA, BPZ, BPE, BPS-MAE, PERGAFAST 201, BPP, BPAP, TCBPA, TBBPA, BPH, BPS, BAGDE, TransCBDO, and positive control perchlorate (ClO4-)) were dissolved in DMSO at stock concentration of 100 mM and stored at –20 ºC until use. To perform the assay, 150,000 cells/cm2 were seeded in 96-wells plates in 0.25 ml complete medium. After 24 hours, the medium was replaced with an uptake buffer containing 100 µM of Iand various concentration of test substances (ranging from 0.01 – 1000 µM). The cells were incubated 1 hour at 37 ºC. Then, washed twice with PBS. Iodine was quantified using the colorimetric Sandell-Kolthoff reaction. Each substance was tested in triplicate and repeated in at least three independent experiments. IC50 values were calculated with a curve-fitting algorithm in GraphPad Prism. All substances were tested in parallel using MDCK1-hNIS and control MDCK1 cells, alongside the potent inhibitor ClO4as a positive control for the assay. Immunotoxicity To determine cytotoxicity and select the concentrations to be tested, PBMC (1x106 cells/mL) were cultured in a complete medium consisting of RPMI 1640 without phenol red, 2 mM L-glutamine, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL gentamycin, and 50 µM 2-mercaptoethanol, supplemented with 5% heatedinactivated delipidated FBS. The cells were treated in a 96-well plate with increasing concentrations of the selected BPA analogues and DMSO as a vehicle control and then incubated at 37°C in 5% CO2 for 24 hours. For the determination of the leukotoxicity, the CyQUANT™ LDH Cytotoxicity Assay Kit was used (InvitrogenTM Corporation, Massachusetts, US) and the manufacturers procedures followed. Based on the results obtained, the CV80 values were selected, and the concentrations to be used in subsequent tests were determined. In table 5b in the results, the CV80 are reported [UMIL (IT)]. To investigate the effect on T-independent antibody production, PBMC (1.26x106 cells/mL) were cultured in a complete medium consisting of RPMI 1640 without phenol red, 2 mM L-glutamine, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL gentamycin, 50 µM 2-mercaptoethanol, and supplemented with 5% heatedinactivated delipidated FBS. The cells were treated in a 48-well plate with increasing concentration selected base on the CV80 (reported in the results) and DMSO as a vehicle control. They were then incubated at 37°C in 5% CO2 for 24 hours. Subsequently, PBMCs were stimulated, or not, with 1 µg/mL of ODN2006 and 100 IU/mL of rhIL-2 for an additional 5 days. To determine the release of total IgG and IgM, after a total of 6 days, PBMCs were centrifuged for 5 minutes at 25°C at 380 g-. Supernatants were collected and stored at -20°C until measurement. The release of immunoglobulins IgG and IgM was assessed using an ELISA technique. The optical densities were measured using the Molecular Devices SpectraMax ABS, and data were collected and analyzed using integrated software. The results were expressed as fold-change of chemical-treated cells versus vehicle-treated cells [UMIL (IT)]. To assess NK-cell lytic activity, PBMCs (5 × 106 cells/mL) were exposed to BPA and its analogues or DMSO for 24 hours. K562 cells, stained with CellTrace™ CFSE, were used as target cells. Briefly, 500 µL of K562 cells at a concentration of 106 cells/mL were centrifuged, CellTrace™ CFSE was added to the cell pellet (diluted 1:1000), and the mixture was incubated for 15 minutes at 37℃, protected from light. After incubation, the reaction was halted by adding culture medium containing 5% heat-inactivated dialyzed fetal bovine serum. The CellTrace™ CFSEstained K562 cells were then adjusted to a concentration of 105 cells/mL and co-cultured with BPA analogues/DMSO-exposed PBMCs. Three different effectors (PBMC) to target (K562) cell ratios were used: 50:1, 25:1, and 12.5:1, while maintaining a constant concentration of K562 cells. The cells are then co-cultured for 4 h at 37℃ in a 5% CO2 incubator. To evaluate the lytic activity of NK cells, the plate contents were transferred to flow cytometry tubes, and PI (5 nM) was added to each tube. The percentage of PI-positive cells among CFSE-stained D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 82 K562 cells was then measured using a Novocyte 3000 flow cytometer. In total, 1,000 CFSE-positive cells were analyzed for PI positivity, indicating the presence of dead K562 cells [UMIL (IT)]. In silico screening [UL FFA] In silico screening of BPA and its 25 substitutes using Endocrine Disruptome (http://endocrinedisruptome.ki.si/, https://pubmed.ncbi.nlm.nih.gov/24628082/) was performed in order to evaluate their effect on nuclear receptors. Since endocrine disruption might be a contributing factor to immunotoxicity, the compounds being predicted to react with several nuclear receptors were expected to exhibit higher immunomodulatory properties. The software is able to estimate the binding affinity of small molecules to 12 nuclear receptors, i.e., androgen receptor (AR), estrogen receptors α (ERα) and β (ERβ), glucocorticoid receptor (GR), liver X receptors α (LXRα) and β (LXRβ), mineralocorticoid receptor (MR), peroxisome proliferator-activated receptors α (PPARα), β (PPARβ) and γ (PPARγ), progesterone receptor (PR), retinoid X receptor α (RXRα), thyroid receptors α (TRα) and β (TRβ). For each receptor (including agonistic and antagonistic mode of action for AR, GR and ERs), compounds were ranked from 1 to 26, where first place represents the highest binding potential. Then, the comprehensive ranking was performed where the sum of all the ranks for each compound was calculated. The compounds with the lower sum present the higher potential for endocrine disruption. In vitro assays As a model of human macrophages, THP-1 cells were differentiated into macrophages with exposure to 80 nM phorbol-myristate-acetate (PMA) for 72 h and used as such for the experiments. Jurkat T cells were used as a model for human T lymphocytes and lymphoblastoid cell lines (LCLs) as a model for B lymphocytes. For the assessment of the BPA alternatives on metabolic activities, the cells were exposed to the increasing concentrations of substitutes or vehicle control for 24 h. Then, their metabolic activity was evaluated by the resazurin method. The IC50 values were calculated using GraphPad Prism. For the assessment of the BPA substitutes on the cytokine release, the cells were pretreated with the substitutes for 2 hours and then appropriately stimulated for 24 h. The released cytokines were measured in the cell culture supernatants using multiplex bead arrays (macrophages, LCLs) of ELISA (Jurkat). Specifically, THP-1 derived macrophages were exposed to 10 nM, 100 nM or 10 μM of the compounds and their effects on IL-1β, IL-6, IL-8 and TNFα were assessed. Jurkat T cells were exposed to the increasing μM concentrations of compounds and the IC50 values of IL-2 inhibitions were determined. LCLs were exposed to 100 nM or 10 μM compounds and their effects on IL-2, IL-6, IL-10 and TNFα were assessed. The cytokines released from THP-1 derived macrophages and LCLs were compared to the vehicle controls and statistical analysis was performed by Dunnett’s multiple comparisons. Immune Cells Calcium Homeostasis [ISS(IT)] Jurkat T cells were employed as a model for human T lymphocytes. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 IU/mL penicillin and 0.1 mg/mL streptomycin) at 37°C in a humidified atmosphere with 5% CO₂. The experimental procedure for [Ca²⁺]ᵢ measurement was adapted from established protocols, and specific SOPs were developed. On the day of the experiment, cells were loaded with the fluorescent calcium-sensitive probe Fura-2, suspended in a HEPES-buffered saline working solution, and kept on ice in the dark until use (for a maximum of 4 hours). An aliquot of approximately 1.5 × 10⁶ cells was then transferred into a spectrofluorometer cuvette containing 1.5 mL of working solution at 37°C under gentle agitation. After signal stabilization (~1 minute), time-based fluorescence acquisition was initiated. To evaluate the acute effects of the analouges on resting [Ca²⁺]ᵢ, cells were challenged with the selected analouge or with the vehicle alone (0.1% DMSO), and fluorescence signals were recorded for the following five minutes. To evaluate the acute effects of the analouges on calcium signaling, cells were exposed to the analouge for two minutes and then stimulated with OKT3, an anti-CD3 monoclonal antibody that induces a calcium response by activating the T cell receptors. At the end of each experiment, the signal was calibrated to convert arbitrary fluorescence units into calcium concentration units ([Ca2+]i). Finally, cell responses were quantified in terms of area under the curve. Long-term effects were assessed by incubating cells with the analouges (or 0.1% DMSO) in complete cell culture medium without phenol red for 24 hours at 37°C, 5% CO₂, in a humidified atmosphere. At the end of the incubation, basal [Ca²⁺]ᵢ and OKT3-induced calcium responses were measured as described above. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 83 Control experiments without cells were always performed to assess potential interference from autofluorescence and/or scattering phenomena caused by the dispersion of the substances into the buffer. Cell viability under the different assay conditions was assessed using trypan blue. Developmental Neurotoxicology Hippocampal Neurons in vitro screening by [CNRS (FR)] The generation of hippocampal neurons from hiPSCs was set up by adapting previously reported protocols. We performed three differentiations of two distinct hiPSCs clones into hippocampal neural progenitors (hNPCs), which then undergo neuronal maturation. We performed RT-qPCR analyses to confirm successful hippocampal differentiation. We showed that octamer-binding transcription factor 4- (OCT-4) expressing hiPSCs differentiated with high efficiency into hNPCs expressing NPC marker Nestin and hippocampal progenitor markers prospero homeobox 1 (PROX1) and zinc finger and BTB domain containing 20 (ZBT20). Furthermore, the neuronal marker microtubule associated protein 2 (MAP2), and hippocampal markers ELAV like RNA binding protein 2 (ELAVL2) and glutamate ionotropic receptor kainate 4 (GRIK4) were expressed in mature hippocampal neurons at day 43. These hNPCs were treated chronically with increasing doses (0.001 µM, 0.01 µM, 0.1 µM, 1 µM, 10 µM, 100 µM) of BPA, BPAP, BPE and BPZ over a 14-day period. Cell viability tests were conducted. All the analyzed bisphenols were associated with a concentration-dependent decrease in cell viability, with a significant decrease observed at doses of 100 µM for BPA, BPAP and BPZ and from 10 µM for BPE. On the basis of the results of these tests, non-toxic doses (0.001 µM, 0.01 µM, 0.1 µM, 1 µM) were chosen to treat the NPC with bisphenols for 14 days. Immunocytochemistry for β-III tubulin has been performed, and analysis of neuronal morphology (number of neurites per neuron, length and number of branches) is currently underway. In addition, protein levels of presynaptic (synaptophysin, synapsin 1) and postsynaptic (PSD95, gephyrin, glutamate receptors) markers will be quantified by Western blot to measure the impact of treatments on synaptic plasticity. RNA and DNA have been extracted at the end of the treatment for analyses of gene expression and DNA methylation. In parallel with these analyses, cell viability tests for BPA, BPP, BPS-MAE and TCBPA will start soon, before conducting the same analyses as for BPA, BPAP, BPE and BPZ. SOPs for these analyses are under progress. Neurosphere Assay (In Vitro Test Battery for DNT) by [IUF (DE)) The analysis was performed by making use of the Neurosphere assay (Koch et al. 2022), a medium throughput and high content in vitro assay for developmental neurotoxicity (DNT), and an integral part of the developmental neurotoxicity in-vitro testing battery (DNT-IVB; Blum et al. 2023; OECD377 2023). It utilizes primary fetal human neural progenitor cells (hNPCs) cultured as neurospheres, 3-dimensional cell aggregates with the potential of differentiating into various brain effector cells (i.e. neurons, astrocytes, and oligodendrocytes). The assay models seven critical early human neurodevelopmental key events associated with DNT: hNPC proliferation (NPC1); radial glia, neuronal and oligodendrocytes migration (NPC2a/b/c respectively); neuronal differentiation (NPC3); neurite outgrowth (NPC4) and oligodendrocyte differentiation (NPC5). In the NPC1 assay, evaluating the hNPC proliferation, neurospheres were cultured in suspension, over 3 days. The cells were exposed to the tested chemicals from day 0, at 7 different concentrations (20, 6.6, 2.2, 0.74, 0.24, 0.082, 0.027 µM) using four technical replicates each. On day 3, hNPC proliferation was assessed by two methods: increase of sphere area (NPC1a) and 5′-bromo-2′-deoxyuridine (BrdU) incorporation in the DNA (NPC1b). Cytotoxicity and mitochondrial activity were assessed on day 3, in order to discern between unspecific hits (i.e. caused by cytotoxicity) and specific hits (i.e. caused by a substance-related mode of action that differs from cytotoxicity or reduced viability). Unless stated otherwise in table xx, at least three biological replicates were conducted for each substance. The NPC2-5 assay is a multiplexed assay used to evaluate key neurodevelopmental events related to DNT. Neurospheres were cultured on an extracellular matrix and allowed to differentiate for five days. Chemical exposure started at day 0 and continued throughout the assay duration of five days, 7 concentrations were tested (20, 6.6, 2.2, 0.74, 0.24, 0.082, 0.027 µM) with four technical replicates per condition. At day 3, radial glia migration D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 84 (NPCa) and cytotoxicity were assessed, and at the same time, cell culture medium with chemicals was renewed. On day 5 cytotoxicity and mitochondrial activity assessment was performed, after which, the cells were fixated, and immunohistochemical staining was performed to identify differentiated neurons and oligodendrocytes. To calculate NPC2b/c, NPC3, NPC4, and NPC5 endpoints, stained neurons and oligodendrocytes were identified by Omnisphero, an in-house software, in combination with two convolutional neural networks (CNN), based on the Keras architecture implemented in Python 3. Unless stated otherwise in table xx, at least three biological replicates were conducted for each substance. Carcinogenicity Cell Transformation Assay by [INSA (PT), NILU (NO), INRS (FR), FIOH (FI)] This assay was conducted following the OECD Guidance Document on the In Vitro Bhas 42 cells Transformation Assay, 2016. Bhas 42 cells were thawed and grown in DMEM supplemented with 10% FBS until approximately 70% confluence and then were trypsinized and grown in DMEM F-12 supplemented with 10% FBs and 1% Penicilin/Streptomycin (100,000 units/L Penicillin G sodium and 10 mg/mL streptomycin sulfate) until reaching 70% confluence. At day 0, cells were seeded in 6-well plates at 4000 cell/well for the initiation test and at 14,000 cell/well for the promotion test. Six wells were prepared per treatment condition. For the concurrent cell growth assay, 96well plates were also seeded with 200 cells/well for initiation and with 400 cells/well for promotion. Eight wells were prepared per treatment condition. For the initiation test, 24h after seeding (day 1), treatment medium containing bisphenols (máx. DMSO 0.25%) were added to the cultures. 3-Methylcholanthrene (MCA) at 1 µg/mL was used as a positive control for the initiation test. The treatment was continued for 72h. At day 4, all treatment media were removed, and the cells were post-cultivated in fresh medium until day 7 (concurrent cell growth) or until day 21, receiving medium exchanges on day 7, 11 and 14. For the promotion test, at day 4, culture medium was replaced with treatment medium containing bisphenols (máx. DMSO 0.25%). 12-O-tetradecanoylphorbol 13-acetate (TPA) at 0.05 µg/mL was used as a positive control for the promotion test. The treatment was continued for 72h and removed at day 7 (concurrent cell growth) or renewed at day 7 and 11. At day 14, all treatment media were removed, and the cells were postcultivated in fresh medium until day 21. DMSO at 0.1 and 0.25%, as well as 5% H2O and caffeine at 100 µg/mL, worked as negative controls for both initiation and promotion. Concurrent cell growth assay ended at day 7 for both initiation or promotion tests, and viability tests were conducted on these cultures to ascertain cells viability in the concentrations tested. MTT assay, Alamar Blue or WST-1 are some of the viability assays that were performed. Results were expressed as % of viability compared to the vehicle control. At day 21, for initiation and promotion, culture medium was removed and cells were fixed with absolute ethanol, and stained with a 5% Giemsa solution for 30 min. and then rinsed with tap water. After drying overnight, plates were ready for foci scoring. The transformed foci were evaluated according to the criteria described by Sakai et al. (2011), Sasaki et al. (2012) and OECD Guidance Document (2016). The results in the Bhas 42 cell transformation assays were positive when inducing statistically significant increases in the number of transformed foci compared to the respective vehicle control. D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 85 A.3 Methods: BPA alternatives environment Aquatic invertebrates Activities (i) & (ii) tests SLU (SE) IISPV (ES) SDU (DK) INERIS (FR) IEP-NRI (PL) BPI (GR) UG-PL (PL) UAVR (PT) SU (SE) Cefas (UK) Acute toxicity test on Daphnia magna (OECD TG 202) n/r BPS, BPSMAE, BPAP, BPE (scheduled but not finalised yet) n/r Not initially scheduled but it is stated in the report that BPA, BPF, BPS and BPB have been tested No data presented for single compounds (mixtures) BPB, BPF, BPS, BPAF No data presented for single compounds (mixtures) No data presented for single compounds (mixtures) BPA, BPZ, BPE, BPF, BPS, BPAF n/r Daphnia magna reproduction test (OECD TG 211, level 4 of OECD CF) n/r n/r n/r n/r No data presented for single compounds n/r No data presented for single compounds (mixtures) No data presented for single compounds (mixtures) BPA, BPAF, BPS, BPF n/r Short-term Juvenile Hormone Activity Screening Assay using Daphnia magna (JHASA, OECD 253) n/r n/r n/r BPA, BPF, BPS, BPB n/r n/r n/r No data presented for single compounds n/r n/r Lymnaea stagnalis Reproduction Test (OECD TG 243) BPA, BPZ, BPS-MAE n/r BPE BPAP n/r n/r n/r n/r n/r n/r n/r Regeneration assay using the freshwater planarian Girardia tigrina n/r n/r n/r n/r n/r n/r n/r BPA, BPAF, BPS-MAE, BPZ n/r n/r 10-day acute sediment toxicity bioassay with the marine polychaete Arenicola marina n/r n/r n/r n/r n/r n/r n/r n/r n/r BPA, BPAF, BPAP, BPZ 42-day chronic toxicity test using the freshwater amphipod Hyalella azteca, spiking sediment n/r n/r n/r n/r n/r n/r n/r n/r n/r BPAF n/r: not relevant D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 86 Zebrafish Activities (i) & (ii) tests INERIS (FR) NIB (SI) UU (SE) BPI (GR) UAVR (PT) Fish Embryo Acute Toxicity Test (OECD TG 236) BPA, BPB, BPE, BPC, BPC-Cl, BPF, BPS, BPS-MAE, BPS-MPE, BPZ, TCBPA, BPAF, BPAP, 4-4’ODP BPA, BPAF, BPPH, BPAP n/r BPS, BPB, BPF BPE, BPZ, BPSMAE Fish short-term reproduction assay (OECD TG 229, level 3 of OECD CF), or 21-day fish assay: a short-term screening for estrogenic and androgenic activity and aromatase inhibition (OECD TG 230, level 3 of OECD CF) It was initially planned to assess the endocrine and reproductive effect of one bisphenol substitute in the OECD TG N°229 but it was not possible to perform this experiment due to “human factors” beyond partner’s control.” n/r n/r n/r n/r EASZY assay: Detection of substances acting through Estrogen Receptors Using Transgenic cyp19a1b GFP Zebrafish Embryos (OECD TG 250, level 3 of OECD CF) BPA, BPB, BPE, BPC, BPC-Cl, BPF, BPS, BPS-MAE, BPS-MPE, BPZ, TCBPA, BPAF, BPAP, 4-4’ODP n/r n/r n/r n/r n/r: not relevant Alga / aquatic plants Activities (i) & (ii) tests IISPV (ES) IEP-NRI (PL) BPI (GR) UG-PL (PL) SU (SE) Algae growth inhibition test on Raphidocelis subcapitata (formerly known as Selenastrum capricornutum) (OECD TG 201) No data presented yet for single compounds No data presented yet for single compounds BPB, BPS, BPF n/r BPAF, BPS, BPF, BPA Lemna sp. Growth Inhibition Test (OECD TG 221) n/r BPZ, BPP, BPE n/r No data presented yet for single compounds (mixtures) Initially scheduled to test: BPS, BPE, BPA, BPF n/r n/r: not relevant D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 87 Soil organisms n/r: not relevant Amphibians Activities (i) & (ii) tests UAVR (PT) In vitro assays with A6 epithelial-like cells (ECACC 89072613) of Xenopus laevis BPA, BPAF, BPZ, BPE, BPS-MAE Behaviour toxicity assay with larvae of Xenopus laevis and zebrafish eleutheroembryos BPA, BPAF, BPZ, BPS-MAE (results still being analysing for zebrafish) Frog embryo teratogenicity assay (ASTM E1439-12) BPA, BPAF, BPZ, BPE, BPS-MAE Xenopus Eleutheroembryo Thyroid Assay XETA (OECD TG 248, level 3 of OECD CF) No data has been presented yet n/r: not relevant In vitro / high throughput assays Activities (i) & (ii) tests IISPV (ES) IEP-NRI (PL) BPI (GR) UAVR (PT) Eawag (CH) UFZ (DE) Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay (OECD TG249) n/r n/r n/r n/r BPA, BPE, BPF, BPS, BPS-MPE, BPS-MAE, BPB, BPAP, BPAF, BPZ, n/r Activities (i) & (ii) tests IISPV (ES) INERIS (FR) BPI (GR) Effects on soil micoorganisms: Carbon transformation test (OECD TG 217 and ISO 11274) BPS, BPS-MAE, BPAP, BPE, BPZ, Pergafast 201, BPP, BPPH (no endpoints are available yet since the statistics were not finalized) n/r n/r Effects on soil microorganisms: Nitrogen transformation test (OECD TG 216) BPS, BPS-MAE, BPAP, BPE, BPZ, Pergafast 201, BPP, BPPH (on going) n/r n/r Earthworm reproduction test (OECD 222) n/r n/r BPE, BPZ (on going experiments) D5.4 1ST DATA GAPS REPORT P-A-R-C HORIZON-HLTH-2021-ENVLTH-03 CONTRACT N. 101057014 88 Activities (i) & (ii) tests IISPV (ES) IEP-NRI (PL) BPI (GR) UAVR (PT) Eawag (CH) UFZ (DE) BPP, bisOPP-A, TBBPA, TCBPA* Microtox® Acute Toxicity Test n/r BPAF, BPF, BPSMAE, BPS-MPE, BPPH BPAF, BPF, BPSMAE, BPS-MPE, BPAP, BPP, BPPH, BPE, BPZ, Pergafast n/r n/r n/r YES assay, in vitro screen with Saccharomyces cerevisiae n/r n/r n/r BPA, BPE, BPAF, BPZ, BPAP, BPG, BPS, BPS-4-allyl ether n/r n/r Reporter gene assays and mammalian cell models of neurotoxicity for high-throughput screening n/r n/r n/r n/r n/r n/r n/r: not relevant