SaferWorldbyDesign Webinar: SaferNeuron Case Study: Discussion of Tier 1 Safety Assessment Methodology and Initial Results on Acrylamide

As an example focus case study, we shared our initial Tier-1 assessment results on acrylamide exposure from French fries in fast-food preparation & consumption scenarios, integrating AI-assisted knowledge review, exposure estimation, benchmark dose data, and Margin-of-Exposure (MOE) calculations.

🔍 Key insights
• Acrylamide concentrations in fried potatoes typically range ~300–1000 µg/kg, strongly influenced by time–temperature and colour.
• Consumer exposure from a 150 g portion results in MOEs of:
• ~80–260 (cancer reference)
• ~200–670 (neurotoxicity reference)
• All MOEs fall below the 10,000 value considered low-concern for genotoxic carcinogens → reinforcing the need for as low as reasonably achievable management.
• Mechanistic consistency across literature, PubChem, QSAR, and regulatory sources supports a bioactivation > DNA/protein adduct > neural toxicity pathway.

✅ Operational implications
Clear high-impact controls for kitchens and food operators:
• Fry at ≤175–180 °C
• Aim for golden-yellow colour, not dark brown
• Proper potato storage and oil management
• Effective ventilation & safe handling practices for workers
• Encourage moderation & lighter-colour choices for consumers
• Consider alternative cooking methods

✅ Consumer implications
• Choose golden-yellow, not dark-brown fries; avoid “burnt fries”
• Balance diet with lower-acrylamide foods across the day
• Consider cooking methods used at home, prcedures at visited restaurants

🚀 Next steps (Tier 2/3)
We will now proceed to consider NAMs-based refinement, including:
• Genotoxicity in metabolically competent human systems
• iPSC-derived neuronal activity assays (MEA)
• Adductomics for translational biomarkers
• PBPK-IVIVE to convert in vitro potency → human-equivalent doses
• Updated MOEs and mechanistic confidence bands

This staged approach helps demonstrate how digital first safety assessment + targeted NAMs can accelerate trusted, human-relevant decisions under Safe-and-Sustainable-by-Design.

📅 We will share the final consolidated case study results in our follow-up webinar in the coming weeks — stay tuned!

1. Case Study Webinar 29 October 2025 Description of problem formulation for Cases and Tier 1 Assessment Approaches Focus today on SaferNeuron Cases

2. Summary 1. Compounds and Selection 2. Scenarios and Assessments 3. Tier 1 Case Study Methods and Results 4. Discussion of Results 5. Next Steps, Tier 2 recommendations

3. Compound Selection Criteria Based on these Selection criteria 1. Neurotoxicity concern 2. Mechanistic spread across AOPs 3. Regulatory relevance 4. NAMs potential (in silico, in vitro ) 5. SSbD substitution potential 6. Practical case relevance

4. Current Shorter Compound List selected from Longer List ( SaferNeuron ) 1. Rotenone 2. Tebufenpyrad 3. Carboxin 4. Pyraclostrobin 5. Domoic acid 6. Acrylamide 7. n-Hexane 8. Manganese 9. Bisphenol A (BPA). 10. Bisphenol S (BPS) 11. Tetrabromobisphenol A (TBBPA) 12. Nicotine 13. Dimethyltin dichloride (recently added to the SIN list as a neurotoxicant) 14. Methyltin mercaptide (recently added to the SIN list as a neurotoxicant) 15. Sodium pyrithione ( recentl yadded to the SIN list as a neurotoxicant)

5. Cases and Exposure Scenarios ( Neurotoxcity focus here) A. Tebufenpyrad Scenario (Occupational - Pesticide Application on Grapes) Consumer (Eating Grapes) B. Domoic Acid Scenario (Consumer Risk Assessment for Food Consumption with pesticide) C. Acrylamide Scenario (Preparation of French Fries in a Fast Food Restaurant leading to acrylamide formation) D. n-hexane Scenario (occupational exposure in a shoe/handbag workshop – applying spray adhesive) E. Nicotine Scenario (Occupational - small e-liquid filling room) Scenario (Consumer - Vaping) F. BPA Substitution Scenario ( SSbD for BPA substitution on food metal can coating) G. Dimethyltin dichloride - DMTCl ₂ (Recent Addition to SIN List for neurotoxicity concern) Scenario (Occupational exposure to plastic additive)

6. NAMs-based Solutions (?) <- Test Business Cases on Risk Assessment Problem Formulations with Resource Limitations https://risk-hunt3r.net/

7. Solution Overview ASPA Workflows for Next Generation Risk Assessment (NGRA) An alternative safety profiling algorithm (ASPA) to transform next generation risk assessment into a structured and transparent process, Marcel Leist et al ; https://zenodo.org/records/16993943 Published article: https://www.altex.org/index.php/altex/article/view/3041

8. Solution Overview Biological Data (Biostudies, EdelweissData , Notebooks) Knowledge Graphs Compound Database Methods Database ( ToxTemp ) Prediction and Analysis Tools (QSAR, Molecular Simulations, Metabolism, Omics) Toxicokinetics and Exposure protocols OpenModel Repository Case Study Documentation AI assisted AI assisted AI assisted Life Cycle Assessment AI assisted In our model we treat AI as an Assistance Tool to Human Knowledge Work organised in Workflows of Tasks and Decision Points supported by (ideally) FAIR sources of data and knowledge

9. PubMed ChemInsight: Automated literature mining PubChem Pal: Automated chemical data extraction AI Assistance of collection, extraction and curation of data from safety data sheets and other legacy documents AI Assistance Resource Examples AI Assistance of data from literature AI Assistance of collection and curation of data from databases AI Assistance of data from documents

10. Application Product knowledge base ( SmartSafety ) – processing documents, generating reports on formulations and ingredients with AI-Assistance into End User Software designed with and for Risk Assessors AI assist on processing and extracting information from documents into ingredient and formulation profiles AI assist on generating reports (e.g., batches responding to a new or modified regulation) Human – AI collaboration e.g, on curation, interpretation https://smartsafety.edelweissconnect.com/

11. SaferSkin Integration with SmartSafety supporting NAMs-based Skin Safety Assessment of Ingredients in Formulation https:// saferworldbydesign.com / saferskin /in-silico/skin-sensitization-app/app/

12. SSbD Framework proposed by the EC JRC

13. ©Furxhi et al., RSC Sustain., 2023,1, 234-250 CC BY-NC 3.0 Safe and Sustainable by Design ( SSbD ) framework Safety and sustainability integrated in the chemical/product development Key Principles Safety Ensuring that products and processes do not pose risks to human health and the environment. Sustainability: Minimizing environmental impact and promoting resource efficiency throughout the lifecycle.

14. SETAC Europe 2024 Workshop on SSbD, Seville and associated article ( https://academic.oup.com/ieam/article/21/4/735/8139937 ) SSbD – screening and filtering candidates

15. SSbD Framework – hazard scoring

16. SSbD Framework – life cycle assessment scoring

17. ECHA DB & Notebooks Retrieve data from other resources SDS Toolbox ACCORDs KI SmartSafety Perform read-across Sufficient information Read-across applicable Criteria passed Determination of required NAM experiments Namastox ToxTemp/ Protocols DB Defining physicochemical properties data from partners ToxTemp/ Protocols DB Cut-off criteria Continue SSbD assessment DTU QSAR Model ZZS Similarity Tool STOPTOX OECD QSAR TB JANUS RDKit OperaQSAR SaferSkin Retrieve SMILES/InChl Selection of compounds to be substituted & alternative compounds STEP 1 No No Yes Yes No Yes No Yes Alerts for any defined endpoint No Yes Tools developed under SSbD4CheM Sharing Knowledge Portal Tools under development PubMed ChemInsight PubChemPal EPA CompTox Integrated Risk Assessment Data Sources SSbD Hazard Characterization Workflow

18. From databases From in silico tools SSbD Hazard Characterization – Example results

19. Continue SSbD Assessment SSbD Hazard Characterization Workflow: Example Scoring result

20. Example Case Study Summaries with Exposure Scenarios

21. A. Tebufenpyrad (Pesticide Application on Grapes) Occupational Scenario A professional vineyard worker is preparing and spraying a pesticide mixture to protect grapevines from mites. The product contains tebufenpyrad , a chemical that targets the nervous system of small insects and mites. The worker mixes the pesticide in a tank and uses a handheld or backpack sprayer to apply it along vine rows for several hours during the morning. Although they wear gloves, a hat, and overalls, it’s a warm day and there is moderate wind, so some spray mist and residues contact the worker’s hands, forearms, and face. Small amounts can also be inhaled with the mist. These short-term exposures happen several times each growing season. This scenario represents a typical occupational situation where protective gear and good practices minimize risk but do not eliminate it completely. Input Data Vineyard or greenhouse pesticide application (mixing/loading/spraying) Exposure Routes Dermal (hands, forearms, face), Inhalation (fine droplets) Exposure Dermal: 0.02 mg/kg bw/day Inhalation: 0.001 mg/kg bw/day Total Systemic Exposure ≈ 0.021 mg/kg bw/day Toxicological Reference (AOEL) 0.025 mg/kg bw/day

22. A. Tebufenpyrad (Pesticide Application on Grapes) Occupational Risk Assessment Summary Total systemic exposure : 0.021 mg/kg bw /day Dermal: 0.020 (≈ 95% of total) <- risk driver Inhalation: 0.001 (≈ 5% ) Toxicological reference (AOEL) : 0.025 mg/kg bw /day Risk quotient (RQ = Exposure/AOEL) : 0.84 Margin of safety (MOS = AOEL/Exposure) : 1.19 Interpretation: Exposure is below the AOEL (RQ < 1), indicating acceptable risk for daily operator exposure under typical conditions— but with a narrow safety margin . Decision Acceptable with targeted risk mitigation. Prioritise controls that reduce dermal exposure to increase the MOS above ~2 where practicable. Follow-up / Monitoring Aim to halve dermal exposure through the above controls (target total ≤ 0.010 mg/kg bw /day → MOS ≳ 2.5 ). Keep a simple exposure log (weather, PPE used, task duration) and perform a seasonal review . If available, consider biomonitoring or task-based exposure measurements to validate assumptions.

23. A. Tebufenpyrad (Pesticide Application on Grapes) Consumer Exposure (Eating Grapes) A parent buys a bag of table grapes at the supermarket during peak season. The grower used a mite control product containing tebufenpyrad earlier in the season, and tiny traces can remain on the fruit by harvest. After a quick rinse under the tap, the family eats the grapes as a snack. Food authorities set legal residue limits to keep these traces very low, and they regularly tighten them when new data arrives. Even so, how much a person takes in depends on how many grapes they eat, their body weight, and the residue level found in that batch. The quick check below shows what a typical adult and a child would take in if residues are at the current EU limit level for grapes, and whether that’s within health-based guidance values. Input Data Residue level used for screening: 0.30 mg/kg Health-based values for consumers (EU/EFSA): ADI (chronic): 0.01 mg/kg bw /day (order of magnitude used in EFSA assessments for tebufenpyrad) ARfD (acute): 0.10 mg/kg bw (EFSA peer-review era; used for acute screening)

24. A. Tebufenpyrad (Pesticide Application on Grapes) Assumptions (conservative but realistic) Adult: 70 kg bw , 200 g grapes (single snack) Child: 20 kg bw , 100 g grapes (single snack) Rinse scenario: simple tap rinse → processing factor ~0.8 (20% reduction) Health-based values: ADI = 0.01 mg/kg bw /day , ARfD = 0.10 mg/kg bw Results (per snack at residue 0.30 mg/kg) MOS (margin of safety) = Reference value / exposure. The Acceptable Daily Intake (ADI) is the maximum amount of a chemical substance (usually a pesticide, food additive, or veterinary drug residue) that can be ingested daily over a lifetime without appreciable risk to health. ADI = NOAEL/Safety Factors. The Acute Reference Dose ( ARfD ) is the estimated amount of a substance that can be ingested in a single meal or within 24 hours without appreciable health risk to the consumer. ARFD = Acute NOAEL/Safety Factors. Consumer Portion Intake (mg/kg bw) %ADI Chronic MOS %ARfD Acute MOS Adult 200 g 0.000857 8.6% 11.7 0.86% 116.7 Child 100 g 0.00150 15% 6.7 1.5% 66.7 Adult (rinsed) 200 g 0.000686 6.9% 14.6 0.69% 145 Child (rinsed) 100 g 0.00120 12% 8.3 1.2% 83.3

25. C. Acrylamide (Preparation of French Fries in a Fast Food Restaurant) Consumer Exposure Scenario A fast-food outlet prepares French fries from par-fried frozen potatoes. During high-heat frying, natural sugars (glucose/fructose) react with the amino acid asparagine in the potato and form acrylamide. Staff aim for a “golden-yellow” color, but on busy shifts some batches run darker. A customer orders a medium portion (about 150 g). Even though there’s no “legal limit” for acrylamide in fries, the EU has benchmark levels to encourage restaurants to keep levels low through good practices (choice of potatoes, storage, blanching, oil temperature/time, color target). Because acrylamide is a probable carcinogen formed during cooking, risk can be assessed by comparing the person’s intake with a scientific reference point, using a margin of exposure (MOE): the higher the MOE, the better. Input Data Typical acrylamide concentrations: mean ≈ 300 µg/kg (range often <20–1,000+ µg/kg in food-service). EU benchmark level (performance target): 500 µg/kg (French fries, ready-to-eat). Toxicology reference for MOE: BMDL10 = 0.17 mg/kg bw /day = 170 µg/kg bw /day (neoplastic effects). MOE guidance: values ≪10,000 indicate a health concern for genotoxic carcinogens and call for minimisation .

26. C. Acrylamide (Preparation of French Fries in a Fast Food Restaurant) Calculations (adult, per portion). Exposure (µg/kg bw) = C×P/BW; MOE = 170 / exposure Scenario (C, µg/kg) Exposure (µg/kg bw) MOE Light (100) 0.214 793 Mean (300) 0.643 264 Benchmark (500) 1.071 159 Dark (800) 1.714 99 Very dark (1000) 2.143 79 Interpretation: All MOEs are far below 10,000 , so acrylamide should be kept as low as reasonably achievable . Darker fries sharply reduce MOE. Practical risk-reduction For outlets (EU “Acrylamide Toolbox” good practices): • Use low-sugar potato varieties; avoid cold storage (<6 °C) that raises sugars. • Blanch/soak cut potatoes; control oil temperature and frying time. • Target “golden-yellow” color; discard over-browned batches. • Prefer thicker-cut fries; maintain fryer calibration and turnover. For consumers: • Choose golden-yellow, not dark-brown fries; avoid the crispiest/darkest bits. • Balance with lower-acrylamide foods across the day.

27. Literature

28. Literature We searched the databases by the following key-words: Neuro, Brain, Acute, Chronic, Development, LD50, NOEL, LOEL, NOAEL, LOAEL.

29. PubMed Literature Combines chemical identifiers (name + CAS) with risk-related keywords to retrieve targeted literature. Captures evidence across all risk assessment stages: Hazard Identification: Detects health and environmental hazards (e.g., carcinogenicity, endocrine, neurotoxicity). Dose–Response Assessment: Extracts thresholds (NOAEL, LOAEL, BMD, RfD , ADI) for quantitative modeling. Exposure Assessment: Identifies human and environmental exposure data (biomonitoring, routes, modeling). Risk Characterization: Integrates hazard and exposure evidence for quantitative risk estimation. PubMed serves as a scientific evidence engine connecting research findings to every stage of risk assessment. https://pubmed-cheminsight.edelweissconnect.com/

30. https://pubmed-cheminsight.edelweissconnect.com/

31. Pub Med - Example Result (Acrylamide) NOTE: Article A b stracts did not contain BMDL, NOAEL or MOE values that could be used in risk assessment. Need to access tables in full articles.

32. Pub Med - Example Result (Acrylamide) Albert Sebastià, Carmen Fernández- Matarredona , Juan Manuel Castagnini , Francisco J. Barba, Houda Berrada, Juan Carlos Moltó , Olga Pardo, Francesc A. Esteve- Turrillas , Emilia Ferrer, Acrylamide content in popcorn from Spanish market: Risk assessment, Food and Chemical Toxicology, Volume 196, 2025, 115145, ISSN 0278-6915, https:// doi.org /10.1016/j.fct.2024.115145. ( https://www.sciencedirect.com/science/article/pii/S0278691524007117 ) Abstract: Snacks, including popcorn, are increasingly consumed in Spain and are susceptible to acrylamide (AA) formation. AA, classified as a probable human carcinogen by the International Agency for Research on Cancer (IARC), is produced via the Maillard reaction between reducing sugars and amino acids, particularly glucose, and asparagine, when foods are heated above 120 °C. This study aims to analyze the AA content in 91 popcorn samples, categorized by flavor (salted, butter, caramel, flavored , colored , unflavored ) and cooking method (ready-to-eat, popcorn maker, microwave), and assess dietary AA exposure in the Spanish population. Samples were collected from supermarkets, grocery stores, and cinemas across Spain and analyzed using solid-liquid extraction (SLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The average AA concentration in the samples was 277 ± 119  μ g kg−1, with only two samples below the limit of quantification (LOQ, 60  μ g kg−1). At the same time, no significant correlation between flavor and AA content was found. Whereas microwave cooking notably increased AA levels. Estimated AA intake for adults and children ranged from 0.011 to 0.045  μ g kg⁻1 day⁻1, depending on the exposure scenario. In children, a margin of exposure (MOE) below 10,000 was observed for Harderian gland tumors in realistic and pessimistic scenario. Keywords: Acrylamide; Popcorn; Dietary intake; Risk assessment “AA is a toxic formed during Maillard reaction, occurring between reducing sugars and amino acids, especially glucose and asparagine, when foods are heated to more than 120 °C. The International Agency for Research on Cancer (IARC) classifies AA as a probable carcinogen ( Hamzalıoğlu et al., 2019; IARC, 1994). The European Food Safety Authority (EFSA) concluded in 2015 that AA may be linked to an increased risk of developing cancers, including kidney, endometrial, and ovarian cancers, as well as potential interference with fetal development. Studies in animals have shown a connection between AA exposure and the development of genetic mutations, tumors , and adverse effects on the nervous system (EFSA, 2015). AA exhibits neurotoxic effects by damaging nerve fibers , impairing neurotransmitter release, and inducing oxidative stress in neurons. It can lead to peripheral neuropathy, characterized by muscle weakness, numbness, and motor dysfunction. Prolonged exposure may also disrupt central nervous system function, contributing to cognitive and behavioral deficits (Park et al., 2021).” “Risk assessment is a critical process used to evaluate the potential health hazards posed by exposure to toxic substances such as AA. According to Joint FAO/WHO Expert Committee on Food Additives (JECFA) and EFSA, one key approach in this assessment is the application of the Margin of Exposure (MOE) concept. The MOE is calculated by comparing the level of AA exposure to the benchmark dose lower confidence limit (BMDL10). The BMDL10 is the level of exposure to AA with the dose known to cause harmful effects, typically in animal studies. An MOE of 10,000 or higher represents a low concern for public health regarding cancer risk (EFSA, 2005). A previous study suggests a health problem for the adolescent population relate” “To calculate exposure levels, it is first necessary to know the concentration of the contaminant in the food. Some of the foods most studied for their high levels of AA are coffee, potato products and bakery products (Sebastià et al., 2023). The Spanish Agency for Food Safety and Nutrition (AESAN) published a report in 2017 on AA levels in various food groups. According to the report, average AA concentrations were found to be 753 μ g kg−1 in potato chips, 247 μ g kg−1 in cookies, and 10 μ g kg−1 in bread.”

33. S ources for Acrylamide Concentrations in French Fries Acrylamide in French fries prepared at primary school canteens Check for updates Marta Mesias, ORCID logo *a Cristina Delgado-Andrade, ORCID logo a Francisca Holgado ORCID logo a and Francisco J. Morales https://pubs.rsc.org/en/content/articlehtml/2020/fo/c9fo02482d ? Children are one of the most exposed groups to dietary acrylamide with ‘potato fried products’ that account for up to half the total exposure to this contaminant. Acrylamide was measured in French fries prepared in 31 primary school canteens randomly recruited from different Spanish regions. The mean content was 329 μ g kg−1 (from <20 to 4000 μ g kg−1). French fries prepared from frozen par-fried potatoes reported a lower acrylamide content than those from fresh potatoes, 229 and 460 μ g kg−1, respectively. Only 15.7% of samples were above the benchmark levels established by the EU Regulation 2017/2158 (500 μ g kg−1). Significant differences were found according to the color of toasted (2274 μ g kg−1), dark-golden (463 μ g kg−1), golden (134 μ g kg−1) and light-golden (52 μ g kg−1) French fries . All the samples that were golden and light-golden showed acrylamide content below the threshold. The chromatic parameter a* was used as a rough classification of the French fries according to the benchmark level. Both educational initiatives intended for food operators and managerial-technical criteria that include the code of frying practices at public food service establishments should consider the golden color as the target for the end-point of frying. Acrylamide exposure will be reduced and, therefore, the risk linked to French fries consumption. This fact is especially relevant in establishments that prepare food for schoolchildren and would help to provide healthier diets, not only from a nutritional point of view but also from the reduction of chemical contaminants.

34. S ources for Acrylamide Concentrations in French Fries A Burhan Başaran, Hulya Turk, The influence of consecutive use of different oil types and frying oil in French fries on the acrylamide level, Journal of Food Composition and Analysis, Volume 104, 2021, 104177, ISSN 0889-1575, https://doi.org/10.1016/j.jfca.2021.104177 . (https:// www.sciencedirect.com /science/article/ pii /S088915752100377X) Abstract: In the literature, the effects of different frying oil types and consecutive frying sessions on acrylamide formation are still controversial. In this study, 8 consecutive frying sessions were applied with 4 different commercial oil products (sunflower oil, olive oil, corn oil, hazelnut oil) known to be used in French fries, and the formed acrylamide level in French fries was determined by LC–MS/MS. The relationship between variables was analyzed statistically. Acrylamide levels were measured as 890−1200 μ g/kg in sunflower oil, 892−1163 μ g/kg in olive oil, 981−1299 μ g/kg in corn oil, and 779−1120 μ g/kg in hazelnut oil. The poly-unsaturated fatty acid ratio of frying oil is thought to have a positive effect on acrylamide formation. The consecutive use of frying oil significantly affected acrylamide formation in French fries. In this study, consumers may be advised to use hazelnut and olive oil, and avoid corn oil, according to acrylamide levels detected in the French fries. Nevertheless, frying oil should not be used consecutively in French fries. These results can be used in studies to reduce acrylamide exposure from French fries. Keywords: Acrylamide; French fries; Oil types; Consecutive frying; Unsaturated fatty acid; Hazelnut oil

35. QSAR

36. QSAR OECD QSAR Toolbox v4.7 - Neurotoxicity

37. QSAR OECD QSAR Toolbox v4.7 - Metabolism evaluation

38. QSAR VEGA - NOAELs and LOAELs 1. Rotenone 2. Tebufenpyrad 3. Carboxin 4. Pyraclostrobin 5. Domoic acid 6. Acrylamide 7. n-Hexane 8. Manganese 9. Bisphenol A (BPA). 10. Bisphenol S (BPS) 11. Tetrabromobisphenol A (TBBPA) 12. Nicotine

39. QSAR VEGA - NOAELs and LOAELs COMPARISON This CONCERT model is based on the dataset as described by Gianluca Selvestrel , Giovanna J. Lavado e al. In the work of this paper in silico models were developed for predicting NOAEL and LOAEL for the sub-chronic toxicity data (90 days of exposure) . VEGA uses a second model for NOAEL predictions which is the CORAL model. CORAL uses a different dataset as base for predicting NOAEL values as it is described by Andrey J. Selvestrel , Alla P. Toropova et al.22. In this work only d ata referring to 90 days of oral administration in rats was considered and reproductive toxicity studies were rejected. Values for 28 days of treatment were considered but, in order to have consistent data, they were divided by a factor of 3 as specified by the scientific committee on consumer safety (SCCS) in order to approximate the 90-day NOAEL. NOAEL A = NOAEL [mg/kg bdwt /d] CONCERT/Coral 1.0.0, NOAEL B = NOAEL [mg/kg bdwt /d] IRFMN-CORAL 1.0.1, Liver NOAEL [mg/kg bdwt /d] CORAL 1.0.2, LOAEL A = LOAEL [mg/kg bdwt /d] 1.0.0 CONCERT/CORAL 1.0.0 and Liver LOAELL [mg/kg bdwt /d] CORAL 1.0.2. Compounds 1. Rotenone 2. Tebufenpyrad 3. Carboxin 4. Pyraclostrobin 5. Domoic acid 6. Acrylamide 7. n-Hexane 8. Manganese 9. Bisphenol A (BPA). 10. Bisphenol S (BPS) 11. Tetrabromobisphenol A (TBBPA) 12. Nicotine

40. AI-assisted OECD Toolbox Ivo Djidrovski LinkedIn Post Introducing O-QT Assistant, an AI-powered multi-agent system that bridges the OECD QSAR Toolbox with next-generation reasoning and automation GitHub https:// github.com /VHP4Safety/O-QT-OECD-QSAR-Toolbox-AI-assistant/

41. O-QT Assistant – Acrylamide Example

42. O-QT Assistant – Acrylamide Example Executive Summary Acrylamide exhibits high water solubility, moderate environmental mobility, and rapid biotransformation, resulting in low environmental persistence. Its physicochemical profile indicates high bioavailability potential, especially via water and dermal exposure routes. Mechanistically, QSAR profiling suggests that acrylamide possesses electrophilic features associated with genotoxicity, carcinogenicity, and skin sensitization, primarily driven by its alpha,beta -unsaturated amide moiety. Experimental data support its mutagenic potential in vitro, though in vivo mutagenicity results are mixed. Carcinogenicity studies in rats indicate possible low-dose carcinogenic effects, while developmental toxicity data are generally reassuring at higher doses but highlight neurodevelopmental concerns at low concentrations in zebrafish. Ecotoxicity assessments reveal low to moderate toxicity to aquatic organisms, with NOECs typically exceeding 2 mg/L. The predicted metabolites suggest detoxification pathways involving oxidative hydrolysis and conjugation, reducing reactive potential. A comprehensive read-across strategy, integrating structural and mechanistic similarities, is recommended to address data gaps, particularly in long-term carcinogenicity, neurotoxicity, and mechanistic pathways.

43. O-QT Assistant – Acrylamide Example Toxicity Profile and Mechanisms Experimental (Toolbox) Data Summary Mutagenicity: Bacterial assays (OECD Guideline 471) consistently report negative results. Mammalian cell mutation tests are also negative, but some in vivo transgenic assays show positive mutagenic activity, indicating potential mechanistic concerns. Carcinogenicity: In rat studies, positive carcinogenic effects are observed at low doses (~0.5 mg/kg bw /day), though some data are filtered or inconsistent. The overall evidence suggests a potential carcinogenic hazard. Developmental Toxicity: In rats, NOAELs 0.5 mg/kg bw /day show no adverse developmental effects. Zebrafish studies at low mg/L concentrations report neurodevelopmental and skeletal effects, with some statistically significant findings, indicating possible neurotoxicity at environmentally relevant concentrations. Neurotoxicity: Repeated dose assessments rank neurotoxicity as Category C, aligning with known neurotoxic effects of acrylamide. Other Endpoints: • Skin permeability is high, increasing dermal exposure risk. • Blood-brain barrier permeability is poor, limiting CNS exposure via systemic routes. • Structural alerts indicate electrophilic reactivity, supporting mechanisms involving covalent binding to proteins and DNA.

44. O-QT Assistant – Acrylamide Example Profiling (QSAR Toolbox) Insights • Structural features ( alpha,beta -unsaturated amide) underpin alerts for genotoxicity, carcinogenicity, and skin sensitization. • Protein binding alerts suggest covalent interactions with skin proteins and nucleophilic amino acids, consistent with electrophilic reactivity. • The absence of DNA binding alerts from OECD models indicates that DNA interaction may be indirect or mediated via reactive metabolites. • Reactivity with GSH (moderately reactive) supports potential for conjugation and detoxification pathways. Implications: Acrylamide’s electrophilic nature underpins its genotoxic and carcinogenic potential, with additional concerns for skin sensitization and neurotoxicity. Its rapid biotransformation and high water solubility facilitate systemic and dermal absorption, but environmental

45. O-QT Assistant – Acrylamide Example Ecotoxicity and Environmental Impact Summary: Acrylamide exhibits low to moderate acute toxicity to aquatic organisms, with NOECs generally above 2 mg/L for algae and invertebrates, and above 10 mg/L for fish. Its high water solubility and mobility suggest potential for widespread environmental distribution, emphasizing the need for environmental monitoring.

46. PubChem Chemical Identity & Structure 🧪 Descriptors, identifiers, physicochemical data → QSAR & property modeling. Biological & Mechanistic Data 🧬 Reactions, genes, pathways → Mechanistic links & AOP mapping. Toxicity, Safety & Disease ☣️ Toxicity endpoints, hazards, diseases → Hazard identification & dose–response. Use & Manufacturing 🏭 Industrial categories, production volumes → Exposure potential. Literature & Patents 📚 Scientific & regulatory evidence → Weight-of-evidence validation. Analytical & Reference Data 🔬 Spectra, reference sources, QC → Data verification & reliability. https://pubchempal.edelweissconnect.com/

47. PubChem - Example Result on Toxicity (Acrylamide)

48. PubChem - Example Result on Toxicity (Acrylamide) The dataset lists acute toxicity (LD₅₀) data for multiple species and exposure routes compiled from historical literature. Interpretation • Oral LD₅₀s across species range roughly 100–150 mg/kg bw , indicating moderate acute toxicity (Globally Harmonized System (GHS) Category 3). • Intraperitoneal routes show higher potency (LD₅₀ ≈ 90 mg/kg bw ), consistent with systemic distribution bypassing first-pass metabolism. • Dermal exposures are less toxic (LD₅₀ ≈ 400 mg/kg in rats; ~1,700 µL/kg in rabbits), reflecting lower absorption through intact skin. • The dataset includes observed effects such as tremor, convulsions, hallucinations, and blood chemistry changes, consistent with acrylamide’s neurotoxic mechanism. Context with Chronic / Benchmark Data Compared with acute LD₅₀s, chronic studies (EFSA, JECFA) yield: • BMDL₁₀ (tumours): 0.17–0.31 mg/kg bw /day • BMDL₁₀ (neurotoxicity): 0.43 mg/kg bw /day This demonstrates that chronic critical effect levels are ~300×–700× lower than acute lethal doses, aligning with acrylamide’s classification as a genotoxic carcinogen and neurotoxicant.

49. PubChem - Example Result on Bioassays (Acrylamide) Overall conclusion The PubChem bioassay profile of acrylamide confirms broad but mechanistically coherent biological activity, dominated by: • DNA-damage and stress-response signaling (PARP1, TP53 ) • apoptosis and oxidative stress (FAS, NR3C1 ) • secondary endocrine and cytotoxic effects at higher concentrations (ESR1, CCND1) This dataset reinforces acrylamide’s categorisation as a genotoxic neurotoxicant Overview of the dataset • Total assays: ≈ 80 bioassays • Assay types: Predominantly Confirmatory (high-confidence) assays with measurable dose-response curves (≈ 80 %). • Activity metric: Activity Value represents reported bioactivity concentration or effect level (e.g. µM, % effect). • Targets covered: ≈ 70 unique human and mammalian gene/protein targets. • Range of reported activity: 0.0012 → ~7,700 (µM or equivalent effect units).

50. PubChem - Example Result on Pathways (Acrylamide) The dataset links Acrylamide to four primary biological pathways across human and microbial systems, mainly derived from WikiPathways and BioCyc databases. 1. Human Pathway: Acrylamide Biotransformation and Biomarkers Source: WikiPathways WP4233 (PMID 29302712; DOI 10.1007/s00204-017-2143-2) Associated CIDs: 6287 ( glycidamide ), 6579 (acrylamide), 91550 (mercapturic acid conjugates), 124886 ( hemoglobin adducts) Mechanistic outline: 1. Absorption → Acrylamide enters via diet, inhalation, or dermal uptake. 2. Metabolic activation via CYP2E1 → formation of glycidamide , an epoxide metabolite responsible for genotoxicity. 3. Detoxification pathways: o Glutathione conjugation → mercapturic acids (excreted in urine) o Hemoglobin adduct formation → biomarkers of exposure 4. DNA and protein adduction → genotoxic and neurotoxic outcomes. Key gene link: CYP2E1 (Gene 1571) — central to oxidative metabolism of acrylamide. Toxicological implication: this pathway directly supports the Mode of Action (MOA) involving bioactivation, DNA reactivity, and cumulative neurotoxicity.

51. PubChem - Example Result on Pathways (Acrylamide) Microbial and Environmental Degradation Pathways Sources: BioCyc acrylonitrile degradation superpathways These describe microbial metabolic routes converting acrylonitrile → acrylamide → acrylic acid → propionate , involving: Nitrile hydratase (EC 4.2.1.84) and amidase enzymes. The enzymes P27763 and P27764 (from Rhodococcus spp.) catalyze conversion of acrylonitrile to acrylamide. Environmental or biotechnological relevance: microbial bioremediation of acrylamide precursors , not direct human toxicity. Toxicological context: While not human pathways, these microbial routes demonstrate potential environmental detoxification processes—important for SSbD life-cycle risk assessment of acrylamide and related nitriles. Interpretation • The human biotransformation pathway (WP4233) provides mechanistic grounding for your Tier-1 in silico models and Tier-2 in vitro validation, linking molecular initiating events (CYP2E1 activation → glycidamide formation → DNA/protein adducts) to observed neurotoxic and carcinogenic effects. • The microbial pathways support SSbD environmental dimensions, highlighting possible biodegradation routes for mitigation and circularity analyses. • Together, these confirm that acrylamide’s risk profile is defined by a balance between bioactivation (toxicity) and biotransformation (detoxification).

52. EPA CompTox 🧪 Hazard Identification Bioactivity Assays Data: Reveal chemical effects on biological systems and target mechanisms. Bioactivity Data: Provide potency and activity profiles across assays, supporting mode-of-action analysis. Cancer Data: Contain carcinogenicity and tumor information for chronic hazard evaluation. ⚗️ Dose–Response Assessment Exposure HTTK Data: Provide toxicokinetic parameters (e.g., clearance, partition coefficients) for linking in vitro to in vivo effects (IVIVE). Bioassay and potency data: Support derivation of NOAEL, LOAEL, and benchmark doses for quantitative modeling. https:// comptox.epa.gov /dashboard/

53. EPA CompTox 🌍 Exposure Assessment Chemical Data: Include physical–chemical properties (e.g., solubility, logP) for estimating environmental and biological distribution. Chemical Fate Data: Describe degradation, persistence, and bioaccumulation behavior in the environment. Exposure Functional Use Category Data: Identify product types and industrial uses where chemicals appear. Exposure Functional Use Data: Specify detailed use patterns across consumer and occupational settings. Exposure Functional Use Probability Data: Quantify likelihood of use in each category for probabilistic modeling. Exposure List Presence Data: Show occurrence of chemicals in regulatory and consumer product inventories. 🧩 Risk Characterization Integrates hazard , dose–response , and exposure information. Supports estimation of risk metrics such as Margin of Exposure (MOE) or Reference Dose (RfD). Enables chemical prioritization for regulatory action or further testing. Reduces uncertainty by combining biological, toxicokinetic, and exposure data.

54. EPA CompTox - Example TK Results (Acrylamide)

55. SDS ToolBox ✅ Reliable & Traceable: Uses verified manufacturer SDS sources ensuring legal and scientific credibility. 🔄 Standardized & Integrated: Harmonizes diverse chemical data for use with regulatory and toxicological databases. 🧩 Comprehensive Coverage: Combines hazard, exposure, and management data for complete risk evaluation. 🤖 Supports Automation & AI: Enables digital workflows, predictive modeling, and machine-readable data use. ⚙️ Efficient Decision-Making: Speeds up chemical prioritization, classification, and regulatory reporting. 🧾 Regulatory Compliance: Aligns with REACH, GHS, and global safety documentation requirements. 🌱 Sustainability & SSbD: Aids in identifying safer alternatives and supporting sustainable product design. https://sds-toolbox.edelweissconnect.com/

56. SDS Toolbox - Example Result (Acrylamide)

57. SDS Toolbox - Example Result (Acrylamide) Overview of the dataset • Records analysed: 8 individual SDS (Safety Data Sheet) entries • Sources: Fisher Scientific, FUJIFILM Wako, Sciencelab , Serva, Spex Certiprep , etc. • Products covered: Acrylamide solids, solutions (30–40 %), and mixtures (acrylamide + bisacrylamide ). • Chemical identity: 2-Propenamide / Ethylenecarboxamide • Main use classes: Laboratory reagent, electrophoresis gel component, R&D or analytical reference material. • Typical physical form: White crystalline solid or aqueous solution.

58. SDS Toolbox – Hazard Result (Acrylamide) Classification domain Common entries across SDSs Summary interpretation GHS hazard class Acute Toxicity (Oral, Category 3); Acute Toxicity (Dermal Category 4); Skin Irritation Category 2; Eye Irritation Category 2A; Germ cell mutagenicity Category 2; Carcinogenicity Category 2; Reproductive Toxicity Category 2; Specific Target Organ Toxicity (Repeated Exposure) Category 2 Confirms moderate acute toxicity and clear chronic hazard potential (mutagenic, carcinogenic, neurotoxic). Signal word Danger High-concern substance requiring restricted professional use. Hazard statements H301, H312, H315, H319, H331, H340, H350, H361, H373, H401/H410 Reflects oral and inhalation toxicity , mutagenicity , carcinogenicity , and chronic organ effects . Transport classification UN 2074; Class 6.1 (Toxic Substance); Packing Group III Managed as a toxic solid/liquid under ADR/IMDG/IATA.

59. SDS Toolbox – Toxicology Result (Acrylamide) Endpoint Typical SDS entry Interpretation LD₅₀ (oral, rat) 107–124 mg/kg bw Confirms GHS Acute Tox. Cat. 3 (consistent with PubChem). LD₅₀ (dermal, rat/rabbit) ~400 mg/kg bw Moderate dermal toxicity. Inhalation toxicity LC₅₀ > 3 mg/L (4 h, rat) Low volatility but toxic vapours possible at high temperature. Carcinogenicity “Suspected of causing cancer” (IARC Group 2A) Aligns with EFSA/JECFA evaluation via glycidamide metabolite. Mutagenicity / Genotoxicity Positive in vitro and in vivo Mechanistic driver for carcinogenic risk. Reprotoxicity / Neurotoxicity Possible reproductive effects; neurotoxicity through axonal degeneration Mechanistic link to Tier-1 AOP (neurotoxicity). Aquatic toxicity H401 / H410 — harmful to aquatic organisms Assigns Aquatic Acute 2 / Chronic 2 ; relevant for SSbD life-cycle analysis.

60. ECHA Db (CLP) compound Acute Tox. Aquatic Acute Aquatic Chronic Asp. Tox. Carc. Eye Dam. Eye Irrit. Flam. Liq. Muta. Repr. STOT RE STOT SE Skin corr. Skin Irrit. Skin Sens. n-Hexane 2 1 2 2 1 3 2 Pyraclostrobin 3, 4 1 1 2 2 3 2 Carboxin 1 1 2 1 Micotine 2, 2, 2 2 Acrylamide 3, 4, 4 1B 2 1B 2 1 2 1 Tetrabromobisphenol A (TBBPA) 1 1 1B Bisphenol A (BPA) 1 1 1 1B 3 1 Bisphenol S (BPS) 1B Rotenone 3 1 1 2 3 2 Dimethyltin dichloride 2, 3, 3 2 1 1B Methyltin mercaptide 4 2 1 1A Sodium pyrithione No data was found for sodium pyrithione.

61. ECHA Db ( REACH dossiers ) Filtered fields: Key study GLP compliance: yes Reliability: 1 (reliable without restrictions) or 2 (reliable with restrictions) Avoided endpoints for skin sensitisation , corrosion, irritation, eye irritation compound Toxicological endpoint Effect levels Acrylamide Acute toxicity: oral LD0: 200 mg/kg bw (rat) LD50: 354 mg/kg bw (rat) Carcinogenicity: oral NOAEL: 0.5 mg/kg bw/day (rat) Chronic toxicity: oral NOAEL: 0.5 mg/kg bw/day (rat) 2-generation reproductive toxicity NOAEL: 2 mg/kg bw/day (rat) n-Hexane Carcinogenicity: inhalation NOAEC: 9016 ppm (rat) NOAEC: 3000 ppm (mouse female) LOAEC: 9018 ppm (mouse female) NOAEC: 9018 ppm (mouse male) In vitro gene mutation study negative 2-generation reproductive toxicity negative NOAEC 9000 ppm (inhalation) Manganese Acute toxicity: inhalation LC50: >5.14 mg/L air (rat) Acute toxicity: oral LD50: >2000 mg/kg bw (rat) Sub-chronic toxicity: inhalation NOAEL: 0.5 ug/L (rat) Bisphenol A (BPA) Carcinogenicity: oral Between 2.7 and 300 mg/kg bw/day (rat) Carcinogenicity: oral NOAEL: 25,000 mg/kg bw/day (rat) Sub-chronic toxicity: inhalation LOEC: 10 mg/m3 air (rat) NOEC: 10 mg/m3 air (rat) RDT: oral NOEL: 30 ppm (mouse) NOAEL: 300 ppm (mouse) NOEL: 75 ppm (rat) NOAEL: 750 ppm (rat) Short-term RDT: oral LOAEL: 600 mg/kg bw/day 1-generation reproductive toxicity NOAEL: 2.7 mg/kg bw/day (rat) 2-generation reproductive toxicity NOAEL: 0.2 mg/kg bw/day (rat) Bisphenol S (BPS) Sub-chronic toxicity: oral No effects (rat, 90 days) Extended 1-generation reproductive tox. NOAEL: 20 mg/kg bw/day (rat) Tetrabromobisphenol A (TBBPA) Acute toxicity: oral LD50: >5000 mg/kg bw/day (rat) Acute toxicity: dermal LD50: >2 g/kg bw (rat) Sub-chronic toxicity: oral NOAEL: >1000 mg/kg bw/day (rat) Nicotine Acute toxicity: dermal LD50: 70.4 mg/kg bw (rabbit) Short-term RDT: inhalation No effects observed Reproductive/developmental toxicity NOAEC: 20 mg/m3 air (rat) Dimethyltin dichloride Acute toxicity: dermal LD50: 404 mg/kg bw (rabbit) Acute toxicity: oral LD50: 164 mg/kg bw (rat) Sub-chronic toxicity: oral NOAEL: 0.98 mg/kg bw/day (rat male) NOAEL: 1.02 mg/kg bw/day (rat female) Methyltin mercaptide Acute toxicity: oral LD50. 1150 mg/kg bw/day (rat) Sub-chronic toxicity: oral NOAEL: 60 ppm (rat) Short-term RDT: oral NOAEL: 175 ppm (rat) Sodium pyrithione Acute toxicity: dermal LD50: 1800 mg/kg bw (rabbit) Acute toxicity: inhalation LC50: 1.08 mg/L air (rat) Acute toxicity: oral LD50: 1208 mg/kg bw (rat) RDT: dermal NOEL: 5 mg/kg bw/day (mouse) RDT: oral LOAEL: 1.5 mg/kg bw/day (rat) NOAEL: 0.5 mg/kg bw/day (rat) Sub-chronic toxicity: inhalation LOAEL: 8.1 mg/m3 air (rat) NOAEL: 0.46 mg/m3 air (rat)

62. NeuroToxicity pLD50 Results (Tomaz Mohoric )

63. NeuroToxicity pLD50 Results (Tomaz) Model training and test set: All the tested compounds fall roughly in the centre of the training set’s pLD50.

64. NeuroDeRisk Results Results included additional of predicted metabolites

65. MultiCase Results

66. MultiCase Model Results (Example sub-section - Part 1)

67. MultiCase Model Results Results (Example sub-section - Part 2)

68. MultiCase Model Results Results (Example sub-section - Part 3)

69. Kreatis Results ( MechoA )

70. Kreatis Results ( MechOA )

71. SSbD

72. SSbD- Step 1 Scoring system JRC (2023).Technical report- Application of the SSbD framework to case studies; doi:10.2760/329423 JRC (2024). Safe and Sustainable by Design chemicals and materials - Methodological Guidance; doi: 10.2760/28450

73. SSbD- Step 1 Results Carc (carcinogenicity), Repr (Reproductive toxicity), Skin Sen (Skin sensitization), STOT RE (Specific Target Organ Toxicity - Repeated Exposure) National Institute of Technology and Evaluation (NITE) of Japan GHS Classification Results Canada: Canada CNESST Workplace Hazardous Materials Information System This is a first-tier assessment. Some chemicals are not yet classified for specific hazard, and not all relevant endpoints are currently covered.

74. AI-based Assessment Acrylamide Case (ChatGPT 5)

75. AI-based Assessment (Acrylamide Case) 1) Hazard summary (what acrylamide does) • Forms during high-temperature frying via Maillard reactions (asparagine + reducing sugars). • Genotoxic & carcinogenic potential (via bioactivation to glycidamide ) and neurotoxicity with repeated exposure. • For risk characterisation, a Tier-1 benchmark approach typically uses BMDL10 ≈ 0.17 mg/kg bw /day (cancer) and ≈ 0.43 mg/kg bw /day (neurotoxicity) and evaluates MOE (Margin of Exposure); for genotoxic carcinogens, MOE ≳ 10,000 is generally considered of low concern.

76. AI-based Assessment (Acrylamide Case) 2) Tier-1 exposure & MOE (illustrative) Assumptions (typical quick-service context; replace with your own measurements if available): • Acrylamide in fries: ~300–1000 µg/kg (golden to dark; varies by potato, storage, time/temperature). • Portion: 150 g (0.15 kg). • Adult body weight: 70 kg. Step-by-step: • Intake per portion = concentration × portion = 300–1000 µg/kg × 0.15 kg = 45–150 µg (= 0.045–0.150 mg). • Dose = intake / bw = 0.00064–0.00214 mg/kg bw . • MOE (cancer) = 0.17 / dose ≈ 260–80. • MOE (neuro) = 0.43 / dose ≈ 670–200. Interpretation: these MOEs are far <10,000, indicating that keeping acrylamide “as low as reasonably achievable (ALARA)” is appropriate. Note: the MOE framework addresses chronic average exposure; a single portion has little absolute impact, but frequent consumption can materially increase average exposure. Children’s sensitivity (illustrative): a 30 kg child, 100 g portion at 500 µg/kg → dose ≈ 0.00167 mg/kg bw → MOE(cancer) ≈ 100 (also well below 10,000).

77. AI-based Assessment (Acrylamide Case) 3) Worker exposure (preparation) • Primary routes: inhalation of cooking fumes/aerosols; incidental dermal contact with oils/surfaces. Dietary intake off-shift will typically dominate total acrylamide exposure, but worker inhalation is chronic and should be controlled. • Co-pollutants (aldehydes/particulates) co-form in hot-oil fumes—good ventilation controls both. 4) Risk-reduction measures (practical and high-impact) For kitchen operations (process controls) • Time–temperature: keep fry oil ≤175–180 °C; avoid over-frying; use timers and batch mass limits to prevent darkening. • Colour target: train crews to “go for golden, not dark brown”; remove very dark pieces. • Potato management: o Use low-sugar varieties and supplier-mitigated products (asparaginase/processing steps upstream). o Do not refrigerate raw potatoes (cold sweetening raises sugars). Prefer storage around 8–12 °C. o Rinse/blanch cut chips to wash out surface sugars (where workflow allows). • Oil quality: avoid over-used oil; filter/refresh per manufacturer guidance—limits thermal degradation and colour drift. • Ventilation: ensure effective capture hoods over fryers; maintain filters and airflow; keep lids/screens fitted when possible.

78. AI-based Assessment (Acrylamide Case) • Verification: periodically spot-check acrylamide with supplier certificates or occasional lab tests on finished fries; fold results into SOPs. For workers (exposure & hygiene) • Keep hoods on; avoid leaning into plume; rotate tasks on long shifts. • Gloves for cleaning residues/filters; prompt hand-washing before eating. • Maintain clean, cool fry stations; remove carbonised residues that promote darkening. For consumers (simple guidance) • Prefer golden-coloured fries, not overly crisp/dark. • Moderate portion size and frequency across the week. • For children, share portions or choose smaller sizes. • Overall diet matters: diversify starchy foods and cooking methods (boiling/baking at lower colour). 5) Communication & monitoring • Embed a one-page SOP + colour chart at the station; train new staff within onboarding. • Include acrylamide minimisation in supplier QA (specs for potato sugar content, pre-treatments). • Track oil temperature, change-out intervals, and colour compliance as process KPIs.

79. AI-based Assessment (Acrylamide Case) Bottom line • Acrylamide cannot be eliminated from high-temp frying, but simple process controls (time-temperature, colour target, potato management, ventilation) materially reduce levels. • From a Tier-1 MOE perspective, keeping fries “golden” and moderating portion/frequency offers the clearest benefit for consumers, while good ventilation and SOP discipline protect workers. • For higher assurance, pair these measures with periodic product testing and supplier mitigation to demonstrate ALARA and continuous improvement.

80. Initial Tier 2/3 NAMs Assessment Acrylamide Case (BASED ON CHATGPT ANSWER)

81. Tier 2/3 Assessment (Acrylamide Case) What NAMs already tell us (evidence snapshot) Genotoxicity (metabolically mediated) • In human TK6 cells, glycidamide (GA)—the CYP2E1-activated epoxide of acrylamide—produces DNA strand breaks (comet), micronuclei, and TK mutations; acrylamide (AA) is weak/negative unless metabolic activation is provided. This establishes GA as the proximate genotoxicant . • EFSA’s 2015 opinion and a 2022 EFSA statement confirm AA is a genotoxic carcinogen via GA, supporting an MOE-based risk framework (no safe threshold). Neurotoxicity / developmental neurotoxicity • Human mixed neuron–astrocyte cultures show developmental neurotoxic perturbations from AA/GA at concentrations near those seen in biomonitoring-informed ranges, affecting neurite outgrowth/synaptogenesis-related endpoints (Tier-2 relevance). • Reviews converge on distal axonopathy / presynaptic dysfunction as key events; human-relevant mechanistic coherence is strong. • Human iPSC-derived neuronal systems (MEA and cytotoxicity panels) are feasible for detecting AA-class neurotoxicants and comparing potencies across neuronal subtypes.

82. Tier 2/3 Assessment (Acrylamide Case) High-throughput bioactivity ToxCast /Tox21-style assays (as we saw in PubChem) flag DNA-damage response (PARP1, TP53), apoptosis (FAS), stress/glucocorticoid signalling, consistent with the GA-mediated genotoxic MOA and secondary stress pathways. (Program overview for context.) What this adds to Tier-1 (uncertainty reduction) U1 — Causation clarity: NAMs resolve the AA vs GA question: GA drives genotoxicity; AA needs bioactivation. This reduces mechanistic uncertainty behind the cancer POD (your BMDL₁₀s). U2 — Human relevance: Human neuronal/glial cultures reproduce neurodevelopmental KEs seen in vivo (neurite/synapse effects), strengthening biological plausibility for your SaferNeuron case studies. U3 — Potency anchoring for IVIVE: In vitro AC₅₀/benchmark concentration bands from genotoxicity and neurofunction assays can be translated via PBPK (CYP2E1-linked) to human-equivalent doses, narrowing exposure → effect extrapolation uncertainty in your MOE calculus. (EFSA confirms MOE framing.) U4 — Weight-of-evidence: Agreement across genotoxic NAMs, neuronal NAMs, and HTS stress pathways yields a convergent mechanistic narrative, improving confidence in your Tier-1 conclusions.

83. Tier 2/3 Assessment (Acrylamide Case) Tier-2 / Tier-3: what to run now (maximum value) 1. Genotoxic NAMs with metabolic competence (Tier-2) • Human HepaRG or primary hepatocyte co-cultures + comet & micronucleus (TG-aligned methods) to capture CYP2E1-driven GA formation in-system; optionally add hOGG1/ Fpg -modified comet to fingerprint oxidative/epoxide lesions. Outcome: human-relevant potency (BMC/BMD- conc ) for IVIVE. 2. Human iPSC neuronal networks on MEA (Tier-2/3) • Record spike/burst metrics, synchrony, and neurite morphology after AA vs GA ± metabolic supply (microsomes/S9 or hepatocyte co-culture). Outcome: functional NOEC/BMCs for presynaptic dysfunction KE; strengthens neuro AOP link. 3. Developmental neurotoxicity battery (Tier-3) • Mixed neuron–astrocyte human cultures across neurodevelopmental windows (proliferation → differentiation → network formation) to derive benchmark concentrations for neurite/synapse endpoints. Outcome: human DNT potency ranking; compare AA vs GA. 4. Targeted adductomics / biomarkers (Tier-3) • Hb/N-terminal valine adducts (ex vivo spiking) and DNA/protein adduct profiling in NAM systems to bridge to human biomonitoring used by EFSA; aligns in vitro potency with exposure biomarkers. Outcome: tighter TK linkage to real-world exposure. 5. PBPK-IVIVE module (Tier-3 integration) • Build/extend a CYP2E1-parameterised PBPK model for AA→GA with glutathione conjugation; translate NAM BMCs → human-equivalent doses; produce NAM-anchored MOE bands for your scenarios.

84. Tier 2/3 Assessment (Acrylamide Case) Does the NAM package improve uncertainty & interpretation? • Yes—Mechanism: Confirms bioactivation-driven genotoxic MOA; clarifies why cancer PODs are appropriate and why MOE is the right decision metric. • Yes—Potency: Provides human-cell BMCs to constrain dose–response slopes used in IVIVE (narrower credible intervals vs animal-only). • Yes—Human relevance in neurotoxicity: Functional neuronal readouts (MEA) anchor the SaferNeuron KE and reduce cross-species extrapolation. How to report in your Case dossier • Tier-2 summary table: assay → cell system → endpoint → BMC (with CI) → IVIVE HED → derived MOE vs your fast-food scenarios. • Tier-3 integration figure: AOP schematic showing AA → (CYP2E1) → GA → DNA/protein adducts → p53/PARP activation → apoptosis/axonopathy, annotated with NAM potency bands and EFSA BMDL₁₀ lines.

85. Below here – additional cases to work on

86. A. Tebufenpyrad (Pesticide Application on Grapes) Occupational Scenario A professional vineyard worker is preparing and spraying a pesticide mixture to protect grapevines from mites. The product contains tebufenpyrad , a chemical that targets the nervous system of small insects and mites. The worker mixes the pesticide in a tank and uses a handheld or backpack sprayer to apply it along vine rows for several hours during the morning. Although they wear gloves, a hat, and overalls, it’s a warm day and there is moderate wind, so some spray mist and residues contact the worker’s hands, forearms, and face. Small amounts can also be inhaled with the mist. These short-term exposures happen several times each growing season. This scenario represents a typical occupational situation where protective gear and good practices minimize risk but do not eliminate it completely. Input Data Vineyard or greenhouse pesticide application (mixing/loading/spraying) Exposure Routes Dermal (hands, forearms, face), Inhalation (fine droplets) Exposure Dermal: 0.02 mg/kg bw/day Inhalation: 0.001 mg/kg bw/day Total Systemic Exposure ≈ 0.021 mg/kg bw/day Toxicological Reference (AOEL) 0.025 mg/kg bw/day

87. A. Tebufenpyrad (Pesticide Application on Grapes) Occupational Risk Assessment Summary Total systemic exposure : 0.021 mg/kg bw /day Dermal: 0.020 (≈ 95% of total) <- risk driver Inhalation: 0.001 (≈ 5% ) Toxicological reference (AOEL) : 0.025 mg/kg bw /day Risk quotient (RQ = Exposure/AOEL) : 0.84 Margin of safety (MOS = AOEL/Exposure) : 1.19 Interpretation: Exposure is below the AOEL (RQ < 1), indicating acceptable risk for daily operator exposure under typical conditions— but with a narrow safety margin . Decision Acceptable with targeted risk mitigation. Prioritise controls that reduce dermal exposure to increase the MOS above ~2 where practicable. Follow-up / Monitoring Aim to halve dermal exposure through the above controls (target total ≤ 0.010 mg/kg bw /day → MOS ≳ 2.5 ). Keep a simple exposure log (weather, PPE used, task duration) and perform a seasonal review . If available, consider biomonitoring or task-based exposure measurements to validate assumptions.

88. A. Tebufenpyrad (Pesticide Application on Grapes) Consumer Exposure (Eating Grapes) A parent buys a bag of table grapes at the supermarket during peak season. The grower used a mite control product containing tebufenpyrad earlier in the season, and tiny traces can remain on the fruit by harvest. After a quick rinse under the tap, the family eats the grapes as a snack. Food authorities set legal residue limits to keep these traces very low, and they regularly tighten them when new data arrives. Even so, how much a person takes in depends on how many grapes they eat, their body weight, and the residue level found in that batch. The quick check below shows what a typical adult and a child would take in if residues are at the current EU limit level for grapes, and whether that’s within health-based guidance values. Input Data Residue level used for screening: 0.30 mg/kg Health-based values for consumers (EU/EFSA): ADI (chronic): 0.01 mg/kg bw /day (order of magnitude used in EFSA assessments for tebufenpyrad) ARfD (acute): 0.10 mg/kg bw (EFSA peer-review era; used for acute screening)

89. A. Tebufenpyrad (Pesticide Application on Grapes) Assumptions (conservative but realistic) Adult: 70 kg bw , 200 g grapes (single snack) Child: 20 kg bw , 100 g grapes (single snack) Rinse scenario: simple tap rinse → processing factor ~0.8 (20% reduction) Health-based values: ADI = 0.01 mg/kg bw /day , ARfD = 0.10 mg/kg bw Results (per snack at residue 0.30 mg/kg) MOS (margin of safety) = Reference value / exposure. The Acceptable Daily Intake (ADI) is the maximum amount of a chemical substance (usually a pesticide, food additive, or veterinary drug residue) that can be ingested daily over a lifetime without appreciable risk to health. ADI = NOAEL/Safety Factors. The Acute Reference Dose ( ARfD ) is the estimated amount of a substance that can be ingested in a single meal or within 24 hours without appreciable health risk to the consumer. ARFD = Acute NOAEL/Safety Factors. Consumer Portion Intake (mg/kg bw) %ADI Chronic MOS %ARfD Acute MOS Adult 200 g 0.000857 8.6% 11.7 0.86% 116.7 Child 100 g 0.00150 15% 6.7 1.5% 66.7 Adult (rinsed) 200 g 0.000686 6.9% 14.6 0.69% 145 Child (rinsed) 100 g 0.00120 12% 8.3 1.2% 83.3

90. B. Domoic Acid (Consumer Risk Assessment) Scenario (Consumer food exposure) Imagine someone buying a portion of fresh blue mussels from a local fish market and preparing a generous seafood meal at home. The mussels come from an area where naturally occurring marine algae sometimes produce a toxin called domoic acid . When people eat mussels that contain this toxin, it can affect the brain and cause nausea, dizziness, or in severe cases, memory problems — a condition known as amnesic shellfish poisoning . Although food safety authorities set legal limits to prevent harmful levels from reaching consumers, a person who eats a large meal — for example, a full 350-gram plate — could still take in more of the toxin than is considered completely safe for a single sitting. This example shows how portion size and contamination level together determine whether a seafood meal is safe, and why public health agencies test shellfish regularly and may issue warnings or harvest closures during algal blooms. Input Data Measured concentration (C): 8 mg/kg shellfish meat (compliant sample) Portion size (P): 350 g cooked mussels (large adult meal) Body weight (BW): 70 kg adult Toxicological reference: ARfD = 30 µg/kg bw (EFSA) Regulatory limit (EU/US): 20 mg/kg shellfish meat (action/maximum level)

91. B. Domoic Acid (Consumer Risk Assessment) %ARfD = 40/30×100= 133% MOS (ARfD/Dose) = 30/40= 0.75 Result: For a 350 g meal at 8 mg/kg, the ARfD is exceeded (133%); acute risk not acceptable for that meal size, despite the sample being compliant. • A 350 g meal at 8 mg/kg exceeds the ARfD for a 70-kg adult; portion control is required. • Children reach the ARfD at much smaller portions; large servings are not advisable when domoic acid is present.

92. D. n-hexane (occupational exposure) Scenario (occupational exposure in a shoe/handbag workshop – spray adhesive) A small leather workshop uses a fast-drying spray adhesive to bond shoe uppers and linings. The adhesive contains n-hexane, a very volatile solvent. On busy days, a worker spends a couple of hours spraying inside a bench-top booth but often finishes gluing at the worktable when the booth is occupied. The room is warm, and the local exhaust is weak, so a noticeable solvent smell builds up. N-hexane can damage the peripheral nerves after repeated exposure (through its metabolite 2,5-hexanedione), causing tingling, numbness and weakness in hands and feet. Good ventilation, limiting spray time in open areas, and choosing safer products are key to keeping exposure low. Input Data Acute hazard/flammability: Very volatile; LEL 1.1% (11,000 ppm); NIOSH IDLH 1,100 ppm (set at 10% LEL for safety). Class IB flammable liquid. Key OELs for comparison ACGIH TLV-TWA: 50 ppm (8-h). NIOSH REL-TWA: 50 ppm (10-h). OSHA PEL-TWA (legal US): 500 ppm (8-h) — widely viewed as outdated/insufficiently protective. Biomonitoring: ACGIH BEI for free 2,5-hexanedione in urine = 0.4 mg/L (end-of-shift/end-of-week). Example exposure profile Task 1 – Spray in booth (2 h): 150 ppm (weak capture) Task 2 – Hand spraying at bench (1 h): 200 ppm (no LEV) Task 3 – Open adhesive use/assembly (5 h): 20 ppm background

93. D. n-hexane Tasks and concentrations (given): • Spray in booth (2 h): 150 ppm • Hand spray at bench (1 h): 200 ppm • Open use/assembly (5 h): 20 ppm TWA = (2 x 150) + (1 x 200) + (5 x 20) / 8h = 75 ppm Comparison to limits / guidance • ACGIH TLV-TWA (8 h): 50 ppm → Exceeded (75 ppm = 150% of TLV) • NIOSH REL-TWA (10 h): 50 ppm → Exceeded • OSHA PEL-TWA (8 h): 500 ppm → Below (widely regarded as outdated / not sufficiently protective) Health interpretation: At 75 ppm TWA, chronic neurotoxicity risk (via 2,5-hexanedione) is elevated; control measures are warranted . TWA = The Time-Weighted Average (TWA) is the average exposure concentration to a chemical or physical agent over a specified reference period, typically an 8-hour work shift (for daily exposure) or a 40-hour work week (for long-term exposure).

94. D. n-hexane (risk management) Engineering: · Do all spraying in effective Local Exhasut Ventilation (LEV.) Set booth face velocity around 0.5 m/s (~100 fpm) ; add capture hood/slot extraction at the bench for any hand spray. · Fix weak capture : verify airflow, duct sizing, filter loading, and make-up air; keep workpiece inside hood capture zone . · General ventilation : increase Air Changes per Hour (ACH) and ensure directional flow away from workers. · Substitution : move to hexane-free or low-n-hexane adhesives (cyclohexane-free is also preferable) or water-borne/hot-melt systems where feasible. Administrative: · Eliminate open-area spraying ; schedule tasks so the booth is available when needed. · Limit total spray time per shift; lid containers and use small transfer cups to reduce evaporative background. · Training on spray technique, hood positioning, and housekeeping (rags, waste). · Hot-work controls and no ignition sources; store class IB liquids properly. PPE (as a back-up, not a substitute for LEV): · Respiratory protection when spraying until engineering fixes verified: half-mask APR with organic vapor (OV) cartridges ; implement a cartridge change-out schedule (solvent breakthrough can be rapid). · Gloves with solvent resistance (e.g., laminate/PE-EVOH or thick nitrile ); prompt change if contaminated. · Safety glasses/face shield for splash; protective clothing to prevent skin contact. Biomonitoring / health surveillance: · Use ACGIH BEI : free 2,5-hexanedione in urine = 0.4 mg/L (end-of-shift / end-of-week) to verify controls. · Periodic checks for peripheral neuropathy symptoms (tingling, numbness, weakness) and function.

95. E. Nicotine (occupational exposure) Scenario (Occupational - small e-liquid filling room) A two-person team fills and caps nicotine e-liquid pods in a back-room workshop. They dilute a nicotine concentrate and then run a semi-automatic filler for several hours. The room has a bench hood, but one operator often mixes on the open bench when the hood is busy. You can smell solvent and a faint tobacco-like odor. During the shift, a few droplets splash on a glove cuff and, once, a small splash reaches bare wrist skin before it’s wiped away. Nicotine can pass through the skin quickly and also be inhaled as vapor or fine mist; repeated exposures can cause nausea, dizziness, headache, and, at high doses, more serious poisoning. Good ventilation, strict “all mixing in the hood,” and skin protection are the main defenses. Input Data Exposure limits: ACGIH Threshold Limit Value - Time Weighted Average (TLV-TWA): 0.002 ppm (0.013 mg/m³); Short-Term Exposure Limit - 15 minutes (STEL) 0.05 mg/m³; skin. National Institute for Occupational Safety and Health - Recommended Exposure Limit (Time-Weighted Average) for 10h work day, 40h work week NIOSH REL-TWA: 0.5 mg/m³; skin U.S. Occupational Safety and Health Administration, Permissible Exposure Limit, maximum average concentration during an 8h work day, (older) legal limit; OSHA PEL-TWA (US): 0.5 mg/m³; skin NIOSH Immediately Dangerous to Life or Health - 30 minutes or less (IDLH): 5 mg/m³ Critical point: NIOSH skin profile shows dermal uptake can dominate; computed skin-to-inhalation dose ratio ≈ 998 at typical limits → skin exposure can overwhelm inhalation controls if not managed. Example exposure profile: Air concentrations (personal): mixing at hood 0.01 mg/m³ (2 h); open-bench mixing 0.04 mg/m³ (1 h); automated filling/room background 0.008 mg/m³ (5 h). 8-h TWA = (2×0.01+1×0.04+5×0.008)/8= 0.014 mg/m³

96. E. Nicotine (occupational exposure) Exposure summary (inhalation) Given profile: 0.01 mg/m³ (2 h) + 0.04 mg/m³ (1 h) + 0.008 mg/m³ (5 h) → 8-h TWA = 0.014 mg/m³ . Limits (skin notation applies): ACGIH TLV-TWA: 0.013 mg/m³ (≈0.002 ppm); STEL: 0.05 mg/m³ NIOSH/OSHA TWA: 0.5 mg/m³ (less protective) Comparison: Versus TLV-TWA : 0.014 / 0.013 = 1.08 → 108% of TLV (slight exceedance). Short-term peak ( 0.04 mg/m³ ) is below STEL (0.05 mg/m³) , but STEL is a 15-min average ; verify with short-term sampling. Interpretation: Inhalation alone is marginally above the most protective limit.

97. E. Nicotine (occupational exposure) Dermal exposure Nicotine has a skin notation ; dermal uptake can dominate total dose (your note: skin-to-inhalation dose ratio ≈ ~10³ at typical limits). Observed splash to bare wrist and droplets on glove cuff = material risk even if air levels meet limits. Risk characterization Overall risk: Not acceptable as currently operated , due to (a) TLV exceedance on TWA and (b) credible dermal dosing events . Key contributors: open-bench mixing , inadequate hood availability/discipline , glove/forearm contamination , and background vapour/aerosol during filling.

98. E. Nicotine (occupational exposure - risk management (1)) Engineering (priority) All mixing in containment. Add a second bench hood or dedicate one strictly to mixing so operators never mix on open bench. Verify hood performance: face velocity ~ 0.5 m/s (~100 fpm) , sash height limits, smoke-pattern checks, alarms for low flow. Local capture at the filler (slot/backdraft hood or snorkel over nozzles) to cut background 0.008 mg/m³. Closed transfer for nicotine concentrates (sealed bottles, luer-lock syringes, quick-connects), spill-resistant beakers with lids. General ventilation: ensure adequate ACH (≥8–12) and clean make-up air; keep room slightly negative to adjacent spaces. Administrative Zero open-bench mixing policy; schedule work so a hood is always free. SOPs for decanting, filling, decontamination, and waste. Immediate decon for any skin contact: remove contaminated PPE, wash with soap/water ≥15 min; document event. Glove change rules: after any splash or 60–90 min of use, whichever first. No nicotine/tobacco use on site (avoids biomonitoring confounding). Training: skin hazards, symptoms (nausea, dizziness, headache), spill response, don/doff. Emergency : eyewash and tepid water within 10 s; stocked spill kit.

99. E. Nicotine (occupational exposure - risk management (2)) PPE (back-up to engineering) Gloves: laminate film (e.g., PE/EVOH “4H/SilverShield”) over or under thick nitrile ; long cuffs under a liquid-resistant forearm sleeve . Replace immediately if contaminated. Clothing: disposable lab coat or chemical-resistant gown with knit cuffs ; consider apron during mixing. Eye/face: chemical splash goggles; face shield for decanting. Respiratory (interim, until engineering fixes verified): half-mask APR with P100/OV combo if solvents present and any aerosol generation; fit-tested, with change-out schedule. Monitoring & targets Air: repeat personal sampling (full-shift + 15-min STELs during mixing/filling). Target TWA ≤ 0.006–0.008 mg/m³ (≥ factor-2 below TLV to account for skin). Dermal wipes at glove cuffs and wrists during pilot runs to confirm control of surface/splash. Biomonitoring: urinary cotinine (and trans-3′-hydroxycotinine) pre- and post-shift in confirmed non-users ; track trends vs. background. (No ACGIH BEI set for nicotine; use as internal performance metric , not pass/fail. Surface contamination mapping (wipe tests) to enforce cleaning standards around benches, fillers, and bottle capper Expected impact If engineering/admin controls cut: hood mixing 0.01→0.005 mg/m³, open-bench mixing eliminated, filling/background 0.008→0.004 mg/m³: New 8-h TWA ≈ (2×0.005+1×0+5×0.004)/8=0.0036mg/m³ → ~28% of TLV (comfortable). With dermal controls (no bare-skin events, clean cuffs), overall risk becomes acceptable.

100. E. Nicotine (Consumer exposure) Scenario (Consumer - Vaping) An adult uses a refillable pod e-cigarette after dinner. The pod holds 2 mL of liquid at 20 mg/mL nicotine (the EU maximum for consumer e-liquids), and they take about 15 puffs over ~10–15 minutes. Nicotine from the aerosol is absorbed mainly through the lungs; how much actually gets into the body depends on puff intensity, device type, and the user’s technique. In general, a short session like this delivers a fraction of a cigarette’s nicotine, while longer sessions or newer high-performance disposables can approach cigarette-like delivery. EU rules also require child-resistant packaging and spill-resistant refilling to reduce accidental exposure. Input Data Device/liquid assumptions (EU context) Nicotine concentration: 20 mg/mL (max under the EU Tobacco Products Directive). Pod size: 2 mL (max for cartridges/pods under the TPD). Benchmark nicotine delivery Typical nicotine per puff reported in earlier e-cig studies: 0–35 µg/puff (device/user dependent). For screening, use 10–35 µg/puff. Cigarette systemic intake (for comparison): about 1–2 mg nicotine per cigarette to the smoker. Newer disposable e-cigs can reach cigarette-like blood nicotine profiles in some studies. Session exposure (15 puffs) Lower delivery case (10 µg/puff): 150 µg = 0.15 mg systemic. Upper delivery case (35 µg/puff): 525 µg = 0.525 mg systemic. Interpretation vs cigarette A 15-puff session ≈ 0.1–0.5 of a cigarette worth of nicotine (very device/user dependent). Multiple sessions or higher-output devices can approach ~1 cigarette per session.

101. E. Nicotine (Consumer exposure) Risk narrative notes Key drivers of exposure: device generation/power, puff topography (length/intensity), nicotine strength, and session length. Range justification: legacy lab work shows tens of µg/puff ; recent human PK data indicate some modern disposables produce cigarette-like Cmax and rapid uptake. ( https://pmc.ncbi.nlm.nih.gov/articles/PMC4018182/ ) Regulatory safeguards (EU): max 20 mg/mL nicotine; 2 mL pod / 10 mL bottle limits; child-resistant & tamper-evident packaging; spill-resistant refilling. These don’t fix exposure (that’s behavior/device), but they cap concentration and volume and reduce accidental poisonings . ( https://health.ec.europa.eu/tobacco/product-regulation/electronic-cigarettes_en ?) A short, 15-puff session on an EU-compliant 20 mg/mL pod typically delivers about 0.15–0.53 mg nicotine (≈0.1–0.5 cigarettes), but newer high-output disposables can reach cigarette-like delivery—so exposure hinges more on device and puffing behavior than on liquid strength alone. Accidental ingestion risk scenario (child) Because EU packaging is child-resistant, severe incidents are rarer—but if a toddler accessed a 10 mL bottle at 20 mg/mL, that’s 200 mg nicotine in total; even small swallowed amounts can cause poisoning. This is why child-resistant packaging and safe storage out of reach are essential. (If you include this in communications, keep it preventive, not procedural.) https://health.ec.europa.eu/tobacco/product-regulation/electronic-cigarettes_en ?

102. E. Nicotine (Consumer exposure) Consumer (acute) risk screen for nicotine from a 15-puff vaping session, using EFSA’s acute reference dose as a conservative benchmark. Inputs Nicotine per puff (screening range): 10–35 µg/puff → session dose = 0.15–0.525 mg (15 puffs) Adult body weight: 70 kg EFSA acute reference dose ( ARfD ): 0.0008 mg/kg bw (0.8 µg/kg bw ) for nicotine. ( https://pmc.ncbi.nlm.nih.gov/articles/PMC9483820/ ) EU TPD context: max 20 mg/mL ; 2 mL pod/cartridge; child-resistant, spill-resistant refilling. ( https://health.ec.europa.eu/system/files/2016-11/dir_201440_en_0.pdf ) Calculation (per session) Systemic dose per kg = (0.15–0.525 mg) / 70 kg = 0.00214–0.00750 mg/kg . % ARfD = dose / 0.0008 mg/kg × 100 = ~268%–938% . Interpretation: Using the dietary ARfD as a conservative toxicological screen, the 15-puff session exceeds the ARfD range. (For context, a cigarette delivers ~1–2 mg systemic nicotine, which would also exceed the ARfD by a large margin; the ARfD was set for food incidents, not to regulate tobacco products.) ( https://pubmed.ncbi.nlm.nih.gov/6744784/ )

103. E. Nicotine (Consumer exposure) What this means (practical take) The ARfD comparison signals pharmacologically active dosing in a short session; nicotine-naïve or sensitive users may experience acute effects (nausea, dizziness, tachycardia). This does not imply poisoning at the stated doses, but supports minimisation of peak intake and prevention of dermal/ingestion accidents during refilling. Risk-reduction options Lower delivered dose: fewer puffs per session; use devices/settings with lower aerosol output ; choose lower nicotine strength (where lawful). Spacing: avoid rapid chain-vaping; space sessions to limit peaks. Refilling hygiene: keep all mixing/refilling away from skin ; wipe drips; wash hands; store refills child-resistant (TPD requirement). ( https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX%3A52016DC0269 ) Avoid dermal exposure: nicotine readily penetrates skin; any spill → immediate soap-and-water wash. Notes & caveats Applicability of ARfD : EFSA’s 0.0008 mg/kg ARfD was derived for acute dietary exposure incidents (e.g., nicotine detected in foods). It is a conservative benchmark when used to screen inhalation use of nicotine products; tobacco product regulations instead rely on product standards (e.g., TPD limits) and broader public-health policy. https:// pmc.ncbi.nlm.nih.gov /articles/PMC9483820/

104. F. BPA Substitution Scenario ( SSbD for BPA substitution on food metal can coating) A food brand sells chopped tomatoes in metal cans. The inside of each can is coated with a BPA-based epoxy to stop the acidic tomatoes from reacting with the metal during heat processing and storage. The company wants to remove BPA without compromising food safety, taste, or shelf life. The R&D team evaluates bisphenol-free linings (polyester-epoxy “BPA-NI”, acrylic-phenolic, and plant-based oleoresin) and non-coating solutions (a thin PP insert or switching the product to glass jars). They compare hazard, exposure (migration), technical performance in retort, environmental footprint, worker safety, cost, and recyclability. After lab tests, one bisphenol-free polyester-epoxy lining passes migration and heat-shock tests, keeps flavor stable for 24 months, and is compatible with existing lines. The company plans a controlled roll-out with shelf-life, migration, and sensory monitoring, and a supplier spec that bans BPA/BPF/BPS outright to avoid “regrettable substitution.”

105. F. BPA Substitution Input Data Product & function • Product: 400 g canned chopped tomatoes (pH ~4.2), retorted at 121 °C. • Baseline technology: BPA-epoxy can lining. • Functional requirements: corrosion barrier in acid matrix, heat resistance (retort), 24-month shelf life, no taint/odor, food-contact compliance. Potential Substitution candidates 1. BPA-epoxy (baseline) — high performance; endocrine activity concerns. 2. BPS/BPF-epoxies 3. Bisphenol-free polyester-epoxy (“BPA-NI”) 4. Acrylic-phenolic 5. Oleoresin (plant-based) 6. PP insert 7. Format switch: glass jar + BPA-NI lid Consumer exposure (migration) Assume one 400 g can; adult 70 kg; once per day consumption for screening. Baseline BPA-epoxy Migration of 10 µg/kg food (tomato) Intake per can 4 µg Daily dose (µg/kg bw ) 0.057 Include reduction in exposure from migration in substitution candidate analysis

106. F. BPA Substitution Input Data Product & function • Product: 400 g canned chopped tomatoes (pH ~4.2), retorted at 121 °C. • Baseline technology: BPA-epoxy can lining. • Functional requirements: corrosion barrier in acid matrix, heat resistance (retort), 24-month shelf life, no taint/odor, food-contact compliance. Potential Substitution candidates 1. BPA-epoxy (baseline) — high performance; endocrine activity concerns. 2. BPS/BPF-epoxies 3. Bisphenol-free polyester-epoxy (“BPA-NI”) 4. Acrylic-phenolic 5. Oleoresin (plant-based) 6. PP insert 7. Format switch: glass jar + BPA-NI lid Consumer exposure (migration) Assume one 400 g can; adult 70 kg; once per day consumption for screening. Baseline BPA-epoxy Migration of 10 µg/kg food (tomato) Intake per can 4 µg Daily dose (µg/kg bw ) 0.057 Include reduction in exposure from migration in substitution candidate analysis

107. F. BPA Substitution Input Data Product & function • Product: 400 g canned chopped tomatoes (pH ~4.2), retorted at 121 °C. • Baseline technology: BPA-epoxy can lining. • Functional requirements: corrosion barrier in acid matrix, heat resistance (retort), 24-month shelf life, no taint/odor, food-contact compliance. Potential Substitution candidates 1. BPA-epoxy (baseline) — high performance; endocrine activity concerns. 2. BPS/BPF-epoxies 3. Bisphenol-free polyester-epoxy (“BPA-NI”) 4. Acrylic-phenolic 5. Oleoresin (plant-based) 6. PP insert 7. Format switch: glass jar + BPA-NI lid Consumer exposure (migration) Assume one 400 g can; adult 70 kg; once per day consumption for screening. Baseline BPA-epoxy Migration of 10 µg/kg food (tomato) Intake per can 4 µg Daily dose (µg/kg bw ) 0.057 Include reduction in exposure from migration in substitution candidate analysis

108. F. BPA Substitution Hazard screen (illustrative “traffic light”) BPA: ⚠️ Endocrine activity (ER/AR, thyroid; developmental effects). BPS/BPF: ⚠️ Similar concern profile → exclude by policy. Polyester-epoxy (bisphenol-free): 🟢 No bisphenols; screen oligomers/ photoinitiators . Acrylic-phenolic: 🟡 Generally acceptable; check monomer residuals. Oleoresin: 🟡 Natural origin, but performance risk at high temp/acid (potential for higher migration of natural constituents). PP insert / glass: 🟢 Inert contact surface; different footprint/logistics. Policy gate: Adopt a “No bisphenols (A, S, F, BADGE, BFDGE, NOGE)” supplier clause for all candidates.

109. F. BPA Substitution - candidate comparison Case Measured migration (BPA or total bisphenols) Intake per can Daily dose (µg/kg bw) Baseline BPA-epoxy 10 µg/kg food (tomato) 4 µg 0.057 Candidate A (polyester-epoxy, bisphenol-free) <0.5 µg/kg (target; ND for bisphenols) <0.2 µg <0.003 Candidate B (acrylic-phenolic) 1 µg/kg 0.4 µg 0.006 Candidate C (oleoresin) 2 µg/kg 0.8 µg 0.011 Candidate D (PP insert) <0.2 µg/kg <0.08 µg <0.001 Candidate E (glass jar) <0.2 µg/kg (from lid) <0.08 µg <0.001 Interpretation: All bisphenol-free options reduce consumer BPA exposure by ~10–50× relative to a typical BPA-epoxy case; PP insert and glass give the lowest migration but may have line change and logistics impacts.

110. F. BPA Substitution - performance and safety comparison Technical performance & quality (go/no-go tests) Retort survivability: 3× cycles at 121 °C; no blisters, no delamination (A, B pass; C borderline). Sensory: triangle test at 0, 6, 12, 24 months; no metallic/phenolic taint (A, E strong). Corrosion: 6/12/24-month stack at 30 °C; iron pick-up < threshold (A, B good; C risky for acid foods). Adhesion: cross-hatch & pasteurization shock (A excellent; B good). Worker safety (manufacturing) Baseline : epoxy lines handling BPA/BADGE resins (dermal, dust). Substitutes : check resin/solvent SDS (styrene, solvents, isocyanates if present); ensure LEV and dermal protection remain adequate; update exposure register . Environment & circularity snapshot (screening) GHG & energy : Glass has higher transport energy; metal cans highly recyclable; PP inserts add material complexity . Recyclability : Metal cans with thin polymer linings remain widely recyclable; keep coating <1% weight , avoid chlorine. Chemicals of concern : Adopt supplier declaration + analytical verification for No Bisphenols and No SVHCs above de minimis. SSbD decision (candidate) Select Candidate A: Bisphenol-free polyester-epoxy for current can format. • Why?: Meets barrier/retort needs; bisphenol-free; low migration; drop-in for existing lines; maintains metal-can recyclability. • Guardrails: Contractually ban A/S/F analogues; set QC migration spec ≤1 µg/kg; monitor non-intentionally added substances (NIAS).

111. G. Dimethyltin dichloride - DMTCl ₂ (Recent Addition to SIN List for neurotoxicity) Scenario (Occupational exposure to plastic additive) At a plastics additives plant, a technician weighs out dimethyltin dichloride powder to make a tin-mercaptide stabilizer used for PVC. For each batch they open drums, scoop the powder into a hopper, and charge it into a reactor. Most of the day the system is closed, but during weighing and charging a visible dust cloud can form. The material is highly toxic by inhalation and corrosive to skin/eyes, and it can also release acidic fumes if it contacts moisture. Good local exhaust, fully enclosed charging, and strict skin/eye protection are essential to avoid acute poisoning and burns.

112. G. Dimethyltin dichloride - DMTCl ₂ (Occupational Exposure) Input Data Occupational limits for organotins (as Sn): ACGIH TLV-TWA 0.1 mg/m³ (as Sn); STEL 0.2 mg/m³ (as Sn); Skin notation. NIOSH REL 0.1 mg/m³ (as Sn); IDLH 25 mg/m³ (as Sn) for organic tin compounds. (These group limits apply to organotins like DMTCl₂ when expressed as tin.) Tin conversion factor (compound → “as Sn”): Molecular weight Me₂SnCl₂ ≈ 219.7 g/mol; tin is 118.7 g/mol → tin fraction ≈ 0.54. So 0.1 mg/m³ (as Sn) ≈ 0.185 mg/m³ of dimethyltin dichloride (compound). (0.1 / 0.54) Example 8-h shift exposure (personal air) Tasks & concentrations (compound, mg/m³): Weigh/charge (30 min): 0.60 mg/m³ (brief dust peak) Closed mixing (2 h): 0.05 mg/m³ General background (5.5 h): 0.01 mg/m³ 8-h TWA (compound): (0.6×0.5+0.05×2+0.01×5.5)/8=0.0569 mg/m^3 Convert to “as Sn”: 0.0569 × 0.54 = 0.0307 mg/m³ (as Sn) Compare to limits: TWA: 0.0307 vs 0.1 mg/m³ (as Sn) → Below TLV/REL. Short-term: the 0.60 mg/m³ peak equals 0.324 mg/m³ (as Sn) → exceeds STEL 0.2 mg/m³ (as Sn) during charging. Sampling/analytics: Use NIOSH 5504 (organotin compounds, as Sn) for personal air monitoring; working range 0.015–1 mg/m³ (as Sn).

113. G. Dimethyltin dichloride - DMTCl ₂ (Input Data and Comparison to Limits) Input Data Occupational limits for organotins (as Sn): ACGIH TLV-TWA 0.1 mg/m³ (as Sn); STEL 0.2 mg/m³ (as Sn); Skin notation. NIOSH REL 0.1 mg/m³ (as Sn); IDLH 25 mg/m³ (as Sn) for organic tin compounds. (These group limits apply to organotins like DMTCl₂ when expressed as tin.) Tin conversion factor (compound → “as Sn”): Molecular weight Me₂SnCl₂ ≈ 219.7 g/mol; tin is 118.7 g/mol → tin fraction ≈ 0.54. So 0.1 mg/m³ (as Sn) ≈ 0.185 mg/m³ of dimethyltin dichloride (compound). (0.1 / 0.54) Example 8-h shift exposure (personal air) Tasks & concentrations (compound, mg/m³): Weigh/charge (30 min): 0.60 mg/m³ (brief dust peak) Closed mixing (2 h): 0.05 mg/m³ General background (5.5 h): 0.01 mg/m³ 8-h TWA (compound): (0.6×0.5+0.05×2+0.01×5.5)/8=0.0569 mg/m^3 Convert to “as Sn”: 0.0569 × 0.54 = 0.0307 mg/m³ (as Sn) Compare to limits: TWA: 0.0307 vs 0.1 mg/m³ (as Sn) → Below TLV/REL. Short-term: the 0.60 mg/m³ peak equals 0.324 mg/m³ (as Sn) → exceeds STEL 0.2 mg/m³ (as Sn) during charging. Sampling/analytics: Use NIOSH 5504 (organotin compounds, as Sn) for personal air monitoring; working range 0.015–1 mg/m³ (as Sn).

114. G. Dimethyltin dichloride - DMTCl ₂ (Controls to being peaks under STEL) Engineering / containment Closed, dust-tight charging (split-butterfly valve/bag-in-bag-out or drum tipper with flexible enclosure). Local exhaust ventilation at the point of weighing/charging (capture ≥0.5 m/s); dedicated dry room to avoid moisture/HCl. HEPA extraction and negative pressure in the weighing booth; interlocks so charging cannot proceed unless LEV is “OK”. Substitution / form factor Buy or make encapsulated or granulated dimethyltin intermediates (or move to dimethyltin mercaptide in liquid/paste where feasible) to eliminate powder peaks. Administrative / work practice Pre-weigh in sealed containers; minimize open handling time; slow pour rates; anti-static measures; moisture control. Two-person rule during charging; keep others out of the area for 15 minutes post-charge (worst-case airborne decay). PPE (last line, for residual risk) Chemical splash goggles + face shield ; acid-resistant suit/apron ; nitrile (or butyl) gloves with frequent change-out; disposable sleeves ; S-class cartridge/APR or PAPR for charging until peaks are demonstrably ≤ STEL. Emergency eyewash/shower within 10 s travel time; no skin exposure tolerated. SDSs emphasize severe acute hazards. ( https://www.spectrumchemical.com/media/sds/D1453_AGHS.pdf ?) Verification Personal sampling on charging days (task + full-shift) using NIOSH 5504 ; demonstrate TWA ≤ 0.1 mg/m³ (as Sn) and 15-min STEL ≤ 0.2 mg/m³ (as Sn) . Routine LEV checks (smoke test + velocity log). Incident protocol for spills/splash (corrosive): isolate area, neutralize per SDS, medical eval for any suspected inhalation/dermal contact. https://www.cdc.gov/niosh/docs/2003-154/pdfs/5504.pdf ?