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- 1. La direttiva 2010/63/UE e i laboratori di riferimento dell’UE per la convalida dei metodi alternativi C. Rovida CAAT Europe – University of Konstanz costanza.rovida@chimici.it 8 febbraio 2018 RICERCA E INNOVAZIONE NELL’AMBITO DEI METODI SOSTITUTIVI
- 8. Article 4 Principle of replacement, reduction and refinement Principio della sostituzione, della riduzione e del perfezionamento • 1. Member States shall ensure that, wherever possible, a scientifically satisfactory method or testing strategy, not entailing the use of live animals, shall be used instead of a procedure. Gli Stati membri assicurano che, ove possibile, un metodo o una strategia di sperimentazione scientificamente soddisfacente che non comporti l’uso di animali vivi possa essere utilizzato in sostituzione di una procedura
- 9. Article 47 - Alternative approaches 1. The Commission and the Member States shall contribute to the development and validation of alternative approaches which could provide the same or higher levels of information as those obtained in procedures using animals, but which do not involve the use of animals or use fewer animals or which entail less painful procedures, and they shall take such other steps as they consider appropriate to encourage research in this field. 2. Member States shall assist the Commission in identifying and nominating suitable specialised and qualified laboratories to carry out such validation studies. 3. After consulting the Member States, the Commission shall set the priorities for those validation studies and allocate the tasks between the laboratories for carrying out those studies. 4. Member States shall, at national level, ensure the promotion of alternative approaches and the dissemination of information thereon. 5. Member States shall nominate a single point of contact to provide advice on the regulatory relevance and suitability of alternative approaches proposed for validation. 6. The Commission shall take appropriate action with a view to obtaining international acceptance of alternative approaches validated in the Union.
- 15. DECRETO LEGISLATIVO 4 marzo 2014, n. 26 Attuazione della direttiva 2010/63/UE sulla protezione degli animali utilizzati a fini scientifici. (14G00036) (GU Serie Generale n.61 del 14-03-2014) note: Entrata in vigore del provvedimento: 29/03/2014 Articolo 1(2). E' consentito l'utilizzo degli animali ai fini scientifici o educativi soltanto quando, per ottenere il risultato ricercato, non sia possibile utilizzare altro metodo o una strategia di sperimentazione scientificamente valida, ragionevolmente e praticamente applicabile che non implichi l'impiego di animali vivi. Articolo 5(2). Non possono essere autorizzate le procedure: … e) per le ricerche sulle sostanze d'abuso Articolo 13. Scelta dei metodi (1). Non sono autorizzabili le procedure che prevedono l'impiego di animali vivi per le quali esistono altri metodi o strategie di sperimentazione, riconosciute dalla legislazione dell'Unione europea, ovvero prevedono metodi vietati dalla normativa vigente nazionale. “non sono autorizzabili”???
- 17. Uso dei conigli a scopo scientifico • Irritazione dermale e oculare – Sostituzione con modelli di epidermide ricostituitIrritazione dermale e oculare – Sostituzione con modelli di epidermide ricostituita completamente accettati • Seconda specie per test di sviluppo – Tante possibilità, ma poco esplorate • Test dei pirogeni – LAL, come il coniglio positive solo – MAT, applicabilità universale completamente accettata
- 18. Research and development ALTERNATIVE TEST METHOD PREDICTION MODEL TEST SYSTEM= + WHAT IS AN ALTERNATIVE (REPLACEMENT) TEST ?
- 19. Reliability (reproducibility) Relevance: scientific basis Relevance: Predictive capacity Research and development ALTERNATIVE TEST METHOD PREDICTION MODEL TEST SYSTEM= +
- 20. (Animal) Model Test Validated Test Accepted Test “Face validity” Based on phenomenological similarity and/or current scientific understanding Pre-validation & Validation
- 21. (Animal) Model Test Validated Test Accepted Test A definition of the scientific purpose of the method A description of its mechanistic basis The case for its relevance The availability of an optimized protocol, including: - standard operation procedures - specification of endpoints and endpoint measurements - derivation, expression and interpretation of results (preliminary prediction model) - the inclusion of adequate controls An indication of limitations (preliminary applicability domain) Quality assurance (GLP, Good Cell Culture Practice) Pre-validation & Validation
- 22. https://eurl-ecvam.jrc.ec.europa.eu/test-submission Step 1: Presubmission - is based on the EURL ECVAM Test Presubmission Form (TPF) and is a mandatory requirement for a test method to be eventually considered for the EURL ECVAM validation process. The completed TPF filled in all its parts will allow EURL ECVAM to perform a preliminary assessment of the status of development, optimisation and/or validation of the test method and its potential relevance with regard to the 3Rs (replacement, reduction, refinement of animal testing).
- 23. 1. INTRODUCTION 2. PURPOSE 3. METHOD OUTLINE 4. LIMITATIONS 5. DEFINITIONS/ABBREVIATIONS 6. MATERIALS 6.1. Cell 6.2. Technical Equipment 6.3. Reagents, Media, Sera, Culture Plates 6.4. Preparation of media and solutions 7. METHODS 7.1. Cell maintenance and culture conditions 7.2. Quality check of cells 7.3. Handling of chemicals 7.4. Cell viability 7.6. Measure of endpoints 7.7. Data Analysis 7.8. Final Report 8. HEALTH SAFETY AND ENVIRONMENT 9. REFERENCES 10. APPENDIX Protocol Template Sens-it-iv Newsletter 23, http://www.sens-it-iv.eu https://ecvam-dbalm.jrc.ec.europa.eu/home/contribute
- 24. Annex 1 Good Cell Culture Practice (GCCP) Annex 2 Good Cell Culture Practice for stem cells and stem-cell derived models [Good Cell Culture Practice: human (stem) cells and organoids] OECD GUIDANCE DOCUMENT ON GOOD IN VITRO METHOD PRACTICES (GIVIMP) FOR THE DEVELOPMENT AND IMPLEMENTATION OF IN VITRO METHODS FOR REGULATORY USE IN HUMAN SAFETY ASSESSMENT http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2018)19&doclanguage=en
- 25. (Animal) Model Test Validated Test Accepted Test Validation is the independent assessment of the scientific basis, the reproducibility and the predictive capacity of a test. Prerequisite since 2005 for all new OECD test guidelines also for animal tests Pre-validation & Validation
- 26. EURL ECVAM Validation Process
- 28. (Animal) Model Test Validated Test Accepted Test = Post-validation Test guideline development (typically much broader than SOP) Increasingly bottleneck for availability International Harmonization Implementation Pre-validation & Validation
- 30. 428 Skin Absorption: In Vitro Method 430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER) 431 In Vitro Skin Corrosion: Human Skin Model Test 432 In Vitro 3T3 NRU Phototoxicity Test 435 In Vitro Membrane Barrier Test Method for Skin Corrosion 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants 438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method 471 Bacterial Reverse Mutation Test 472 Genetic Toxicology: Escherichia coli, Reverse Assay 473 In Vitro Mammalian Chromosome Aberration Test 474 Mammalian Erythrocyte Micronucleus Test 476 In Vitro Mammalian Cell Gene Mutation Test 477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster 479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay 481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay 482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro 483 Mammalian Spermatagonial Chromosome Aberration Test 487 In Vitro Mammalian Cell Micronucleus Test OECD Methods
- 31. 428 Skin Absorption: In Vitro Method 430 In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER) 431 In Vitro Skin Corrosion: Human Skin Model Test 432 In Vitro 3T3 NRU Phototoxicity Test 435 In Vitro Membrane Barrier Test Method for Skin Corrosion 437 Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants 438 Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method 471 Bacterial Reverse Mutation Test 472 Genetic Toxicology: Escherichia coli, Reverse Assay 473 In Vitro Mammalian Chromosome Aberration Test 474 Mammalian Erythrocyte Micronucleus Test 476 In Vitro Mammalian Cell Gene Mutation Test 477 Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster 479 Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells 480 Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay 481 Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay 482 Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro 483 Mammalian Spermatagonial Chromosome Aberration Test 487 In Vitro Mammalian Cell Micronucleus Test 442c In Chemico Skin Sensitisation (DPRA) 442d In Vitro Skin Sensitisation ARE-Nrf2 Luciferase Test Method OECD Methods
- 32. OECD 437 BCOP Test H318 Causes serious eye damage
- 33. NAMs: Origin Two motivating drivers for the workshop were: • A better understanding of the underlying biology behind how chemicals cause adverse effects to human health; and • New tools and techniques that provide a huge amount of data to be used in solving regulatory issues.
- 34. In 2025 the Netherlands could be world leader animal-free innovations! “NCad, I urge you to develop a roadmap for the phasing out of the use of animals in scientific research”
- 36. Phasing out of animal testing is impossible: We need animals Animal testing should be banned immediately 3R’s are important but cannot replace animals Scientists: Society, NGO’s, other organisations, news, social media, and political pressure
- 37. Validity status of reprotox testing 1) Not robust (about 25% equivocal studies), [Bailey et al., 2005] 2) 74 industrial chemicals tested in New Chemical Database: 34 showed effects on offspring, but only 2 chemicals have been classified as developmental toxic [Bremer & Hartung, 2004] 3) 55% of positives in screening studies not in multi-generation studies [Bremer & Hartung, 2004] 4) Group size limits statistical power [Hotchkiss 2008] 5) 61% inter-species correlation [Hurrt 2003, Bailey 2005] Moreover a) Lack of objectivity of in vivo tests b) Tests at high concentration ALTEX 2018, 35:139-162
- 38. Report del National Research Council of the National Academies Published on 2007 (Google: "Toxicity Testing in the 21st Century")
- 39. Biologic Inputs Normal Biologic Function Morbidity and Mortality Cell Injury Adaptive Stress Responses Early Cellular Changes Exposure Tissue Dose Biologic Interaction Perturbation Low Dose Higher Dose Higher yet (Courtesy of Mel Andersen) A New Paradigm: Activation of Toxicity Pathways
- 42. Il passato prossimo: Metodo tradizionale GPMT e Buehler test 30 cavie Applicazione con un irritante per 8 giorni sulla spalla Verifica di una eventuale reazione allergica al 20° giorno
- 43. Il passato prossimo: Metodo tradizionale GPMT e Buehler test 30 cavie Applicazione con un irritante per 8 giorni sulla spalla Verifica di una eventuale reazione allergica al 20° giorno Magnusson B. and Kligman A.M. (1969). The identification of contact allergens by animal assay. The guinea pig maximisation test. J. Invest. Dermatol., 52, 268
- 44. Il passato prossimo: Metodo tradizionale GPMT e Buehler test 30 cavie Applicazione con un irritante per 8 giorni sulla spalla Verifica di una eventuale reazione allergica al 20° giorno Magnusson B. and Kligman A.M. (1969). The identification of contact allergens by animal assay. The guinea pig maximisation test. J. Invest. Dermatol., 52, 268 OECD TG 406
- 45. Primo metodo "alternativo" LOCAL LYMPH NODE ASSAY Days 1, 2 & 3 Apply Chemical Day 6 - Inject H-Thymidine3 5 hours Later Remove Lymph Nodes Make Cell Suspension Determine H-Thymidine Incorporation by Liquid Scintillation Counting 3
- 46. LLNA Kimber, I. and Basketter, D.A. (1992), The murine local lymph node assay; collaborative studies and new directions: A commentary, Food Chem. Toxicol., 30, 165-169 OECD (2002), Skin Sensitisation: Local Lymph Node Assay. OECD Guideline for the Testing of Chemicals No 429, Paris. http://www.oecd.org/env/testguidelines
- 47. … intanto 11 settembre 2004: proibizione di testare prodotti cosmetici sugli animali 11 marzo 2009: proibizione di testare ingredienti cosmetici sugli animali (con qualche eccezione) 11 marzo 2013: proibizione completa di testare sugli animali qualunque ingrediente/prodotto cosmetico
- 48. Sensibilizzazione della pelle: in vitro Assorbimento attraverso la pelle Metabolismo Attivazione delle cellule dendritiche Migrazione verso i linfonodi Risposta dei linfonodi
- 50. First Edition Publication: January 2008 "vitro" mentioned 36 times
- 51. Scientific American article (translated into Arabian, Chinese, German, Italian, Japanese, Korean, Polish, Portugese, Spanish…) Humane science is the best science!
- 52. Non si conoscono Sono più difficili da applicare Spesso sono più costosi Non si è sicuri che vengano accettati Si è sempre fatto in un altro modo Altex 27, 3/10
- 54. Molecular properties Molecular initiating events Cellular responses Organ response Organism response Skin absorption and access to the viable epidermis OECD 428 Q(SAR)s Binding to protein to form a complete antigen (haptenation) Electrophilic reactivity (direct or via auto- oxidation or metabolism) Activation of keratinocytes (KC) and dendritic cells (DC) Antigen presentation and specific T cell activation ADVERSE OUTCOME PATHWAY Allergic contact dermatitis DPRA (Gerberick et al., 2008) Human T cell priming assay (Martin et al., 2010)Q(SAR)s (Patlewicz et al., 2007) ARE reporter gene assays (Natsch et al., 2007) KC/EE and IL-18 (Corsini et al., 2009; Gibbs et al., in press) KC DC KC and gene profile (Vandebriel et al., 2010) hCLAT (Sakaguchi et al., 2009) GARD (Johansson et al., 2011) MUSST (Python et al., 2007) Primary DC (see dos Santos et al., 2009 for review) VitoSens (Hooyberghs et al., 2008)
- 55. Existing data on physico-chemical properties 1 Is the substance a strong acid (pH≤ 2.0) or base (pH≥ 11.5), corrosive to the skin or (spontaneously) flammable in air at room temperature? Existing human data 2 Are there adequate existing human data, which provide evidence that the substance is a skin sensitiser? Existing animal data from sensitisation studies 3 Are there data from existing studies on skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitiser, or non-sensitiser? Existing (Q)SAR data and read-across 4 Do “read-across” from structurally and mechanistically related substances and do suitable (Q)SAR predictions reliably indicate
- 56. ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)? (Key event 2 of the AOP) In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met. 7c Does the substance demonstrate induction of the cell surface markers (CD54 and/or CD86) on monocytic cells in an validated in vitro test (e.g. h CLAT)? (Key event 3 of the AOP) In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met. 7d Is any additional testing/generation of data considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained in previous elements or to address the Key event 4 of the AOP (T cell proliferation) with an in vitro test? d
- 57. Weight-of-Evidence analysis 8 The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-7) into account, is there sufficient information to meet the respective information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted? For specific guidance on Weight of Evidence see below. Generation of new in vivo data for sensitisation as a last resort (Annex VII to the REACH Regulation) 9 Does the substance demonstrate sensitising properties in an EU/OECD adopted in vivo test, the LLNA (EU B.42/OECD TG 429, EU B.50/442A or EU B.51/442Be)? →
- 58. Allegato VII – revisione 20 settembre 2016
- 59. Allegato VII – revisione 20 settembre 2016
- 60. The reproductive cycle Courtesy of Michael Schwarz
- 61. • https://aopwiki.org/ • https://aopwiki.org/aops Solo teoria? 2012
- 63. Aop: 3 AOP Title: Inhibition of the mitochondrial complex I of nigro-striatal neurons leads to parkinsonian motor deficits Short name: Mitochondrial dysfunction and Neurotoxicity ? https://aopwiki.org/aops/3 AOP: un esempio
- 65. Regenerative medicine-based strategies for the treatment of Parkinson's disease Development 2015 142: 1918-1936; doi: 10.1242/dev.097394 X Commercialmente disponibili (20 anni!) Dal paziente Mouse Embryo
- 66. Cell Biological Processes necessary for Brain Development With courtesy from William Mundy, U.S. Environmental Protection Agency and John Havel, SRA International, Inc. Endpoint Cell Death Proliferation Migration Differentiation Neurite Outgrowth GFAP Network Formation Myelination
- 67. Mini-brains Hogberg et al., Stem Cell Res 2013 Pamies et al., Exp Biol Med 2014 Pamies et al., Altex 2017
- 69. Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth Tang, H. et al. Cell Stem Cell 18, 1–4, May 5, 2016 Neural stem cells (NSCs) Ventricular zone (VZ) Subventricular zone (SVZ) Inner and Outer SVZ (ISVZ and OSVZ) Intermediate zone (IZ) Cortical plate (CP) Kelava and Lancaster (2016). Stem Cell Models of Human Brain Development. Cell Stem Cell Toxicology and Applied Pharmacology 354 (2018) 3–6
- 70. Not our goal!
- 72. Microphysylogical Systems (Human on a chip)
- 74. Summary Traditional approach in vitro methods 3D system Species specific correlation - + / - +++ Reproducibility + / - +++ + Time /money demand - + + + + Applicability domain + (-) - - (+) Test on intact organisms + - + / - Well accepted by Regulators + + / - ? + / - ? Quantity of test item - + + Mechanistic elucidation + / - + + +
- 75. Vision for the future of toxicity testing Altex 31, 3/14
- 76. Vision for the future of toxicity testing Altex 31, 3/14
- 79. Test in vivo
- 81. AOP
- 83. Nessun pallone e nessun aeroplano avranno mai successo in pratica (1902) Le macchine volanti più pesanti dell'aria non sono possibili (1895) William Thomson, noto anche come Lord Kelvin (1824-1907), presidente della Royal Society e padre della scala di temperatura che porta il suo nome.
Editor's Notes
- CAAT Europe is located at the University of Konstanz I Germany. It is within the department of in vitro toxicology in the Faculty of Biology
- Aanpak voor de beantwoording van ons advies was: De ‘snelkookpan’. In korte tijd hebben we vele experts en maatschappelijke groeperingen geconsulteerd op allerlei gebied. Van transitiedenkers, tot CEO’s van innovatieve bedrijven, van fundamenteel wetenschappelijk onderzoekers tot beoordelingsautoriteiten op het gebied van geneesmiddelen. En nog vele anderen (die natuurlijk allemaal genoemd en bedankt zijn achterin het advies) Natuurlijk hebben we ook literatuuronderzoek gedaan. Niet alleen wetenschappelijke literatuur, ook kranten en nieuwbladen hebben we hierin betrokken. En zoals dat hoort in deze tijd, hebben we discussies gepoogd te entameren buiten de groep van bekende stakeholders door op social media via een Linked Inn groep ene discussie groep te starten. Een groep studenten van de VU heeft gepoogd meer zicht te krijgen op de bereidheid van burgers en patienten om risico’s te ondergaan die verbonden zijn aan afschaffing van dierproeven.
- Limulus amebocyte lysate Assay Lal solo gram + e interferenze con alcune sostanze MAT monocyte activation tests
- That’s the story to get to the concept of NAM Let’s do a step back to understand the evolution from 3Rs to NAMs
- 34 membri, compresa UE, USA Canada e Giappone. Purtroppo ancora non la Cina I metodi della sezione 4 sono quelli relativi alla salute umana. Come vedete I numeri sono molto alti. IL metodo 401 è la tossicità acuta orale, ancora su 100 animali, che ovviamente non usa più nessuno
- Regolamento REACH dice che che posso usare qualunque OECD, ma comunuque BCOP è stato già incluso nel regolamento 440 Se leggo la linea guida OECD vedo che è adatto per distinguere I corrosivi dagli irritanti, anche se il valore molto negativo è accettato. Restano in dubbio gli irritanti leggeri.
- Torniamo al nostro BCOP
- In 2016, ECHA organised a very important workshop that decided a new way to consider the approach. Two motivating drivers… For the first time they used the terminology “NAMs” to indicate something that is disruptive with the past.
- Zowel in de maatschappij als in de politiek wordt regelmatig de vraag gesteld of onderzoek niet zonder dieren of met veel minder dieren kan. Binnen de wetenschap worden vragen gesteld over de voorspellende waarde van diermodellen voor de situatie van mensen. Niet nieuw, want dat is voor wetenschappers altijd een vraag, maar ik vermoed dat het stellen van vragen in zowel de wetenschappelijke, als de maatschappelijke als de politiek hoek over de waarde van dierproeven ertoe heeft bijgedragen dat de staatssecretaris on op 8 april j.l de vraag stelde om een afbouwschema dierproeven op te stellen.
- Commotie nav de vraag van de EZ was ook voor ons NCad merkbaar. Reacties varieerden van… ‘Het is onmogelijk’ tot ‘per direct stoppen! ‘en daar tussenin: ‘misschien kunnen we meer vermindering realiseren in een hoger tempo’. De vervolgragen bij ‘afbouw’ zijn: welke risico’s neem je daar? Voor patienten, burgers en voor de wetenschappelijk in NL? Wat zijn nu echt reele alternatieven?
- It should be acknowledged that in vivo methods have some limitations. Regarding reproductive toxicity… Also the EOGRTS guideline contains no recommendation for human relevance of the animal data. Human exposure is several order of magnitude lower than doses used in animal studies.
- I 2007 the NIH asked for a report to understand how to change the approach to solve two main problemas: Few chemicals have a full toxicological characterization There is concern on some chemicals that do not have explanations What’s written in this book is that the traditional model is based on animals
- 39
- What is at the base of any toxicological process is the adverse outcome pathways ….. OECD is very much committed in this sense
- Sostanza applicata sulla spalla rasata della cavia insieme ad un irritante per 8 giorni Riposo 0 = no visible change 1 = discrete or patchy erythema 2 = moderate and confluent erythema 3 = intense erythema and swelling
- Sostanza applicata sulla spalla rasata della cavia insieme ad un irritante per 8 giorni Riposo 0 = no visible change 1 = discrete or patchy erythema 2 = moderate and confluent erythema 3 = intense erythema and swelling
- Sostanza applicata sulla spalla rasata della cavia insieme ad un irritante per 8 giorni Riposo 0 = no visible change 1 = discrete or patchy erythema 2 = moderate and confluent erythema 3 = intense erythema and swelling
- Unfortunately, I don't know when it will be published. Our work of revision is just finished. Now, we need the approval of some official committees and I am afraid that final approval will arrive next summer.
- The first guideline was published in 2008. I was involved in that because at that time I was in ECVAM. I still remember how much I had to discuss to make in vitro considered. Now the word in vitro is mentioned 36 times. I personally prepared this flow and I really had to fight to keep the box about in vitro methods. Now I am very happy to announce that the wind has changed
- The past century saw also the beginning of animal welfare movements. CAAT was founded in 1981, but the turn arrived with this paper, translated in many languages and written by Alan Goldberg the founder of CAAT and Thomas Hartung the director now of CAAT It was the beginning of the awareness that in vitro tests and alternative strategies may represent a tremendous improvement of toxicology
- What is at the base of any toxicological process is the adverse outcome pathways ….. OECD is very much committed in this sense
- Check the validity of the listed methods – Adverse outcome pathway with potential non-animal methods for contact hypersensitivity. DPRA, direct peptide reactivity assay; EE, epidermal equivalent; Q(SAR), quantitative structure–activity relationship models Questo è un modello meraviglioso, su cui si deve lavorare, ma mantenendo sempre un occhio rivolto al futuro. Questa deve essere la vera ricerca
- Reprotoxicity The reproductive cycle is one of the most complex set of processes that organisms undergo. We currently still rely on animal tests to predict the potential for chemicals to cause reproductive harm in humans. (reproduction/fertility, prenatal development, postnatal development), or as a single protocol (two-generation test).
- The purpose of SAAOP is to promote and advance scientific research that fosters the development and use of adverse outcome pathways (more info). The SAAOP maintains the AOP-Wiki under the guidance of the OECD Expert Advisory Group on Molecular Screening and Toxicogenomics (EAGMST).
- This Adverse outcome Pathway (AOP) describes the linkage between inhibition of complex I (CI) of the mitochondrial respiratory chain and motor deficit as in parkinsonian disorders. Binding of an inhibitor to complex I has been defined as the molecular initiating event (MIE) that triggers mitochondrial dysfunction, impaired proteostasis, which then cause degeneration of dopaminergic (DA) neurons of the nigro-striatal pathway. Neuroinflammation is triggered early in the neurodegenerative process and exacerbates it significantly. These causatively linked cellular key events result in motor deficit symptoms, typical for parkinsonian disorders, including Parkinson's disease (PD), described in this AOP as an Adverse Outcome (AO). Since the release of dopamine in the striatum by DA neurons of the Substantia Nigra pars compacta (SNpc) is essential for motor control, the key events refer to these two brain structures. The weight-of-evidence supporting the relationship between the described key events is based mainly on effects observed after an exposure to the chemicals rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), i.e. two well-known inhibitors of complex I. Data from experiments with these two chemicals reveal a significant concordance in the dose-response relationships between the MIE and AO and within key events (KEs). Also essentiality of the described KEs for this AOP is strong since there is evidence from knock out animal models, engineered cells or replacement therapies that blocking, preventing or attenuating an upstream KE is mitigating the AO. Similarly, there is proved experimental support for the key event relationships (KERs) as multiple studies performed with modulating factors that attenuate (particularly with antioxidants) or augment (e.g. overexpression of viral-mutated α-synuclein) a KE up show that such interference leads to an increase of KE down or the AO. Information from in vitro and in vivo experiments is complemented by human studies in brain tissues from individuals with sporadic Parkinson's disease (Keeney et al., 2006) to support the pathways of toxicity proposed in this AOP.
- Regenerative medicine-based strategies for the treatment of Parkinson's disease. (A) Human fetal ventral midbrain (VM) tissue has been the classical source of tissue for transplantation and has provided proof of principle for cell replacement therapy in Parkinson's disease (PD). (B,C) Midbrain dopaminergic neurons have also been generated from human PSCs, derived either from blastocysts (ESCs; B) or by reprogramming of fibroblasts (iPSCs; C). Protocols involve following the developmental steps to first generate ectoderm, neural plate (NP) and neuroepithelial cells (NEP), followed by midbrain floor plate (mFP), midbrain dopaminergic (mDA) progenitors (mDP), mDA postmitotic neuroblasts (mDNb) and mDA neurons. (D) An alternative way to create mDA neurons is by direct reprogramming of fibroblasts into induced mDA neurons. (E) mDA neurons and iDA cells can be used for transplantation. (F) Direct reprogramming could also be performed in vivo, to generate iDA neurons in situ, from host cells in the adult brain, without the need for cell transplantation. (G) mDA neurons derived from PD patients or with introduced PD mutations can be compared with controls or corrected mutations and used for disease modeling and drug discovery.
- Brain development needs a multitude of cell biological processes and each process is guided by a plethora of signaling pathways. Therefore, translating the new AOP concept to DNT is a challenging approach because there is a multitude of MIE and KE guiding defects in the different developmental processes. Myelination is the process by which a fatty layer, called myelin, accumulates around nerve cells (neurons). Myelin particularly forms around the long shaft, or axon, of neurons. Myelination enables nerve cells to transmit information faster and allows for more complex brain processes. Thus, the process is vitally important to healthy central nervous system functioning.
- The human brain develops through a series of developmental stages that must occur in a particular sequence and at the right time. The current reliance on DNT testing in rodents to predict effects in humans is called into question. It’s not science fiction and first applications are already available For example, it was used to demonstrate the activity of the Zika virus that affects the embryo neural cells causing immediate death or defective maturation. The possibility to isolate the precise step when the effect occurs is also very helpful to investigate the pharmacological solution. There are also studies that demonstrate the difference between human and rats, the latter resembling in vivo experiments, but different than the experiment performed with human material
- We are not 70Kg rats, but we’re also not a 70Kg cells. We need to move towards a higher awareness of the mechanism behind a possible effect
- Non sto a entrare nei dettagli dei tipi di cellule che si possono utilizzare
- 6 millions from EPA to develop The primary goal of the new center is to develop a series of 3-D human cell cultures that are heavily wired up with different sensors to record how they respond when exposed to small concentrations of potentially toxic chemicals.
- include: exposure differences between in vitro and in vivo studies, accumulation of the compound in the fetal compartment, maternal metabolism, altered maternal status (e.g., hypoglycemia, hemodynamic changes), effects on placental function, and a sensitive window or other mechanistic aspect missing from the alternative assay No in vitro model is perfect, but given the everincreasing rate of technologic change (see Kurzweil 2005), one can realistically imagine a time in the not-too-distant future when the bulk of safety assessment work will be done in these sorts of constructs, particularly as they represent more and more of the natural complexity of the in vivo situations, and the results from these models will enable scientists to make reliable predictions of what would happen in vivo
- The current approach is first to test unknown chemicals in animal tests. This limits overall throughput and leads to a high rate of false positives and false negatives. Mixtures are hardly ever tested because of the limitation of resources. High costs in combination with a low predictivity lead to many cases of “no testing”. Mechanistic studies are only carried out in few cases of particular interest to identify the factors causing the toxicity. The new approach, suggested here, is based on 21st century in silico and in vitro methods identifying PoT. This will in most cases lead to an amount of data that is sufficient to decide whether a substance is toxic (positive) or non-toxic (negative) for the intended scenario. Only in few cases, when not enough information can be obtained, will animal tests be performed as an additional source of information. Good information can be provided on all chemicals and, due to the high throughput of the approach, also on mixtures.
- Hypothesis driven testing The current approach is first to test unknown chemicals in animal tests. This limits overall throughput and leads to a high rate of false positives and false negatives. Mixtures are hardly ever tested because of the limitation of resources. High costs in combination with a low predictivity lead to many cases of “no testing”. Mechanistic studies are only carried out in few cases of particular interest to identify the factors causing the toxicity. The new approach, suggested here, is based on 21st century in silico and in vitro methods identifying PoT. This will in most cases lead to an amount of data that is sufficient to decide whether a substance is toxic (positive) or non-toxic (negative) for the intended scenario. Only in few cases, when not enough information can be obtained, will animal tests be performed as an additional source of information. Good information can be provided on all chemicals and, due to the high throughput of the approach, also on mixtures.
- 1895, 8 anni prima del volo dei fratelli Wright