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La rivoluzione REACH

The document discusses the REACH regulation aimed at protecting human health and the environment by promoting alternative hazard assessment methods and ensuring the free circulation of substances in the internal market. It outlines specific guidelines for various types of data to be collected, including in vitro toxicity testing, and emphasizes the importance of existing data and weight of evidence in assessing chemical properties. Additionally, it highlights the EU directive changes focused on animal welfare and the principles of the 3Rs (reduction, refinement, replacement) in research.

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La rivoluzione REACH Costanza Rovida REACH Mastery - Como 23 maggio 2014
La rivoluzione REACH Costanza Rovida Costanza.rovida@chimici.it Center for Alternatives to Animal Testing – Europe (CAAT-Europe) University of Konstanz 23 maggio 2014
Article 1 Aim and scope 1. The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, as well as the free circulation of substances on the internal market while enhancing competitiveness and innovation
Article 1 Aim and scope 1. The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, as well as the free circulation of substances on the internal market while enhancing competitiveness and innovation
List of required endpoints, according to REACH Regulation Physico-chemical information
List of required endpoints, according to REACH Regulation Toxicological information
List of required endpoints, according to REACH Regulation: Ecotoxicological information
IUCLID fields to be completed
Guidelines on Information Requirements
Guidelines on Information Requirements TOTAL of 34 FILES !!! (How many thousands of pages?)
ECHA Practical Guides 1. How to report in vitro data 2. How to report Robust Study Summary 3. How to report Weight of Evidence 4. How to report waiving 5. How to report (Q)SARs 6. How to report read across and categories http://echa.europa.eu/doc/press/na_10_16_practical_guides_20100409.pdf
Article 1 Aim and scope 1. The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, as well as the free circulation of substances on the internal market while enhancing competitiveness and innovation
Articolo 13
REACH Regulation ANNEXES VII-X Before new tests are carried out to determine the properties listed in this Annex, all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) shall be assessed first ANNEXES VII-X – Column 2 Specific rules for adaptation from column 1 ANNEX XI General rules for adaptation of the standard testing regime set out in annexes VII to X
Annex XI TESTING DOES NOT APPEAR SCIENTIFICALLY NECESSARY • Use of existing data • Historical human data PRO and CONS Studies are often very poor described No analytical identification of the substance No identification of the impurities …………….. ………………
Annex XI -Weight of Evidence There may be sufficient Weight of Evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion. • newly developed test methods • not yet included in the test methods referred to in Article 13(3) • different studies with the same reliability, no key study, but consistent to assess the end point
Annex XI - (Q)SAR QUALITATIVE OR QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP ((Q)SAR) PRO and CONS Applicability domain Fit the purpose “Poor” training data sets Difficulties in reporting …………….. ………………
Annex XI - Grouping and Read Across GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH Two approaches: GROUPING IN CATEGORIES (HPV-SIDS approach) chemical similarities functional groups physical chemical properties common environmental fate common metabolites READ ACROSS FROM SIMILAR SUBSTANCES a specific end-point can be assessed with a result on a similar substance different end point can be assessed from different substances
Annex XI ALTERNATIVE METHODS (ECVAM activities) http://tsar.jrc.ec.europa.eu/ Reduction of animals: Sensitisation Acute oral toxicity Acute aquatic toxicity Developing screening metods that can support the reliability of a category or a read across Pavia – 7 maggio 2011
Annex XI GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH Pavia – 7 maggio 2011 Two approaches: GROUPING IN CATEGORIES (HPV-SIDS approach) chemical similarities functional groups physical chemical properties common environmental fate common metabolites READ ACROSS FROM SIMILAR SUBSTANCES a specific end-point can be assessed with a result on a similar substance different end point can be assessed from different substances
Annex XI SUBSTANCE-TAILORED EXPOSURE-DRIVEN TESTING Testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and Annex X may be omitted, based on the exposure scenario(s) developed in the Chemical Safety Report PERFORM a Chemical Safety Assessment complete with Exposure Scenarios even if the substance is NOT DANGEROUS CONS: Companies are often not cooperative even if they save money Pavia – 7 maggio 2011
In vitro methods In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and direct food additives that may or may not taste good. In vitro assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal movement to reduce the use of animals in research, and a desire to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects. www.answers.com
I metodi alternativi: che cosa sono Principio delle 3R: Reduction = Riduzione Refinement = Miglioramento Replacement = Sostituzione Russell, W. M. S. and Burch, R. L. 1959. The principles of humane experimental technique
Direttiva EU 63/2010 Sostituisce la vecchia Direttiva EEC 86/609 con una maggiore attenzione al benessere animale e un esplicito riferimento alle 3R Maggiore controllo e rigore sugli esperimenti
Element Dir 86/609/EEC Dir 2010/63/EU Ambito di applicazione Vertebrati. Vertebrati, cefalopdi, Feti Compliance with the Three Rs Yes Yes Project evaluation No Yes Project authorisation (Yes/No) Yes Housing and care standards guidelines binding standards Main differences between the current & the new Dir
Main differences between the current & the new Dir Element Dir 86/609/EEC Dir 2010/63/EU Project follow-up (Yes) Yes - Retrospective assessment No Yes Non-technical summaries No Yes Severity classification No Yes Ispezioni periodiche Yes Obbligatorie
Shell Fish Biotoxins Mouse bio-assay
Shell Fish Biotoxins Metodo in vitro
Confronto metodo in vitro e in vivo 140 120 100 80 60 40 20 0 In vitro Assay Mouse Bioassay 17.2% 52.6% 80.0% 1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157 μg STX eq/100 g correlative sample number
In vitro methods In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and direct food additives that may or may not taste good. In vitro assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal movement to reduce the use of animals in research, and a desire to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects. www.answers.com
IN VITRO METHODS Validated methods: Skin corrosion/irritation Skin absorption Eye corrosion/irritation (partially) Screening for genotoxicity and mutagenicity (partially) Acute Toxicity (partially) Pre-validation methods: Sensitisation Carcinogenic potential Reproductive toxicity (embryotoxicity)
In vitro methods: Skin sensitisation LOCAL LYMPH NODE ASSAY Days 1, 2 & 3 Apply Chemical Day 6 - Inject 3 H-Thymidine 5 hours Later Remove Lymph Nodes Make Cell Suspension 3 Determine H-Thymidine Incorporation by Liquid Scintillation Counting
In vitro approach to Skin sensitisation
SKIN Sensitisation DPRA (Direct Peptide Reactivity Assay): OECD 406 (Guinea pig): 2,000€ OECD 429 (LLNA, traditional test): 3,500€ OECD 442b (LLNA, BrdU-ELISA): 2,700€
Strategy: sensitisation Metal salt Metal oxide Pavia – 7 maggio 2011 MO Test with OECD TG 428 Skin Absorption ≥ 0.1% Skin Absorption < 0.1% MX2 LLNA positive (Strong) Negative No classification as skin sensitiser Positive Classified as skin sensitiser H317 Read Across
Strategy: sensitisation IN VITRO STUDIES : An example on how to assess doubtful sensitization results on a basic dye supporting also exposure pattern. No study available on both Oxalate and Chloride forms of the dye Demonstration that the two salts are equivalent is missing There is concern about the skin sensitisation potential Negative result in an old study (1984) Positive in a even older human patch test (1966) • A new study performed on both salts is required • A new study to confirm the sensitisation property is required Pavia – 7 maggio 2011
Strategy: sensitisation Pavia – 7 maggio 2011 ITS proposal: 1. Skin absorption assessment: OECD TG 428 2. In vitro sensitisation: U937 method, which passed the pre-validation and has just started the official validation at ECVAM (European Centre for Validation of Alternative Methods) RATIONALE Skin Absorption: Only adsorbed substances can cause skin sensitisation The results can be also exploited as a comparison between the two salts Sensitisation: a POSITIVE results is accepted (Annex XI:positive results from a suitable in vitro test can be accepted) a NEGATIVE result is also accepted: (peer review literature)
Strategy: sensitisationoss Pavia – 7 maggio 2011 COSTS: OECD TG 428, in GLP: 2,700 €/per substance 4,320 € for the two salts when tested simultaneously U937 Performed for free at the University of Milan, as the validating committee needs new substances to test The final result will be confidential and will not be published (LLNA in vivo method): 7,200 €/per substance 10,800 € for both salts when tested simultaneously
Strategy: full dossier In vitro studies to assess the category of nickel compounds and verify the representative substance to take the existing data in Read Across BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL COMPOUNDS: INHALATION TOXICITY Sample ID Code(s)2 CAS No. Ni Content (%)3 Interstitial Bioaccessibility (% Ni/g sample)4 Pavia – 7 maggio 2011 Lysosomal Bioaccessibility (% Ni/g sample)4 Acute Toxicity (inhalation LC50, mg/L)5 Ni Sulfate Hexahydrate N58-72 10101-97-0 22 12.80 21.35 2.48 Ni Oxide Green N9/N46 (N126) 1313-99-1 77 (81) 0.10 0.82 >5.08 Ni Sub-Sulfide N129 (N18) 12035-72-2 61 (70) 3.60 26.20 1.14 Ni Hydroxy Carbonate N128 (N109) 12122-15-5 49 (49) 1.65 47.206 >2.09 (F); 0.25 (M)7 Ni Chloride Hexahydrate N98 7791-20-0 25 8.10 25.056 NC Ni Acetate Tetrahydrate N103 6018-89-9 24 10.90 24.856 NC Ni Sulfamate N104 13770-89-3 18 8.60 18.306 NC Tetrahydrate Ni Hydroxide N106 12054-48-7 54 0.02 55.80 NC Ni Oxide Black N105 1313-99-1 75 0.56 24.50 NC Ni Sulfide N97 16812-54-7 59 1.08 25.95 NC
Strategy: full dossier BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL COMPOUNDS: ORAL SYSTEMIC TOXICITY Sample ID Code1 CAS No. Ni Content (%)2 Pavia – 7 maggio 2011 Bioaccessibility (% Ni content released)3 In Vivo Acute Toxicity (oral LD50, mg/kg/bw)4 Ni Sulfate Hexahydrate N58-72 10101-97-0 23 90.55% 362 Ni Oxide Green N9/N46 1313-99-1 77 0.33% >11000 Ni Oxide Green N112 1313-99-1 81 <LOD5 >11000 Ni Sub-Sulfide N18 12035-72-2 70 22.65% >11000 Ni Chloride Hexahydrate N98 7791 -20-0 25 89.85% 500 Ni Acetate Tetrahydrate N 103 6018-89-9 24 88.50% 550 Ni Hydroxy Carbonate N109 121 22-1 5-5 49 84.30% 2000 Ni Sulfamate N 104 13770-89-3 18 83.40% 1098 Tetrahydrate Ni Hydroxide N106 12054-48-7 54 26.30% 5000 Ni Oxide Black N105 131 3-99-1 75 29.60% 9990 Ni Sulfide N97 1681 2-54-7 59 9.75% NC6 Ni Fluoride Tetrahydrate7 N111 13940-83-5 32 82.35% 310
European projects OSIRIS Optimised Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Test Information http://www.osiris-reach.eu/ Pavia – 7 maggio 2011
Improvements With the first dossiers many data have been generated, that can be used to improve also the reliability of predictions models in the next years More in vitro metods will be developed / validated Multidisciplinary approach within the working groups Will ECHA be ready? Pavia – 7 maggio 2011
An example for a Strategy IN VITRO STUDIES : An example on how to assess doubtful sensitization results on a basic dye supporting also exposure pattern. No study available on both Oxalate and Chloride forms of the dye Demonstration that the two salts are equivalent is missing There is concern about the skin sensitisation potential Negative result in an old study (1984) Positive in a even older human patch test (1966) • A new study performed on both salts is required • A new study to confirm the sensitisation property is required
ITS proposal: 1. Skin absorption assessment: OECD TG 428 2. In vitro sensitisation: THP-1 method, which passed the pre-validation and has just started the official validation at ECVAM (European Centre for Validation of Alternative Methods) RATIONALE Skin Absorption: Only adsorbed substances can cause skin sensitisation The results can be also exploited as a comparison between the two salts Sensitisation: a POSITIVE results is accepted (Annex XI:positive results from a suitable in vitro test can be accepted) a NEGATIVE result is also accepted: (peer review literature)
COSTS: OECD TG 428, in GLP: 2,700 €/per substance 4,320 € for the two salts when tested simultaneously THP-1 Performed for free at the University of Milan, as the validating committee needs new substances to test (LLNA in vivo method): 7,200 €/per substance 10,800 € for both salts when tested simultaneously
In vitro methods: Strategy Existing data Weight of Evidence GOOD In vitro data SENSE! INTELLIGENT = INTEGRATED Exposure assessment Read across Categories In silico data
Validation modular approach Test definition Within-lab. variability Transferability Between-lab.variability Predictive capacity Applicability domain Minimum performance standards Reprodu-cibility Relevance “Standardised” “Suitable/Adequate” “Validated” “Equivalent” Hartung et al. ATLA 2004, 32:467-472
OECD Member Countries Countries/Economies Engaged in Working OECD Relationships with the OECD
OECD Methods 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 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 437 BCOP Test H318 Causes serious eye damage
Un esempio di una strategia IN VITRO STUDIES : Un esempio di come una strategia in vitro è stata utilizzata per la valutazione di un risultato dubbio sulla sensitization e per avere maggiori dati sull'esposizione. Nessuno studio disponibile su entrambi i Sali in commercio del colorante (Ossalato e cloruro) Mancanza di una dimostrazione che i due Sali fossero equivalenti Dubbio che potesse essere allergenico Risultato negativo in uno studio molto vecchio (1984) Risultato positivo in un human patch test ancora più vecchio (1966) • Necessario uno studio da fare contemporaneamente sui due sali • Necessario uno studio per decidere sulla skin sensitisation
ITS proposal: 1. Skin absorption assessment: OECD TG 428 2. In vitro sensitisation: THP-1 method, che ha già passato la fase di pre-validation and ed è in corso di validazione ufficiale a ECVAM (European Centre for Validation of Alternative Methods) RATIONALE Skin Absorption: Solo le sostanze che vengono assorbite possono provocare allergie. Il risultato può essere utilizzato come base per il confronto tra i due sali Sensitisation: un risultato POSITIVO è accettato (Annex XI: positive results from a suitable in vitro test can be accepted) un risultato NEGATIVO è accettato lo stesso (peer review literature)
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
… Approccio avanzato, ma ancora con impostazione classica…
The reproductive cycle Courtesy of Michael Schwarz
Use of bovine gametes for reproductive toxicity testing 2 cm bovine ovaries In vitro maturation (IVM) of bovine oocytes bovine oocytes Endpoint: % Metaphase II Chemical exposure (24h) during IVM In vitro fertilisation (IVF) of bovine oocytes Chemical exposure (20h) during IVF Endpoint: % 2Pronuclei IVM, 24 h
dall’embrione alle cellule staminali concepimento blastocisti (5 giorni) Coltura di cellule embrionali staminali
Stem Cell Toxicity Approach Developmental Toxicity Acute Toxicty
validation performance predictive confidence in vivo relevance
QIVIVE Quantitative in vitro in vivo extrapolation Target cells: permeability, transport, metabolic competence Extracellular space: proteins, lipids, pH, plastic, other cells Exposure (concentration x time)
Modelli PBPK
Report del National Research Council of the National Academies Pubblicato nel 2007 (Google: "Toxicity Testing in the 21st Century")
A New Paradigm: Activation of Toxicity Pathways 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)
Progetto ToxCast
Toxicity Pathways A cellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect. Set Chemicals Assays Endpoints Completion Available ToxCast Phase I 293 ~600 ~1100 2011 Now ToxCast Phase II 767 ~600 ~1100 03/2013 10/2013 ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014 E1K (endocrine) 880 ~50 ~120 03/2013 10/2013 Tox21 8,193 ~25 ~50 Ongoing Ongoing
Effect of Concentration Response on Polypharmacology 72 Pentachlorophenol
“Non sopravvive chi è più forte o più intelligente, ma chi reagisce più velocemente ai cambiamenti” Charles Darwin

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La rivoluzione REACH

  • 1.
    La rivoluzione REACH Costanza Rovida REACH Mastery - Como 23 maggio 2014
  • 2.
    La rivoluzione REACH Costanza Rovida Costanza.rovida@chimici.it Center for Alternatives to Animal Testing – Europe (CAAT-Europe) University of Konstanz 23 maggio 2014
  • 5.
    Article 1 Aimand scope 1. The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, as well as the free circulation of substances on the internal market while enhancing competitiveness and innovation
  • 6.
    Article 1 Aimand scope 1. The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, as well as the free circulation of substances on the internal market while enhancing competitiveness and innovation
  • 7.
    List of required endpoints, according to REACH Regulation Physico-chemical information
  • 8.
    List of required endpoints, according to REACH Regulation Toxicological information
  • 9.
    List of required endpoints, according to REACH Regulation: Ecotoxicological information
  • 11.
    IUCLID fields tobe completed
  • 12.
  • 13.
    Guidelines on InformationRequirements TOTAL of 34 FILES !!! (How many thousands of pages?)
  • 14.
    ECHA Practical Guides 1. How to report in vitro data 2. How to report Robust Study Summary 3. How to report Weight of Evidence 4. How to report waiving 5. How to report (Q)SARs 6. How to report read across and categories http://echa.europa.eu/doc/press/na_10_16_practical_guides_20100409.pdf
  • 15.
    Article 1 Aimand scope 1. The purpose of this Regulation is to ensure a high level of protection of human health and the environment, including the promotion of alternative methods for assessment of hazards of substances, as well as the free circulation of substances on the internal market while enhancing competitiveness and innovation
  • 16.
  • 17.
    REACH Regulation ANNEXESVII-X Before new tests are carried out to determine the properties listed in this Annex, all available in vitro data, in vivo data, historical human data, data from valid (Q)SARs and data from structurally related substances (read-across approach) shall be assessed first ANNEXES VII-X – Column 2 Specific rules for adaptation from column 1 ANNEX XI General rules for adaptation of the standard testing regime set out in annexes VII to X
  • 18.
    Annex XI TESTINGDOES NOT APPEAR SCIENTIFICALLY NECESSARY • Use of existing data • Historical human data PRO and CONS Studies are often very poor described No analytical identification of the substance No identification of the impurities …………….. ………………
  • 19.
    Annex XI -Weightof Evidence There may be sufficient Weight of Evidence from several independent sources of information leading to the assumption/conclusion that a substance has or has not a particular dangerous property, while the information from each single source alone is regarded insufficient to support this notion. • newly developed test methods • not yet included in the test methods referred to in Article 13(3) • different studies with the same reliability, no key study, but consistent to assess the end point
  • 20.
    Annex XI -(Q)SAR QUALITATIVE OR QUANTITATIVE STRUCTURE-ACTIVITY RELATIONSHIP ((Q)SAR) PRO and CONS Applicability domain Fit the purpose “Poor” training data sets Difficulties in reporting …………….. ………………
  • 21.
    Annex XI -Grouping and Read Across GROUPING OF SUBSTANCES AND READ-ACROSS APPROACH Two approaches: GROUPING IN CATEGORIES (HPV-SIDS approach) chemical similarities functional groups physical chemical properties common environmental fate common metabolites READ ACROSS FROM SIMILAR SUBSTANCES a specific end-point can be assessed with a result on a similar substance different end point can be assessed from different substances
  • 22.
    Annex XI ALTERNATIVEMETHODS (ECVAM activities) http://tsar.jrc.ec.europa.eu/ Reduction of animals: Sensitisation Acute oral toxicity Acute aquatic toxicity Developing screening metods that can support the reliability of a category or a read across Pavia – 7 maggio 2011
  • 23.
    Annex XI GROUPINGOF SUBSTANCES AND READ-ACROSS APPROACH Pavia – 7 maggio 2011 Two approaches: GROUPING IN CATEGORIES (HPV-SIDS approach) chemical similarities functional groups physical chemical properties common environmental fate common metabolites READ ACROSS FROM SIMILAR SUBSTANCES a specific end-point can be assessed with a result on a similar substance different end point can be assessed from different substances
  • 24.
    Annex XI SUBSTANCE-TAILOREDEXPOSURE-DRIVEN TESTING Testing in accordance with sections 8.6 and 8.7 of Annex VIII, Annex IX and Annex X may be omitted, based on the exposure scenario(s) developed in the Chemical Safety Report PERFORM a Chemical Safety Assessment complete with Exposure Scenarios even if the substance is NOT DANGEROUS CONS: Companies are often not cooperative even if they save money Pavia – 7 maggio 2011
  • 25.
    In vitro methods In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and direct food additives that may or may not taste good. In vitro assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal movement to reduce the use of animals in research, and a desire to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects. www.answers.com
  • 26.
    I metodi alternativi:che cosa sono Principio delle 3R: Reduction = Riduzione Refinement = Miglioramento Replacement = Sostituzione Russell, W. M. S. and Burch, R. L. 1959. The principles of humane experimental technique
  • 27.
    Direttiva EU 63/2010 Sostituisce la vecchia Direttiva EEC 86/609 con una maggiore attenzione al benessere animale e un esplicito riferimento alle 3R Maggiore controllo e rigore sugli esperimenti
  • 28.
    Element Dir 86/609/EECDir 2010/63/EU Ambito di applicazione Vertebrati. Vertebrati, cefalopdi, Feti Compliance with the Three Rs Yes Yes Project evaluation No Yes Project authorisation (Yes/No) Yes Housing and care standards guidelines binding standards Main differences between the current & the new Dir
  • 29.
    Main differences between the current & the new Dir Element Dir 86/609/EEC Dir 2010/63/EU Project follow-up (Yes) Yes - Retrospective assessment No Yes Non-technical summaries No Yes Severity classification No Yes Ispezioni periodiche Yes Obbligatorie
  • 30.
    Shell Fish Biotoxins Mouse bio-assay
  • 32.
    Shell Fish Biotoxins Metodo in vitro
  • 33.
    Confronto metodo invitro e in vivo 140 120 100 80 60 40 20 0 In vitro Assay Mouse Bioassay 17.2% 52.6% 80.0% 1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97 103 109 115 121 127 133 139 145 151 157 μg STX eq/100 g correlative sample number
  • 34.
    In vitro methods In vitro toxicity testing is the scientific analysis of the effects of toxic chemical substances on cultured bacteria or mammalian cells. In vitro (literally 'in glass') testing methods are employed primarily to identify potentially hazardous chemicals and/or to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and direct food additives that may or may not taste good. In vitro assays for xenobiotic toxicity are recently carefully considered by key government agencies (e.g. EPA; NIEHS/NTP; FDA), mainly due to a societal movement to reduce the use of animals in research, and a desire to better assess human risks. There are substantial activities in using in vitro systems to advance mechanistic understanding of toxicant activities, and the use of human cells and tissue to define human-specific toxic effects. www.answers.com
  • 35.
    IN VITRO METHODS Validated methods: Skin corrosion/irritation Skin absorption Eye corrosion/irritation (partially) Screening for genotoxicity and mutagenicity (partially) Acute Toxicity (partially) Pre-validation methods: Sensitisation Carcinogenic potential Reproductive toxicity (embryotoxicity)
  • 36.
    In vitro methods:Skin sensitisation LOCAL LYMPH NODE ASSAY Days 1, 2 & 3 Apply Chemical Day 6 - Inject 3 H-Thymidine 5 hours Later Remove Lymph Nodes Make Cell Suspension 3 Determine H-Thymidine Incorporation by Liquid Scintillation Counting
  • 37.
    In vitro approachto Skin sensitisation
  • 38.
    SKIN Sensitisation DPRA(Direct Peptide Reactivity Assay): OECD 406 (Guinea pig): 2,000€ OECD 429 (LLNA, traditional test): 3,500€ OECD 442b (LLNA, BrdU-ELISA): 2,700€
  • 39.
    Strategy: sensitisation Metalsalt Metal oxide Pavia – 7 maggio 2011 MO Test with OECD TG 428 Skin Absorption ≥ 0.1% Skin Absorption < 0.1% MX2 LLNA positive (Strong) Negative No classification as skin sensitiser Positive Classified as skin sensitiser H317 Read Across
  • 40.
    Strategy: sensitisation INVITRO STUDIES : An example on how to assess doubtful sensitization results on a basic dye supporting also exposure pattern. No study available on both Oxalate and Chloride forms of the dye Demonstration that the two salts are equivalent is missing There is concern about the skin sensitisation potential Negative result in an old study (1984) Positive in a even older human patch test (1966) • A new study performed on both salts is required • A new study to confirm the sensitisation property is required Pavia – 7 maggio 2011
  • 41.
    Strategy: sensitisation Pavia– 7 maggio 2011 ITS proposal: 1. Skin absorption assessment: OECD TG 428 2. In vitro sensitisation: U937 method, which passed the pre-validation and has just started the official validation at ECVAM (European Centre for Validation of Alternative Methods) RATIONALE Skin Absorption: Only adsorbed substances can cause skin sensitisation The results can be also exploited as a comparison between the two salts Sensitisation: a POSITIVE results is accepted (Annex XI:positive results from a suitable in vitro test can be accepted) a NEGATIVE result is also accepted: (peer review literature)
  • 42.
    Strategy: sensitisationoss Pavia– 7 maggio 2011 COSTS: OECD TG 428, in GLP: 2,700 €/per substance 4,320 € for the two salts when tested simultaneously U937 Performed for free at the University of Milan, as the validating committee needs new substances to test The final result will be confidential and will not be published (LLNA in vivo method): 7,200 €/per substance 10,800 € for both salts when tested simultaneously
  • 43.
    Strategy: full dossier In vitro studies to assess the category of nickel compounds and verify the representative substance to take the existing data in Read Across BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL COMPOUNDS: INHALATION TOXICITY Sample ID Code(s)2 CAS No. Ni Content (%)3 Interstitial Bioaccessibility (% Ni/g sample)4 Pavia – 7 maggio 2011 Lysosomal Bioaccessibility (% Ni/g sample)4 Acute Toxicity (inhalation LC50, mg/L)5 Ni Sulfate Hexahydrate N58-72 10101-97-0 22 12.80 21.35 2.48 Ni Oxide Green N9/N46 (N126) 1313-99-1 77 (81) 0.10 0.82 >5.08 Ni Sub-Sulfide N129 (N18) 12035-72-2 61 (70) 3.60 26.20 1.14 Ni Hydroxy Carbonate N128 (N109) 12122-15-5 49 (49) 1.65 47.206 >2.09 (F); 0.25 (M)7 Ni Chloride Hexahydrate N98 7791-20-0 25 8.10 25.056 NC Ni Acetate Tetrahydrate N103 6018-89-9 24 10.90 24.856 NC Ni Sulfamate N104 13770-89-3 18 8.60 18.306 NC Tetrahydrate Ni Hydroxide N106 12054-48-7 54 0.02 55.80 NC Ni Oxide Black N105 1313-99-1 75 0.56 24.50 NC Ni Sulfide N97 16812-54-7 59 1.08 25.95 NC
  • 44.
    Strategy: full dossier BIOACCESSIBILITY BASED READ ACROSS ASSESSMENT OF NICKEL COMPOUNDS: ORAL SYSTEMIC TOXICITY Sample ID Code1 CAS No. Ni Content (%)2 Pavia – 7 maggio 2011 Bioaccessibility (% Ni content released)3 In Vivo Acute Toxicity (oral LD50, mg/kg/bw)4 Ni Sulfate Hexahydrate N58-72 10101-97-0 23 90.55% 362 Ni Oxide Green N9/N46 1313-99-1 77 0.33% >11000 Ni Oxide Green N112 1313-99-1 81 <LOD5 >11000 Ni Sub-Sulfide N18 12035-72-2 70 22.65% >11000 Ni Chloride Hexahydrate N98 7791 -20-0 25 89.85% 500 Ni Acetate Tetrahydrate N 103 6018-89-9 24 88.50% 550 Ni Hydroxy Carbonate N109 121 22-1 5-5 49 84.30% 2000 Ni Sulfamate N 104 13770-89-3 18 83.40% 1098 Tetrahydrate Ni Hydroxide N106 12054-48-7 54 26.30% 5000 Ni Oxide Black N105 131 3-99-1 75 29.60% 9990 Ni Sulfide N97 1681 2-54-7 59 9.75% NC6 Ni Fluoride Tetrahydrate7 N111 13940-83-5 32 82.35% 310
  • 45.
    European projects OSIRIS Optimised Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Test Information http://www.osiris-reach.eu/ Pavia – 7 maggio 2011
  • 46.
    Improvements With thefirst dossiers many data have been generated, that can be used to improve also the reliability of predictions models in the next years More in vitro metods will be developed / validated Multidisciplinary approach within the working groups Will ECHA be ready? Pavia – 7 maggio 2011
  • 47.
    An example fora Strategy IN VITRO STUDIES : An example on how to assess doubtful sensitization results on a basic dye supporting also exposure pattern. No study available on both Oxalate and Chloride forms of the dye Demonstration that the two salts are equivalent is missing There is concern about the skin sensitisation potential Negative result in an old study (1984) Positive in a even older human patch test (1966) • A new study performed on both salts is required • A new study to confirm the sensitisation property is required
  • 48.
    ITS proposal: 1.Skin absorption assessment: OECD TG 428 2. In vitro sensitisation: THP-1 method, which passed the pre-validation and has just started the official validation at ECVAM (European Centre for Validation of Alternative Methods) RATIONALE Skin Absorption: Only adsorbed substances can cause skin sensitisation The results can be also exploited as a comparison between the two salts Sensitisation: a POSITIVE results is accepted (Annex XI:positive results from a suitable in vitro test can be accepted) a NEGATIVE result is also accepted: (peer review literature)
  • 49.
    COSTS: OECD TG428, in GLP: 2,700 €/per substance 4,320 € for the two salts when tested simultaneously THP-1 Performed for free at the University of Milan, as the validating committee needs new substances to test (LLNA in vivo method): 7,200 €/per substance 10,800 € for both salts when tested simultaneously
  • 50.
    In vitro methods:Strategy Existing data Weight of Evidence GOOD In vitro data SENSE! INTELLIGENT = INTEGRATED Exposure assessment Read across Categories In silico data
  • 51.
    Validation modular approach Test definition Within-lab. variability Transferability Between-lab.variability Predictive capacity Applicability domain Minimum performance standards Reprodu-cibility Relevance “Standardised” “Suitable/Adequate” “Validated” “Equivalent” Hartung et al. ATLA 2004, 32:467-472
  • 53.
    OECD Member Countries Countries/Economies Engaged in Working OECD Relationships with the OECD
  • 54.
    OECD Methods 428Skin 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
  • 55.
    OECD Methods 428Skin 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
  • 56.
    OECD 437 BCOPTest H318 Causes serious eye damage
  • 57.
    Un esempio diuna strategia IN VITRO STUDIES : Un esempio di come una strategia in vitro è stata utilizzata per la valutazione di un risultato dubbio sulla sensitization e per avere maggiori dati sull'esposizione. Nessuno studio disponibile su entrambi i Sali in commercio del colorante (Ossalato e cloruro) Mancanza di una dimostrazione che i due Sali fossero equivalenti Dubbio che potesse essere allergenico Risultato negativo in uno studio molto vecchio (1984) Risultato positivo in un human patch test ancora più vecchio (1966) • Necessario uno studio da fare contemporaneamente sui due sali • Necessario uno studio per decidere sulla skin sensitisation
  • 58.
    ITS proposal: 1.Skin absorption assessment: OECD TG 428 2. In vitro sensitisation: THP-1 method, che ha già passato la fase di pre-validation and ed è in corso di validazione ufficiale a ECVAM (European Centre for Validation of Alternative Methods) RATIONALE Skin Absorption: Solo le sostanze che vengono assorbite possono provocare allergie. Il risultato può essere utilizzato come base per il confronto tra i due sali Sensitisation: un risultato POSITIVO è accettato (Annex XI: positive results from a suitable in vitro test can be accepted) un risultato NEGATIVO è accettato lo stesso (peer review literature)
  • 59.
    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
  • 60.
    … Approccio avanzato,ma ancora con impostazione classica…
  • 61.
    The reproductive cycle Courtesy of Michael Schwarz
  • 62.
    Use of bovinegametes for reproductive toxicity testing 2 cm bovine ovaries In vitro maturation (IVM) of bovine oocytes bovine oocytes Endpoint: % Metaphase II Chemical exposure (24h) during IVM In vitro fertilisation (IVF) of bovine oocytes Chemical exposure (20h) during IVF Endpoint: % 2Pronuclei IVM, 24 h
  • 63.
    dall’embrione alle cellulestaminali concepimento blastocisti (5 giorni) Coltura di cellule embrionali staminali
  • 64.
    Stem Cell ToxicityApproach Developmental Toxicity Acute Toxicty
  • 65.
    validation performance predictive confidence in vivo relevance
  • 66.
    QIVIVE Quantitative invitro in vivo extrapolation Target cells: permeability, transport, metabolic competence Extracellular space: proteins, lipids, pH, plastic, other cells Exposure (concentration x time)
  • 67.
  • 68.
    Report del NationalResearch Council of the National Academies Pubblicato nel 2007 (Google: "Toxicity Testing in the 21st Century")
  • 69.
    A New Paradigm:Activation of Toxicity Pathways 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)
  • 70.
  • 71.
    Toxicity Pathways Acellular response pathway that, when sufficiently perturbed, is expected to result in an adverse health effect. Set Chemicals Assays Endpoints Completion Available ToxCast Phase I 293 ~600 ~1100 2011 Now ToxCast Phase II 767 ~600 ~1100 03/2013 10/2013 ToxCast Phase IIIa 1001 ~100 ~100 Just starting 2014 E1K (endocrine) 880 ~50 ~120 03/2013 10/2013 Tox21 8,193 ~25 ~50 Ongoing Ongoing
  • 72.
    Effect of ConcentrationResponse on Polypharmacology 72 Pentachlorophenol
  • 74.
    “Non sopravvive chiè più forte o più intelligente, ma chi reagisce più velocemente ai cambiamenti” Charles Darwin

Editor's Notes

  • #4 Un’importante occasione di lavoro
  • #27 http://isogenic.info/html/ethical_considerations.html Russell, W. M. S. and Burch, R. L. 1959. The principles of humane experimental technique Special Edition, Universities Federation for Animal Welfare, Potters Bar, England L'inizio è filosofico-etico
  • #28 Differenza tra Direttiva e regolamento Direttiva non ancora approvata in Italia
  • #29 28
  • #30 29
  • #31 Come si evolvono i metodi alternativi; facciamo un esempio
  • #33 Azaspiracid AZP, comune in Europa
  • #34 Non voglio più parlare di 3R o di metodi alternativi, ma, se vogliamo mantenere la A, di metodi avanzati che siano al passo della tossicologia o della ricerca bio-medica del futuro
  • #52 Si casca sulla predictive capacity
  • #53 OECD è per tutto, non solo per I metodi alternativi Le linee guida della szione 4 sono relative alla salute sull'uomo, le 2 per l'ambiente. Importanti quelle generiche della sezione 0 o 1
  • #55 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
  • #57 Il problema è che tra I composti che non sono classificati nè negativi, nè positivi, non si può prendere una decisione
  • #61 Chiaro che non siamo soddisfatti. Ma si è già iniziato a lavorare su due, forse tre piani separati
  • #62 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).
  • #67 What are equivalent concentrations in vitro and in vivo?
  • #70 69
  • #72 71