Alternative and integrated testing strategies
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The document discusses alternative and integrated testing strategies for toxicity assessment. It describes the development of such strategies over time, from early proposals in 2002 for integrated testing schemes, to the current use of adverse outcome pathways (AOPs) and predictive toxicology approaches using cellular assays. Validation and regulatory acceptance of alternative methods has allowed reduction in animal testing, especially for skin and eye irritation/corrosion hazards.
![Roche Hanging Droplet Culture Plate [HDCP]
• In-house developed injection-molded proprietary
labware for culturing cells in 3D
• Precise engineering allows excellent droplet
stability
• 24well currently used for automated EST assay.
• 96well design finalized & manufactured.
Implementation ongoing.
20 ul 70 ul
Patent pending
Round shape to
avoid EBs sticking
on well corner
Edged shape for
good drop stability
Small ring shape
for good droplet
stability
Patent pending
Slide Claudia McGinnis 36](https://image.slidesharecdn.com/160211a2spielmann-161012154358/85/Alternative-and-integrated-testing-strategies-36-320.jpg)
Alternative and integrated testing strategies
- 1. 1 Horst Spielmann Professor for Regulatory Toxicology Freie Universität Berlin & State Animal Welfare Officer, Berlin Alternative and Integrated Testing Strategies Der Landestierschutzbeauftragte EFSA Symposium “NOVEL CHEMICAL HAZARD CHARACTERISATION APPROACHES “ Section 2: Systems biology approach and predictive toxicology 16 October 2015 – Milano at EXPO2015
- 2. 2 2002 ECVAM - proposal for integrated testing scheme for chemicals 2002 OECD - sequential testing strategy eye & skin irritation/corrosion 2004 BfR - “Concept” for in vitro eye & skin irritation testing 2005 ECVAM - “Top-Down” & “Bottom-Up” approaches for eye irritation testing 2009 ECVAM - WS validation of Integrated Testing Strategies (ITS) 2014 OECD - GD 203 “Integrated approach on testing and assessment (IATA) for skin corrosion & irritation 2012 OECD - AOP for skin sensitization an ITS approach 2012 ROCHE - Embryonic Stem cell Test (EST) an ITS approach 2013 EU - ban on animal testing for cosmetics Topics Horst Spielmann, VTA engl. June 2012
- 3. Action proposed in the EU White Paper 1. Identical amount of testing for new & existing chemicals ►more information on existing chemcials and less on new ones 2. Steps proposed for the testing of 30.000 existing chemicals until 2012 ►high production volume chemicals will be tested first 3. Only in vitro/non-animal methods will be used in the basic test set ► they are faster and cheaper to perform, will the information be sufficient for risk assessment for humans and the environment ? ► CONSEQUENCES in 2002: The 6th Framework Program of the EU Commission includes funding of research for development and validation of new non- animal methods. In 2007 the 7th FP included major funding for alternative methods Principles of the Future Chemicals Policy of the EU (White Paper) 2002 ►REACH !! Principles of the Future Chemicals Policy of the EU (White Paper) 2002 ►REACH !!
- 4. ECVAM WG proposal of an integrated testing scheme for Existing and New Chemicals in the EU 2002 ECVAM WG proposal of an integrated testing scheme for Existing and New Chemicals in the EU 2002 Background Data Name Chemical Structure CAS number Proposed use Scale of production Producers Exposure Transport Physiochemical Properties Volatility, Solubility (aqueous) Melting point, Boiling point Stability, Log P pk a Henry´s partition coefficient UV - visible absorption Surface tension Existing Toxicological Data (Review) Testing required? DATA SUFFICIENT FOR CLASSIFICATION CRITERIA to be defined Is further information required? Cytotoxicity, Genotoxicity S9 (Q)SAR , Predict. metabolism Percutaneous. absorption, Corrosivity / Irritancy, Sensitisation In vitro Testing (Tier 1) Priorities for further testing (Tier 2) YES NO NO YES
- 5. 5 OECD 2002:Sequential Testing Strategy > Agreed stepwise decision logic > Mandatory to follow all steps > Increasingly more complicated > From in vitro to in vivo approaches > Consideration of the hazard after each step > Works best for less complicated hazards (e.g. local effects, single endpoints) > Is a relatively rigid approach > Is relatively easy to harmonise internationally.BfR zebet
- 6. 6 Testing Strategy: Skin and Eye Effects (TG 404, TG 405) (adopted 24 April 2002) Existing human experience (Q)SAR (eye irritation/corrosion) (Q)SAR (skin corrosion) pH and buffering capacity evaluate dermal route toxicity data In vitro Test (eye irritation/corrosion) In vitro Test (skin irritation/corrosion) In vivo 1 Rabbit In vivo 2 Rabbits Reduces by about 90% that eye corrosives damage a rabbit eye
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- 9. 9 PERSPECTIVE: BfR View of a Strategy for Skin and Eye Irritation/Corrosion Assessment without Use of Animals Source: Höfer et al. (2004) Animal Testing and Alternative Approaches... (Arch. Toxicol. 78: 549–564 )
- 10. Conclusions Assessment of the potential of a chemical to induce local lesions We have shown that ► structural alerts for the prediction of a potential to cause local lesions and ► physicochemical limit values (DSS) for the prediction of the absence of such a potential provide testing and assessment strategies which use only (Q)SARs and results of in vitro testing instead of the current employment of test animals for the purpose of classification and labelling of acute local health hazards.
- 11. Horst Spielmann, 9. November 2006, Promega BfR-Position zu REACH Page 11 Alternative Testverfahren und intelligente Teststrategien Rules appropriate for all groups of chemicals: Basis: Evaluation of data of 1627 chemicals with purity > 95% Attention: Rules are valid exclusively for the Risk phrases mentioned within this specific "exclusion rule". This is due to the fact that acute local tissue lesions called "irritation" or "corrosion" and specified by the respective R-phrase of the EU are in reality based on a great variety of totally different biochemical reactions (depending on the chemical reactivity of the molecule which contacts the biological medium or structure first). IF melting point > 200o C THEN NOT (skin corrosion R34 or R35) (is true for 245/252 chemicals tested = 97%) (7 skin corrosive substances are organic salts which release strong inorganic acids or bases when getting in contact with aqueous substrates/organic media) IF log Pow > 9 THEN NOT (lesions R34,R35,R36 or R41) (is true for 32/32 chemicals tested = 100%) IF log Pow < -3.1 THEN NOT (skin corrosion R34 or R35) (is true for 53/53 chemicals tested) = 100%) IF lipid solubility < 0.01 g/kg THEN NOT (skin corrosion R34 or R35) (is true for 58/58 chemicals tested = 100%)) IF aqueous solubility < 0.00002 g/l THEN NOT (eye irritation R41) (is true for 109/109 chemicals tested = 100%)) IF aqueous solubility < 0.000005 g/l THEN NOT (eye irritation R36) (is true for 38/38 chemicals tested= 100%)) IF molecular weight > 650 g/Mol THEN NOT (eye irritation R36) (is true for 139/139 chemicals tested= 100%)) Attention: chemicals with molecular weight > 650 g/Mol may elicit severe tissue damage resulting in local corrosion! Physico-chemical limit values (DSS) for the absence of severely skin and eye irritating potential
- 12. Assessment strategy for local irritation/corrosion EU (& OECD) 2004
- 13. Proposed new BOTTOM-UP Approach BfR 2004
- 14. ECVAM Workshop February 2005 - Publication TIV 2010
- 16. 2009 In the context of safety assessment, an ITS is a methodology which integrates information for toxicological evaluation from more than one source, thus facilitating decision-making. This should be achieved whilst taking into consideration the principles of the Three Rs (reduction, refinement and replacement)”. It was also agreed that, in line with the proposal put forward during the OECD Workshop on Integrated Approaches to Testing and Assessment, held in December 2007, a good ITS should be structured, transparent and hypothesis driven.. 2012 During this workshop it was recognized that there is a fundamental difference between: a) Type 1 ITS, i.e. strategies to gather and analyze a broad range of data coming from different sources (epidemiological studies, animal data, in vitro data, read-across methodologies, etc) and used to draw conclusions based on weight-of-evidence (WoE) approaches; and b) Type 2 ITS: testing strategies composed of e.g. a number of in vitro and in silico methods that, combined and weighted in a fixed way, would serve to replace some or all in vivo experimentation for a given toxicity endpoint. This distinction is essential, when the validation of ITS is under consideration. EPAA ECVAM workshop definitions of ITS (Integrated Testing Strategy)
- 17. EPAA ECVAM workshop on ITS (Integrated Testing Strategy)
- 18. OECD GD 203 - ENV/JM/MONO (2014)19 COMPOSITION OF THE IATA FOR SKIN CORROSION AND IRRITATION 2014
- 19. OECD GD 203 - ENV/JM/MONO (2014)19 COMPOSITION OF THE IATA FOR SKIN CORROSION AND IRRITATION 2014
- 20. 20 H. Spielmann AXLR8 & HTTP: 17-01-2011 BASF Ludwigshafen US National Academy of Sciences 2007 “….. a not-so-distant future where all routine toxicity testing will be conducted in human cells or cell lines in vitro by evaluating perturbations of cellular responses in a suite of toxicity pathway assays…..” Andersen and Krewski (2009). Toxicity Testing in the 21st Century: Bringing the Vision to Life. Tox. Sci., 107, 324-330.
- 21. 21 H. Spielmann AXLR8 & HTTP: 17-01-2011 BASF Ludwigshafen 15 FEBRUARY 2008 VOL 319 SCIENCE www.sciencemag.org We propose a shift from primarily in vivo animal studies to in vitro assays, in vivo assays with lower organisms, and computational modeling for toxicity assessments.
- 22. 22 Spielmann AXLR8 IPM Milano 26 Nov 2010 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 Perturbation of Toxicity Pathways
- 23. 23 Spielmann AXLR8 IPM Milano 26 Nov 2010
- 24. 24 Spielmann AXLR8 IPM Milano 26 Nov 2010
- 25. 25 ● An adverse outcome pathway (AOP) is the sequence of events from chemical structure through the molecular initiating event to the in vivo outcome of interest. ● AOPs are representations of existing knowledge concerning the linkage(s) between a molecular initiating event and an adverse outcome at the individual or population level. ● As such, AOPs delineate the documented, plausible, and testable processes by which a chemical induces molecular perturbation and causes effects at the subcellular, cellular, tissue, organ, and whole animal levels of observation. The Adverse Outcome Pathway (AOP) for Skin Sensitization (draft; Feb 21, 2011) ► ►
- 26. 26 OECD 2012: AOP for skin sensitization initiatiated by covalent binding to protein
- 27. 27 OECD 2012: AOP for skin sensitization initiatiated by covalent binding to protein
- 28. 28 OECD 2012: AOP for skin sensitization initiatiated by covalent binding to protein
- 29. 29 ? Epidermis Epidermis Lymph Node Induction Elicitation 1. Skin Penetration 2. Electrophilic substance: directly or via auto-oxidation or metabolism 3-4. Haptenation: covalent modification of epidermal proteins 5-6. Activation of epidermal keratinocytes & Dendritic cells 7. Presentation of haptenated protein by Dendritic cell resulting in activation & proliferation of specific T cells 8-10. Allergic Contact Dermatitis: Epidermal inflammation following re-exposure to substance due to T cell-mediated cell death Key Event 1 Key Event 2 + 3 Key Event 4 Adverse Outcome Chemical Structure & Properties Organism Response Organ Response Cellular Response Molecular Initiating Event Adverse Outcome Pathway (AOP) for Skin Sensitization
- 30. 30 Strategy for Testing Skin Sensitisation Potential Without Animals Combining Different Methods Addressing the Adverse Outcome Pathway Susanne Kolle and colleagues 2012 BASF
- 31. 31 Test Battery and Weight of Evidence Assessment (Bauch et al. 2012) Sensitivity 93% Specificity 95% Accuracy 94% Test strategy compared to human data Horst Spielmann BB3R Spring School March 2015
- 32. The validated embryonic stem cell test to predict embryotoxicity in vitro hanging drop culture. Embryoid bodies for the differentiation of embryonic stem cells in the embryonic stem cell test are generated by pipetting a single-cell suspension onto the lid of a cell culture dish. The cells aggregate at the bottom of the drop by gravitational force, thereby forming the embryoid body. Nature Protocols Vol. 6, June 2011 Seiler A & Spielmann H The validated embryonic stem cell test to predict embryotoxicity in vitro 32
- 33. Embryonic stem cells develop spontaneously into contracting myocard, this endpoint of prenatal differentiation is used in the mouse EST. Day 0: undifferentiated D3- mouse ES cell line Day 3-5: in vitro differentiation into „embryoid bodies“ Day 5-10: in vitro differentiation into contracting myocard Mouse Embryonic Stem cell Test (mEST)
- 34. Endpoints: assessment from concentration response curves 1. inhibition of differentiation in ES cells ID50 2. cytotoxic effects on ES cells IC50 D3 3. cytotoxic effects on 3T3 cells IC50 3T3 Endpoint 1: inhibition of differentiation ES cells Endpoint 2: cytotoxic effects ES cells Endpoint 3: cytotoxic effects 3T3 cells mouse ES cell line D3 differentiated cells Endpoints of the Embryonic Stem Cell Test (mEST)
- 35. Horst Spielmann, 9. November 2006, Promega BfR-Position zu REACH Page 35 Alternative Testverfahren und intelligente Teststrategien Overall, satisfactory performance of manual EST with > 85% concordance Findings from EST assay used to o rank-order compounds during lead optimization o follow up on in silico flags o frontload in vivo studies Specificity 100.00 % Sensitivity 76.19 % Concordance 87.04 % In Vitro - In Vivo Concordance of Proprietary and Marketed Compounds • 36 Roche proprietary compounds with in house in vivo data (10+/26-) o Diverse set of structures from 24 different projects • 16 Marketed drugs: known teratogens/non teratogens (11+/5-) o HIV drugs, 5HT2 antagonists, kinase inhibitor, anticoagulants, statins... Roche EST assay & Automation Project Manual mEST assay implemented as routine screen since 2004 Slide Claudia McGinnis 35
- 36. Roche Hanging Droplet Culture Plate [HDCP] • In-house developed injection-molded proprietary labware for culturing cells in 3D • Precise engineering allows excellent droplet stability • 24well currently used for automated EST assay. • 96well design finalized & manufactured. Implementation ongoing. 20 ul 70 ul Patent pending Round shape to avoid EBs sticking on well corner Edged shape for good drop stability Small ring shape for good droplet stability Patent pending Slide Claudia McGinnis 36
- 37. Automated EST Platform Set-up at Roche Implemented since Q1, 2012 Compound liquid handling for preparing serial dilutions and compound plates Warming rack for buffer, media, cells RoMa Arm moves plates in/out of incubator 8-channel pipettor uses sterile, disposable tips Centrifuge spins down EB into well for adherence and further differentiation Slide Claudia McGinnis Position for HDCP plate for EB culture/differentiation Staeubli Robotic Arm turns HDCP lid over for liquid handling /media changes Cytomax Incubator for 10 d cytotox/ differentiation incubations 37
- 38. 38 Status of Science 2013 < 3 % 35-50 % < 10 % < 1 % 50-80 % < 3 %