OECD Webinar | OECD Alternatives to in vivo eye irritation testing - David Allen from the Integrated Laboratory Systems (ILS)
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On 27 September 2019, Anne Gourmelon of the Environment Directorate presented an overview of the various alternative test methods developed as OECD Test Guidelines and relevant guidance material to address eye irritation and serious eye damage for hazard classification of chemicals.
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OECD Webinar | OECD Alternatives to in vivo eye irritation testing - David Allen from the Integrated Laboratory Systems (ILS)
- 1. Alternatives for Eye Irritation Testing: Usefulness and Limitations David Allen, PhD ILS, Contractor supporting NICEATM OECD Webinar on Alternatives for Eye Irritation Testing September 27, 2019 Disclaimer: ILS staff provide technical support for NICEATM, but do not represent NIEHS, NTP, or the official positions of any federal agency
- 2. 1. in vitro monolayer cell culture systems – TG 460, 491 2. ex vivo tissues and organ systems – TG 437, 438 3. in vitro reconstructed tissue models – TG 492, 494 4. non-cellular, in chemico test systems – Ocular Irritection™ (draft TG in review) 2 Four major groups of non-animal test methods
- 3. Bovine Cornea and Isolated Chicken Eye 2D Monolayer Cornea epithelium, stroma and endothelium Squamous Epithelium Cells covering cornea 3-D reconstructed human corneal tissue Epithelium Squamous, wing, and basal epithelium 3 Relationship of In Vitro Models to In Vivo Tissues
- 4. http://iivs.org/testing-services/assays/ocular/bcop/ Organotypic Models: BCOP (TG 437) • Ex vivo corneal models assess corneal injury (in vitro irritation score) based on: • Opacity (measure the amount of light transmitted through the cornea); may be due to both epithelial cell changes and/or stromal swelling • Permeability (measure the amount of fluorescein dye that penetrates through the cornea); due to epithelial disruption • Taken from food production animal • Can be used in a top-down or bottom-up approach Limitations • Not recommended for Category 2, 2A, or 2B • Does not consider conjunctival and iridal injuries • Does not evaluate reversibility of corneal injuries • NOTE: histopathological observations can help in identifying test chemicals causing irreversible effects
- 5. Organotypic Models: ICE (TG 438) • Ex vivo corneal models assess corneal injury (in vitro irritation score) based on: • disruption of the corneal epithelium • swelling of the cornea • corneal opacity • Taken from food production animal • Can be used in a top-down or bottom-up approach Limitations • Not recommended for Category 2, 2A, or 2B • Does not consider conjunctival and iridal injuries • Does not evaluate reversibility of corneal injuries • NOTE: histopathology improves performance • NOTE: histopathological observations can help in identifying test chemicals causing irreversible effects Images courtesy of Menk Prinsen
- 6. In Vitro Monolayer Cell Systems • Generally easy to conduct – primary cells and cell lines • Generally quite rapid to execute • Cost effective with batches of test materials – Can use robotics and also scale to medium/high throughput • Machine scored endpoints • Evaluate individual chemicals (ingredients) rather than formulations 6
- 7. • Assess cell damage – Top-Down approach • Madin Darby Canine Kidney (MDCK) cells cultured on permeable inserts • Measures permeability to a marker dye (disruption of tight junctions) • Assumes that chemicals causing eye damage/irritation will disrupt epithelial barrier function in the corneal epithelium and/or conjunctiva • Limitations • Not recommended for Category 2, 2A, or 2B • Only water soluble chemicals • strong acids and bases, cell fixatives and highly volatile chemicals are outside of the applicability domain • colored and viscous chemicals may be difficult to remove • Tight junctions are increasingly compromised with increasing cell passage number Monolayer Culture Systems: Fluorescein Leakage (TG 460)
- 8. • Assess cell damage – top down or bottom up • Rabbit cornea cells cultured on microplates • Measures cytotoxicity (% viability relative to control) • Assumes that chemicals causing eye damage/irritation will induce cytotoxicity in corneal epithelial cells • Limitations • Not recommended for Category 2, 2A, or 2B • not suitable for test chemicals that are insoluble or cannot be uniformly suspended for at least 5 minutes in physiological saline, 5% DMSO in saline, or mineral oil • Solids (other than surfactants) or highly volatile substances with a vapor pressure over 6 kPa are also outside of the applicability domain Monolayer Culture Systems: Short time exposure (STE) test (TG 491) ICE (OECD TG 438)
- 9. Limitations of Dilution–based Assays • Aqueous insoluble materials • Dilution or buffering effects in medium which mask toxicity of the neat material • Pharmacokinetics poorly modeled • No Tissue Barrier Function modeled 9
- 10. 3D Reconstructed HCE Models • Reconstructed corneal tissues better model corneas in vivo (relative to monolayer cell systems) • Can mimic in vivo exposure to substances • Standardized manufacturing ensures standard platforms • Endpoints may be machine scored 10
- 11. • Primary human epidermal keratinocytes or primary human corneal epithelial cells • EpiOcular, SkinEthic HCE, LabCyte CORNEA-MODEL24, MCTT HCETM • Assumes that chemicals causing eye damage/irritation will induce cytotoxicity • Bottom up approach • Limitations: • Don’t discriminate between Category 1, 2, 2A, or 2B • Test chemicals and chemical mixtures that interfere with the cell viability measurements (e.g., direct MTT reducers, color interference) • Gases and aerosols outside of the applicability domain 3D Tissue RhCE Models (TG 492) (https://www.mattek.com/products/epiocular/)
- 12. • Human immortalized corneal epithelial cells fabricated in a collagen vitrigel membrane chamber • Assumes that chemicals causing eye damage/irritation will damage the barrier function of the corneal epithelium (TEER) • Bottom up approach Limitations: • Don’t discriminate between Category 1, 2, 2A, or 2B • Test chemicals with pH<5.0 or solids Vitrigel EIT (TG 494) www.naro.affrc.go.jp
- 13. 13 Non-cellular or In Chemico Methods (Draft TG) • Do not require cell culture facility or cell culture expertise • May be relatively inexpensive to conduct • Some allow exposures as in vivo • Standardized manufacturing or processes ensure reproducible testing platforms • For the VRM: limited to test chemicals with 4<pH<9
- 14. The gap in testing approaches GHS 1 GHS 2A GHS2B Non-Classified BCOP ICE CM (surfactants) STE EIT Ocular Irritection® BCOP ICE FL HET-CAM CM (aqueous soluble) STE EIT Ocular Irritection® How can we address this middle ground? Testing strategy? GHS 2 (2A, 2B): Irritant, Reversible GHS 1: Severe/Corrosive Irritant Chemwatch: December 2014 Webinar14
- 16. • NICEATM analyzed paired in vivo and in vitro data for approximately 200 agrochemical formulations provided by 5 companies • Conclusions: – Insufficient data from multiple assays to establish a defined approach – Prospective testing needed to fill data gaps Background
- 17. • Focus on the most common formulation types – suspension concentrates (SC) – emulsifiable concentrations (EC) – soluble liquids (SL) • Balanced design with respect to hazard classification • Careful consideration of in vivo data • Coded formulations distributed to testing labs by NTP Prospective Testing
- 18. Eye Irritation and Corrosion Testing Labs BCOP Image from Menk Prinsen ICE EpiOcular PorCORA NRR PorCORAEpiOcular OECD TG 492, Time-to-toxicity method; ET50- neat and dilution protocols TG 437 with and w/o histopathology; extended incubation TG 438 with and w/o histopathology;
- 19. Coded formulations and existing data donated by companies Eye Irritation and Corrosion Testing • 2 Phases of testing completed with 16 formulations • 7 EPA Cat. 1 • 1 EPA Cat. II • 1 EPA Cat. III • 7 EPA Cat. IV • Co-organized by NICEATM and the PETA International Science Consortium, with stakeholders from ICCVAM, ODIWG, EURL ECVAM, PMRA, and industry
- 20. • No single test method could be used to assign a correct classification for all 16 pesticide formulations relative to their in vivo classifications. • Results suggest that combining results of multiple tests in an integrated approach may be useful in classification of these formulations (e.g., EpiOcular method correctly classified all the NC formulations and the BCOP with histology correctly classified 6/7 Category 1 formulations). • Ongoing discussions to determine Phase 3 testing plans • Final study outcomes/analyses will be used to suggest endpoints that can form the basis of a defined approach for pesticide formulations testing Prospective Testing - Summary
- 21. Thank You! Disclaimer: ILS staff provide technical support for NICEATM, but do not represent NIEHS, NTP, or the official positions of any federal agency • NTP/NICEATM – Nicole Kleinstreuer – Warren Casey • PETA ISC – Amy Clippinger • ILS/NICEATM – Neepa Choksi – Amber Daniel • IIVS – Slides used in this presentation
- 22. Questions?