Modernizing the “six-pack” testing strategy: influx of modern in vitro techniques Gertrude-Emilia Costin, Ph.D., M.B.A. Institute for In Vitro Sciences, Inc. (IIVS) NorCal SOT Fall Symposium 2017 The 3Rs 28 September 2017, San Francisco, CA, USA Perspectives, challenges, common goals and working together Safety/ Testing Labs Labeling/ Industry/ Regulatory Manufacturer Agency Consumer/ End-user Safety Presentation Outline Current regulatory climate – global acceptance of in vitro methods The reductionist concept of in vitro methods Drivers of in vitro methods advancement Beyond the “six-pack” battery of acute toxicity tests Acute oral toxicity Acute dermal toxicity (oral vs dermal route comparison) Acute inhalation toxicity Ocular irritation (the EPA OPP testing strategy) Skin irritation/corrosion Skin sensitization Modernizing the “six-pack” testing strategy: influx of modern in vitro techniques Current regulatory climate – global acceptance of in vitro methods The reductionist concept of in vitro models 1940s 1990s Whole animal Organ - Eyeball Tissue - Cornea Cell culture (Rabbit) (Enucleated chicken or (Resected bovine (Statens Seruminstitut rabbit eye) cornea) Rabbit cornea cells) “Less is more” Retinal Pigment Epithelium Semi-permeable Membrane Vascular network Nutrient channels Fibrinogen- endothelial cell administration channel Body-on-a-chip Organ-on-a-chip Tissue construct Cell culture (Human organotypic (Human retina) (Human EpiCorneal™ (Normal human microtissues) model) corneal epithelial cells) 2010s 2000s G.-E. Costin and H. A. Raabe. In vitro toxicology models. In: The Role of the Study Director in Non-clinical Studies. Pharmaceuticals, Chemicals, Medical Devices, and Pesticides. (Eds. William Brock, Barbara Mounho and Lijie Fu), John Wiley and Sons (2014). G.-E. Costin. Advances in science: next generation of lab tools, models and testing platforms used in predictive toxicology. Molecular Life 2017; 1(1), 22- 28, doi: 10.26600/MolLife.1.1.3.2017. Available at: http://molecular-life.org/wp-content/uploads/2017/07/Advances-science-next-generation-lab-tools-models- testing-platforms-used-predictive-toxicology.pdf. Drivers of in vitro methods advancement Industry Public CROs R In vitro methods Regulatory Trade Agencies Refinement Associations Animal Academia Welfare Groups Ongoing evolution on so many levels • Improve scientific basis for testing using human-derived test models • Reduce the number of animals for testing • Increase predictivity • Reduce time, price • Harmonize requirements and prediction models http://ntp.niehs.nih.gov/pubhealth/evalatm/test-method-evaluations/project-milestones/index.html#atc-inh. http://alttox.org/mapp/table-of-validated-and-accepted-alternative-methods/. Beyond the “six-pack” battery of acute toxicity tests Acute oral rat Skin Acute irritation dermal rabbit rabbit PESTICIDES Ocular Acute irritation inhalation rabbit rat Skin sensitization guinea pig mouse The modern in vitro toxicology perspective EPA Health Effects Test Guidelines OPPTS 870.1000 Acute Toxicity Testing-Background. Acute oral toxicity – CRO’s perspective Test system: normal human keratinocytes (NHEKs) or Balbc 3T3 mouse fibroblasts Assay endpoints: cell viability [by Neutral Red Uptake (NRU) assay] Data calculation: estimated log LD50 mmol/kg = 0.435 x log mean NRU50 mM + 0.625 For initial dose setting OECD Guidance Document 129 Pre-testing: Solubility Treatment assessment Cell seeding Dilution/Dosing termination Solvent addition NHEK 3T3 NRU addition Plate reading Acute dermal toxicity – Regulatory perspective (oral vs dermal route comparison) Efforts to use a single route • 2013, US EPA OPP, National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM): retrospective analysis of oral and dermal acute lethality studies focused on formulated pesticides products considering the EPA pesticide categorization scheme. Acute dermal toxicity – Regulatory perspective (oral vs dermal route comparison) The dataset of rat acute oral and acute dermal Data analysis LD50 studies included: • For 57% of the 592 formulations, the • 592 formulated pesticide products, representing results of both oral and dermal acute 316 active ingredients toxicity studies fall within the same • all four Toxicity Categories Toxicity Category. • 13 different formulated pesticide product types • For 38% of the formulations, the oral study falls within a lower (i.e., more protective) Toxicity Category. • Thus, for 95% of the formulations in the analysis, if the dermal study had not been available and labelling had been based only on the Toxicity Category for the oral acute toxicity study, the PPE requirements on the labelling would have been equally protective or more protective. • For the remaining 5%, factors other than the dermal acute toxicity may influence PPE labeling requirements. The agency has used this analysis to support a policy statement in Section 3.0 to waive all acute lethality dermal studies for formulated pesticide products. Creton et al. Acute toxicity testing of chemicals – Opportunities to avoid redundant testing and use alternative approaches. Crit. Rev. Toxicol. 40: 50-83 (2010). Seidle et al. Examining the regulatory value of multi-route mammalian acute systemic toxicity studies. ALTEX 28, 2/11 (2011). Moore et al. Can acute dermal systemic toxicity tests be replaced with oral tests? Regul. Toxicol. Pharmacol. 66: 30-37 (2013). Acute inhalation toxicity – CRO’s perspective Efforts for in vitro methods development Cell lines/tissues/explants Endpoints Smoke exposure systems • Bronchial epithelial cells • Cytotoxicity • Reconstructed tissues • mRNA (MUC5AC) • Protein expression: IL-6, IL-8, MMP-1 • Gene expression and microarrays • Cellular glutathione levels EpiAirway model (MatTek Corporation) Normal, human-derived tracheal/bronchial epithelial cells Precision Cut Lung Slice (PCLS) MucilAir model (Epithelix) Alveolar spaces; H&E staining, Primary epithelial cells co- 40x magnification* cultured with human airway fibroblasts Thorne D, Adamson J. A review of in vitro cigarette smoke exposure systems. Exp Toxicol Pathol 65, 1183-1193 (2013). http://www.mattek.com http://www.epithelix.com *Image courtesy of Dr. Khalid Amin, University of Minnesota, Department of Laboratory Medicine and Pathology Acute inhalation toxicity – Regulatory perspective Voluntary pilot program underway where registrants may send the in vivo acute lethality study for oral and inhalation formulation/product testing as currently required and simultaneously submit the calculations using the GHS dose additive mixtures equation. https://www.epa.gov/sites/production/files/2017-04/documents/session-3-2st-century-toxicology-update.pdf. http://www.piscltd.org.uk/wp-content/uploads/2016/09/Acute-Inhalation-Workshop_2016_ALowit.pdf. https://www.epa.gov/pesticides/epa-releases-guidance-voluntary-pilot-program-reduce-animal-testing. Ocular irritation - the EPA OPP testing strategy A continuum of sensitivity *Clorox *Colgate Palmolive *Dial *EcoLabs *JohnsonDiversey (currently SealedAir) *P&G *SC Johnson *The Accord Group *Institute for In Vitro Sciences (IIVS) GHS 1 GHS 2 GHS Non-classified EPA I EPA II EPA III EPA IV Multiple in vitro assays Multiple in vitro assays Extreme Severe Moderate Mild Very Mild Household Color Cosmetics Cleaning Products Industrial Chemicals Currently, one in vitro assay is not sufficient for all eye irritation categories – therefore a bottom-up/top-down strategy was proposed Antimicrobial Cleaning Products (AMCP) Labeling Requirements AMCPs quick facts • Contain ~275 different active ingredients. • Are marketed as sprays, liquids, concentrated powders, and gases. • More than 5000 are currently registered with the U.S. Environmental Protection Agency (EPA) and sold in the marketplace. Antimicrobial claim The vast majority of the household and The product is now EPA regulated commercial cleaning products do not (animal testing for safety is required). have to go through a registration process before they are marketed. Both EPA and industry agreed to work Companies decide how to assure safety together to build a predictive and – generally without using animals (in conservative in vitro strategy designed vitro). to replace the requirement for Draize rabbit eye irritation data with non- animal methods. http://www.epa.gov/opp00001/factsheets/antimic.htm. Ocular irritation - the EPA OPP testing strategy BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) ASSAY Test system: Viable corneas maintained in culture Assay endpoints: opacity and permeability Data calculation: In Vitro Score = Opacity + (15 x Fluor OD490) OECD TG 437 US EPA OPP policy (3-2-2015): Use of an alternate testing framework for classification of eye irritation potential of EPA pesticide products - 40CFR Part 158W for AMCPs (anti-microbial cleaning products) Corneal Excision Initial Test Substance Fluorescein Permeability Fixing the Mounting Opacity Exposure Rinsing Addition Endpoint Corneas BCOP Assay Overall Performance PREDICTIVITY LIMITATIONS LABELING APPLICABILITY • Only 2 of 61 materials (8%) • If the anti-microbial cleaning product • The BCOP assay does were under-predicted. is a High Solvent (>5 solvent) differentiate between EPA formulation, it should be tested in the Category I and II materials, • All of the EPA toxicity Category BCOP assay using a 3 minute so it is most useful in this IV materials are over-predicted exposure instead of the normal 10 higher range. as Category III since the BCOP minute exposure. does