Non-Animal Testing Technologies and Strategies: Supporting Global Product Stewardship. Presentation outline

Transcription

1 Non-Animal Testing Technologies and Strategies: Supporting Global Product Stewardship Gertrude-Emilia Costin, Ph.D., M.B.A. 29 September 2018 Presentation outline Background Current regulatory climate global acceptance of in vitro methods Drivers of in vitro methods advancement The reductionist concept of in vitro methods Placing the Safety in the Safety Data Sheet (SDS) Challenges with the non-animal paradigm Major groups of non-animal test methods Non-animal testing technologies and strategies used for: Chemical hazard and risk assessment Product Development, R&D, Innovation Safety assessment target Dermal Ocular Dermal Ocular Test system In vitro reconstructed tissue model Ex vivo tissues and organ system In vitro reconstructed tissue model Assay setup Single exposure Single endpoint Prediction model Multiple exposures Multiple endpoints No prediction model Assays regulatory status Validated for regulatory purposes Not validated for regulatory purposes Outcome Yes/No answer Ranking of products based on toxicity profile/potential General considerations Limitations Method overview and regulatory status Typical and modified protocols Examples (classifications) Prediction models Integrated Testing Strategies Integrated Approaches to Testing and Assessment

2 Perspectives, challenges, common goals and working together Safety/ Testing Labs Industry/ Manufacturer Labeling/ Regulatory Agency Consumer/ End-user Safety Current regulatory climate global acceptance of in vitro methods

3 Retinal Pigment Epithelium Semi-permeable Membrane Vascular network Nutrient channels Fibrinogenendothelial cell administration channel Drivers of in vitro methods advancement Regulatory Agencies Public Academia Industry R In vitro methods Refinement Animal Welfare Groups CROs Trade Associations Ongoing evolution on so many levels Improve scientific basis for testing using humanderived test models Reduce the number of animals for testing Increase predictivity Reduce time, price Harmonize requirements and prediction models The reductionist concept of in vitro models 1940s Whole animal (Rabbit) Organ - Eyeball (Enucleated chicken or rabbit eye) Tissue - Cornea (Resected bovine cornea) 1990s Cell culture (Statens Seruminstitut Rabbit cornea cells) Less is more Body-on-a-chip (Human organotypic microtissues) Organ-on-a-chip (Human retina) Tissue construct (Human EpiCorneal model) Cell culture (Normal human corneal epithelial cells) 2010s 2000s

4 Placing the Safety in the Safety Data Sheet Standard SDS specified by the Occupational Safety and Health administration (OSHA) Product Stewardship 1. Identification 5. Fire-fighting Measures 2. Hazard(s) Identification 3. Physical and Chemical Properties 6. Accidental Release Measures 7. Handling and Storage 4. First-aid Measures 8. Exposure Controls/ Personal Protection Same/similar needs Various ways to accomplish it Across industries Product types Regulatory requirements Hazard/risk Workers or end-user safety 9. Physical and Chemical Properties 10. Stability and Reactivity 13. Disposal Considerations 14. Transport Information 11. Toxicological Information 15. Regulatory Information 12. Ecological Information 16. Other Information Challenges How are classification and labeling predictions communicated to the regulatory community using the non-animal paradigm? 1. What information is acceptable? 2. Can an ingredient or a formulation be classified without testing? 3. What assays or endpoints are accepted? 4. Can they stand-alone? 5. Is there a hierarchy to follow? 6. How are data gaps addressed? Must meet global expectations of OECD members Mutual Acceptance of Data

5 Further challenges How can the best method be selected? How are data interpreted? 1. By target tissue (eye, skin, systemic toxicity, etc.)? 2. By endpoint? (category specific?) 3. By test system type? (in chemico, cellular 2D, 3D, ex vivo?) 4. By relevance to the test material? (chemicals, formulations, solubility issues) 5. By regulatory acceptance only? (can non-regulatory assays be used in WofE Weight of Evidence?) Plenty of assays to choose from Four major groups of non-animal test methods used in research and regulatory safety testing of chemicals and finished products 1. In chemico test systems Skin Corrosion: Membrane Barrier Test Method Corrositex (OECD TG 435) Eye Irritation: Irritection Test (draft OECD TG) Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) (OECD TG 442C) 2. In vitro monolayer cell culture systems Skin Phototoxicity: Phototoxicity Test (OECD TG 432) Ocular Irritation: Cytosensor Microphysiometer (US EPA AMCP and draft TG) Short-Term Exposure (STE) Assay (OECD TG 491) Skin Sensitization: KeratinoSens (OECD TG 442D) hclat (OECD TG 442E) 3. In vitro reconstructed tissue models systems Skin Corrosion: Reconstructed human EpiDermis (RhE) Corrosion Assay (OECD TG 431) Skin Irritation: RhE Skin Irritation Test (SIT) (OECD TG 439) Eye Irritation: Eye Irritation Test (EIT) (OECD TG 492) 4. Ex vivo tissues and organ systems Ocular irritation: Bovine Corneal Opacity and Permeability Assay (OECD TG 437; US EPA AMCP) Isolated Chicken Eye Test (OECD TG 438) Skin Absorption: In vitro Skin Absorption (OECD TG 428) Skin Corrosion: Rat Skin Transcutaneous Electrical Resistance Test (OECD TG 430)

6 Chemical hazard and risk assessment Safety assessment target Dermal Ocular Test system In vitro reconstructed tissue model Ex vivo tissues and organ system Assay setup Assays regulatory status Single exposure Single endpoint Prediction model Validated for regulatory purposes Acute oral rat Outcome Yes/No answer Skin irritation rabbit Acute dermal rabbit PESTICIDES Ocular irritation rabbit Skin sensitization guinea pig mouse Acute inhalation rat The impact of test substances on human skin Skin corrosive/irritant Vast physical barrier against mechanical, chemical and microbial factors Immune network Unique defense system against UV irradiation Stratum corneum Stratum granulosum Stratum spinosum Basal layer of dividing keratinocytes CORROSION Native human skin IRRITATION SKIN Irreversible damage of the skin following Reversible damage of the skin following exposure to a test substance exposure to a test substance Characterized macroscopically by erythema Visible necrosis through the epidermis and (redness) and oedema into dermis - macroscopically typified by Damage to keratinocytes and dermal cells ulcers, bleeding, etc. leads to inflammation Registration and labeling of chemicals Transport of chemicals Occupational safety Safety of cosmetics, toiletries and household products

7 In vitro Reconstructed human Epidermis (RhE) models validated for regulatory purposes EpiDerm (EPI-200) SkinEthic RHE Native human skin epics EpiSkin (SM) LabCyte EPI-MODEL 1. General model criteria Human keratinocytes are used for construction of the models Multiple layers of viable epithelial cells Functional stratum corneum and barrier 2. Functional model criteria Tissues must be viable (QC from manufacturer, internal controls) Stratum corneum must form sufficient barrier RhE models should exhibit long term reproducibility In vitro reconstructed tissue models systems General considerations Higher order of complexity - Reconstructed tissues better model tissues of interest (relative to monolayer) Exposure to substances as in vivo Relevant mechanisms of action Endpoints may be machine scored Standardized manufacturing expected to ensure reproducibility Limitations Tissue models tend to be costly Reliance on a small number of manufacturers Tissues differ slightly among manufacturers Still relatively simple models, and do not have support of whole body accessory functions How might this impact the toxicity predictions? Care needs to be exercised not to over-interpret (just as in the case of animal models!)

8 RhE test method - skin corrosion assay (OECD TG 431) Brief overview and current regulatory status Test system: RhE models [EpiDerm TM (EPI-200); EpiSkin (SM); SkinEthic RHE and epics ] Assay endpoint: Tissue viability (%) assessed by reduction of the vital dye MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) by viable cells Assay controls: Negative (sterile, deionized water or NaCl solution 9g/L); Positive (8N KOH or glacial acetic acid only for 4 hr exposure).. Applicability: The results can be used for regulatory purposes for distinguishing corrosive from non-corrosive test substances. The method also allows for sub-categorization, i.e., 1A vs. 1B-and-1C vs. non-corrosive test substances. Limitations: The method does not allow discriminating between skin corrosive subcategories 1B and 1C according to the UN GHS due to a limited set of well-known in vivo corrosive Category 1C chemicals... Regulatory status: OECD Test Guideline 431 (TG 431, updated 2016) Tissue Receipt RhE - corrosion: typical protocol Tissue Treatment Tissue Rinsing MTT Reduction Upon receipt, tissues are incubated for at least 1 hr in standard culture conditions ( C in a humidified atmosphere of 5+1% CO 2 in air). Media is refreshed after the initial 1 hr incubation. Duplicate tissues are treated topically with control and test substances for 3 min / 1 hr (4 hr). After exposure, tissues are rinsed to remove the control and test substances. Individual tissues are placed into wells containing unreduced MTT solution and incubated at standard culture conditions for 3 hr. Spectrophotometric Quantification Optical density (OD) at 550 nm (OD 550 ) is determined using a 96-well plate reader. OD values are used to calculate relative viability values presented relative to negative control tissue values. Isopropanol Extraction The tissues are placed in isopropanol at room temperature for 2 hr to extract the reduced MTT. Extracted MTT is thoroughly mixed and transferred to a 96-well plate.

9 Prediction models EpiSkin (SM) Viability measured after exposure time points (3, 60 and 240 minutes) < 35% after 3-minutes exposure 35% after 3-minutes exposure AND < 35% after 60-minutes exposure OR 35% after 60-minutes exposure AND < 35% after 240-minutes exposure 35% after 240-minutes exposure Prediction to be considered UN GHS Category Corrosive: Optional Sub-category 1A Corrosive: A combination of optional Subcategories 1B and 1C Non-corrosive EpiDerm (EPI-200) SkinEthic RHE epics Viability measured after exposure time points (3- and 60-minutes) STEP 1 < 50% after 3-minutes exposure Corrosive 50% after 3-minutes exposure AND < 15% after 60-minutes exposure 50% after 3-minutes exposure AND 15% after 60-minutes exposure STEP 2 Corrosive Prediction to be considered UN GHS Category Non-corrosive <25%; 18%; 15% after 3-minutes exposure Optional Sub-category 1A 25%; 18%; 15% after 3-minutes exposure A combination of optional Sub-categories 1B-and-1C Classification examples: extreme ph mixtures (alkalis) Extreme ph can be a useful predictor of irritation but may lead to overclassifications in weakly buffered systems. 8/12 products tested using the RhE testing system predicted the same skin classification when compared to the in vivo data. The remaining 4 formulas overpredicted the skin classification. There were no under-classifications. Formulas with high levels of solvent (>15%) may result in a more conservative classification when using this in vitro assay.

10 Integrated Approaches to Testing and Assessment (IATA) Dermal corrosion and irritation (self-correcting) Top-Down Strategy Bottom-Up Strategy Test substance expected to be: Corrosive Non-Corrosive Assay to be used: In Vitro Corrosion Assay(s) In Vitro Skin Irritation Assay(s) Test result: Is the test substance predicted as corrosive? Is the test substance predicted as skin irritant? Labeling: Y N Y N Ex vivo tissues and organ systems General Considerations High order of complexity Excised tissues directly correlate to tissues of interest Exposure to substances as in vivo Relevant mechanisms of action Limitations Human tissues of exceptional quality are often difficult to obtain Tissues may differ from trial to trial If the tissue is non-human, is the relevance questionable? Excised tissues no longer have support of whole body accessory functions (inflammatory responses, metabolism, etc.) How might this impact the toxicity predictions? Care needs to be exercised not to over-interpret (just as in the case of animal models!)

11 Bovine Corneal Opacity and Permeability (BCOP) assay (OECD TG 437) Test system: Brief overview and current regulatory status Viable corneas maintained in culture responsive to a large variety of chemical classes and physical forms Assay endpoint: Opacity and permeability (two relevant endpoints measured in a single, one day experiment) Assay controls: Negative (sterile, deionized water); Positive (imidazole solids; ethanol - liquids).. Applicability: The results can be used for regulatory purposes for distinguishing eye corrosive/severe irritants (GHS Category I) from non-irritating test substances (No Category). Adopted as part of a self-correcting strategy to address the eye irritation endpoint as part of the six pack US EPA labeling system (antimicrobial products originally, now extended to conventional pesticides on a case by case basis). Limitations: Cannot assign GHS Category 2 classification Availability/source of eyeballs Cannot address reversibility.. Regulatory status: OECD Test Guideline 437 (TG 437, updated 2017); US EPA OPP policy ( ) Range of protocols Standard Protocols: Liquid test materials: undiluted, 10-minute exposure, 120-minute post-exposure Solid test materials: 20% suspension, 240-minute exposure Specialized Protocols: Surfactant formulations: 10% solution, 60 minute exposure, 60-minute post-exposure (focus on permeability score) Multiple exposures: undiluted, 3 and 10-minute exposure,120-minute post-exposure (for organic solvent-based materials) Extended post-exposure: 10-minute exposure, 4 and 20-hour post-exposures (reactive chemicals such as H 2 O 2 ) Histology may be added to all protocols Decision tree for BCOP testing approach to surfactants. For solid formulations, the protocol should be determined based on the formulation components.

12 If Positive Result: Corneal Excision Mounting Initial Opacity BCOP: typical protocol Test Substance Rinsing/ Exposure Final Opacity Fluorescein Addition Permeability Endpoint Fixing the Corneas In Vitro Score 25 Data calculation: In Vitro Score = Opacity + (15 x Fluor OD 490 ) Predicted Irritation Potential Mild Moderate > 55.1 Severe This model should be used with standard exposures & in conjunction with responses of benchmark materials; may not be appropriate for all classes of materials. Prediction Models Prediction Model Developed by Merck* Prediction Model - OECD TG 437 In Vitro Score UN GHS 3 No Category >3 55 No prediction can be made > 55 Category 1 Adjusted Prediction Model and Tiered Testing Approach: Pharmaceutical Compounds Step 1 ph Determination In Vitro Score Irritation Potential > 55 Severe Irritant ph 2 or 11 2< ph <11 > 25 to 55 Moderate Irritant > 3 to 25 Mild Irritant Step 2 Corrositex Assay If Negative Result: 3 Non-Irritant Packing Group Assignment Step 3a BCOP Assay Evaluate Ocular Corrosion/Irritation Potential Step 3b SIT Assay Evaluate Dermal Irritation Potential Proposed tiered testing strategy for the assessment of ocular and dermal irritation potential of pharmaceutical compounds for the purpose of BMS worker safety Assign Hazard Category

13 Adjusted prediction model and tiered testing approach: Pesticide products registered with US EPA In Vitro Score < 25 US EPA Predicted Category Category III/IV default to Category III and use the self-correcting strategy to discriminate between III and IV > 25 <75 Category II > 75 Category I PREDICTIVITY Only 2 of 61 materials (8%) were under-predicted. All of the EPA toxicity Category IV materials are over-predicted as Category III since the BCOP does not seem to be able to differentiate between materials at this lower end of the toxicity scale. BCOP Assay Overall Performance LIMITATIONS If the anti-microbial cleaning product is a High Solvent (>5 solvent) formulation, it should be tested in the BCOP assay using a 3 minute exposure instead of the normal 10 minute exposure. Testing of ketones and alcohols in the BCOP has been shown to result in high false positive rates for the assay, but not all ketones or alcohols are over-predicted. LABELING APPLICABILITY The BCOP assay does differentiate between EPA Category I and II materials, so it is most useful in this higher range. Ocular irritation - Outline of the in vitro testing strategy Evaluate components BCOP = Bovine Corneal Opacity and Permeability CM = Cytosensor Microphysiometer EO = EpiOcular Oxidizing chemistry? Yes No Expected severe or moderate? Yes No Water soluble? Yes No BCOP CM EO <25 Default Category III; To distinguish Category IV from III, conduct CM or EO In vitro score > 25 <75 >75 Category I Category II In vitro score <2 mg/ml >80 mg/ml Category IV >2 but < 80 mg/ml >4 but < 70 min Category III Default Category I; To distinguish Category II from I, conduct BCOP In vitro score >70 min < 4 min

14 Navigating the dermal and ocular corrosion/irritation continuum Corrosive Severe Moderate Mild to Non-irritant Household Industrial Chemicals Cleaning Products Color Cosmetics In vitro skin corrosion assay In vitro Skin Irritation Test In vitro Skin Irritation Test BCOP ICE IRE HET-CAM CM (aqueous soluble) STE EIT Ocular Irritection BCOP ICE CM (surfactants) STE EIT Ocular Irritection Product Development, R&D, Innovation Safety assessment target Dermal Ocular Test system Assay setup Assays regulatory status Outcome In vitro reconstructed tissue model Multiple exposures Multiple endpoints No prediction model Not validated for regulatory purposes Ranking of products based on toxicity profile/potential Concept Product: concept Proof of concept Efficacy testing Safety Testing Raw ingredient/ Final formulation Efficacy Testing Safety testing Product: production and launching Finished product Product: postmarket follow-up Anti-inflammatory action Skin tone modulation Pre-clinical testing (in vitro) Skin irritation Skin sensitization Co-cultured cellbased models Cell-based models Ex-vivo models Reconstructed tissue models Clinical testing

15 Tissue Viability (% of Control) 2 hours 3 hours Time-to-Toxicity Assay: Typical Protocol Single Short Exposure and Cytokine Analysis Assay: Typical Protocol ~16 hours Overnight pre-incubation in Hydrocortisone Free Maintenance Media (HCF-MM) 1 hour Incubation in fresh Assay Medium (AM) 1 hour Incubation in fresh HCF-MM 1-24 hours Dosing of test materials (100 ml) and incubation Rinsing of tissues 24 hours Adding fresh media and post-exposure incubation for cytokine expression Collection of media for cytokine analysis MTT reduction Isopropanol extraction Spectrophotometric quantification IL-1a Immunoassay Data analysis and interpretation (Time-to-Toxicity), examples Non-irritant The correlation of the results of this in vitro assay with expected in vivo has not been firmly established Severe irritant Moderate irritant However, MatTek suggests the following as a guideline for assigning verbal descriptors for expected in vivo irritation based on the ET 50 scores using the EpiDerm TM (EPI-200) model Exposure Time (Hours) VOC (volatile organic compounds)-containing products EpiDerm Assay Consumer ET50 (hours) Clinical Results Test Materials Experience 100 µl Dose 30 µl Dose Product > hr Derm-NP 0 Product > 24 RIPT - 50 person NP 0 Product > 24 RIPT person NP 0 Product > 24 RIPT person NP +" Product > 24 RIPT person NP 0 Product > 24 RIPT person NP 0 Product > 24 RIPT person NP 0 Product > 24 RIPT person NP 0 Chemical Concentrated nitric acid 1% Sodium Dodecyl Sulfate 1% Triton X-100 Expected in vivo irritation Strong/severe, possibly corrosive ET 50 (hrs) <0.5 Moderate Moderate to mild 4-12 Baby shampoo Very mild % Tween 20 Non-irritating 24

16 Data analysis and interpretation (Single Short Exposure and Cytokine Analysis assay), examples Surfactants-containing products Distribution of the tissue viability response (%) for the 224 surfactant-based test articles in bins of tissue viability. Distribution of the IL-1α response for the 224 surfactant-based test articles in bins of IL-1α. Correlation between in vitro IL-1a concentration and the change in TEWL (trans-epidermal water loss) In vitro reconstructed human tissue models used for ocular safety assessment The surface is covered with non-keratinizing stratified epithelium. Keratocytes are the mesenchymal (fibroblast-like) cells in the stroma. Dense matrix membrane of Descemet s membrane locates beneath the epithelial basement membrane. HCE (EpiSkin) Native human cornea EpiOcular (MatTek Corporation) Immortalized human corneal epithelium cells EpiCorneal (MatTek Corporation) Upper squamous cells Central squamous cells Lower rounded cells Normal human corneal epithelial cell Normal human-derived epidermal keratinocytes

17 Non-surfactant haircare (100%) Creams/ Lotions (100%) Eye area cosmetics (100%) Surfactant formulas (10%) Data analysis and interpretation (Time-to-Toxicity), examples Benchmarks database Draize Test EpiOcular Test Classification Score range Non-irritating 0.0 Minimal ET 50 range (minutes) >60 Mild Example PEG 75 3% SDS Moderate % Triton-X-100 Severe Extreme <3 5% BAK Correlation: in vivo in vitro (Draize-EpiOcular) Comparative analysis: the results provide a reference database of in vitro ocular irritation scores for a crosssection of currently marketed cosmetic and personal care products. Formulation Category (Concentration) Adult shampoo 27.0 Children shampoo 58.2 Baby shampoo 74.9 Baby wash Liquid eyeliner Eye cream Mascara Pencil eyeliner 1440 Adult lotion/cream Sunscreen Baby lotion 1200 Children styling gel Adult conditioner Children conditioner Children hair detangler Mean ET 50 value (minutes) Perspectives, challenges, common goals and working together Industry/ Manufacturer Safety/ Testing Labs Labeling/ Regulatory Agency Trade Associations Animal Welfare Groups High-throughput Sensitive Academia Specific Reproducible Consumer/ End-user Safety Reliable Relevant Transferable Easy to perform Affordable Validated

18 References Slide #5: Slide #6: 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. Molecular Life, 1(1), Available at: (2017). Slide #12: Credit to: Office of Environment, Health & Safety at the University of California Berkeley, ehs.berkeley.edu. Slide #17: Desprez B. et al., Toxicol. In Vitro, 29, (2015). Slide #17: Burrows-Sheppard A.M. et al., The Toxicologist, 114 (1), 106 (2010). Slide #19: Scott L. et al., Toxicol. In Vitro, 24, 1-9 (2010). Calufetti S. et al., The Toxicologist, 138, 268 (2014). Wilt N. et al., The Toxicologist, 144, 89 (2015). Slide #22: Bader J.E. et al., The Toxicologist, 132, 210 (2013). Slide #23: *Sina, J.F. et al., Fundament. Appl. Toxicol. 26, (1995). OECD. OECD guideline for the testing of chemicals. Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (OECD 437). Organisation for Economic Co-operation and Development (OECD) Slide #24: Graham, J.C. et al., Cutan. Ocul. Toxicol. doi: / [Epub ahead of print] (PMID ) (2018). Slides #25: US EPA OPP policy ( ): 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). Also for slide #26. References - continued Slide #27: Adapted in part from: Joao Barroso, Kimberly Norman, ChemWatch Webinar Series, Serious Eye Damage and Eye Irritation: 04.pdf. Slide #28: Modified in part from: Gertrude-Emilia Costin and Kimberly G. Norman. Application of In Vitro Methods in Preclinical Safety Assessment of Skin Care Products, Springer-Verlag Berlin Heidelberg 2015 M.A. Farage et al. (eds.), Textbook of Aging Skin, DOI / _130-1; and Gertrude-Emilia Costin, Decoding and modulating the color of human skin. Cosmetic Chemist Available at: man_skin.html. Slide #29: Costin, G.-E. et al., Rom. J. Biochem. 46 (2), (2009); also for slide #30. Bernhofer, L. P. et al., Toxicol In Vitro. 13 (2), (1999). Fentem, J.H. et al., Toxicol. In Vitro, 15, (2001). Slide #30: MatTek Corporation MTT Effective Time-50 (ET-50) Protocol For Use with Epiderm Skin Model (EPI-200) (2005). Vavilikolanu P. et al., ALTEX 26, 194 (2009). Slide #31: Walters R.M. et al. Altern Lab Anim. 44, (2016). Slide #32: Saika, S. Lab Invest. 86 (2), (2006). Slide #33: Modified in part from: Stern M. et al. Toxicol In Vitro 12, (1998). Kay, J.H. and Calandra, J.C., J. Soc. Cosmetic Chem., 13, (1962). MatTek Corporation MTT Effective Time-50 (ET-50) Protocol For Use with EpiOcular Model (OCL-200) (2005).

19 Other resources Fall-Symposium_GECostin.pdf ESTIV Applied In Vitro Toxicology Course, Bucharest, Romania, April 14-19, 2018, IIVS Team Acknowledgments US EPA OPP Organizers S A E F Industry Consortium The Accord Group Thank you! Gertrude-Emilia Costin, Ph.D., M.B.A. Manager of Scientific Services, Study Director ecostin@iivs.org