1. MB Research Laboratories New Photosensitization and Alternative Phototoxicity Methods George L. DeGeorge, Ph.D., DABT MB Research Laboratories

2. Read OD at 540 nm 3T3 NRU Phototoxicity Test Seed 96 well plates with 3T3 cells Dose 2 plates with 8 conc. of TA Incubate 37ºC, 5% CO 2, 1 hr Plate A: Expose to UVA 5 J/cm 2 /50 mins Plate B: Keep in the dark, 50 mins Add Neutral Red media; incubate 3 hrs; Rinse & Fix Return to 37ºC CO 2 incubator for 24 hrs Plate BPlate A Determine IC 50UVA and IC 50Dark Calculate PIF ratio; if >6 then + phototoxin

3. MB Research Laboratories 3T3 NRU Phototoxicity Test IC 50 : The concentration of test material which reduces cell viability to 50% compared to untreated controls –Determined for +UV and –UV cultures Calculation of Photo-Irritation Factor (PIF) –PIF = IC 50 (-UV)/IC 50 (+UV)

4. MB Research Laboratories In Vitro/Alternatives 3T3 NRU Phototoxicity Assay– (OECD) Other applicable cell types –Primary Human Keratinocytes –Melanocytes –Langerhans Cells In Ovo Phototoxicity Assays 3-D human tissue constructs

5. MB Research Laboratories With H1 Filter: UVA+VIS+IR With H2 Filter: UVB + UVA + VIS + IR With NO Filter: UVC+UVB+UVA+VIS+IR Spectral Distributions Possible Weight: 11 kg Dimensions: 34 x 30 x 40cm Bulb: 400W Metal Halide “S” Bulb SOL 500 Solar Simulator

6. MB Research Laboratories 3T3 NRU Phototoxicity Assay Evaluates photo-cytotoxicity Mouse Fibroblast Cell Lines OECD Guideline (TG 432) New Required Test; No Animal PT Test

7. MB Research Laboratories EpiDerm™: Human Skin Equivalent  Normal Human Keratinocytes  Stratified, Differentiated Morphology with Barrier Function  Cell culture inserts – allow topical application  Normal Ceramide and Lipid Profile  Ultrastructure of Intercellular Lamellar Lipid Sheets Transmission Electron Micrograph of Intercellular Lamellar Lipid Sheets: Broad- Narrow-Broad-Spacing (150K X) Histological Cross Section of EpiDerm 200. Mag = 440X Courtesy of MatTek Corp.

8. MB Research Laboratories The Air/Liquid Interface (ALI) Tissue Culture Technique Tissue Culture Well Culture Insert ALI Tissue Medium Membrane Courtesy of MatTek Corp.

9. MB Research Laboratories EpiDerm Phototoxicity Assay Allows Topical Dosing TA need not be Aqueous- Soluble Human Keratinocyte-based Tissues If: Viability (no UVR) – Viability (+UVR) > 30% Then: Phototoxic If: Viability difference is < 30% Then: Non-phototoxic

10. MB Research Laboratories In Ovo Phototoxicity Assay (IOPA) Based on modified CAM-VA Eggs dosed i.a., i.v., air sac, or topically Functional hepatic and CV systems Pre-phototoxins such as 5-ALA (PPIX) and Nabumetone (via P450) can be characterized.

11. MB Research Laboratories Photosensitization Assays Photo-LLNA: Uses mice instead of GP Validation by ECVAM and ICCVAM EPA and OECD list as “Default preferred method”

12. MB Research Laboratories Negative SI and SI UVA < 3 SI or SI UVA ≥ 3 Photoallergen SI UVA / SI ≥ 1.25 Apply Test Substance to Mouse Ears (+/– UVA) Inject Thymidine / BrdU Excise Nodes Analyze Proliferating LNC (Calculate SIs) Sensitizer (+/– UVA) Basic Photo-LLNA

13. MB Research Laboratories Irritating Sensitizer Irritant Negative SI and SI UVA < 3 SI or SI UVA ≥ 3 negative Ear Swelling >25%  >25%  Photoallergen Apply Test Substance to Mouse Ears (+/– UVA) Inject Thymidine / BrdU Excise Nodes Analyze Proliferating LNC (Calculate SIs) Irritating ? (Ear Swelling) Sensitizer Activation Markers: % CD69+, %I-A k + Immunophenotyping % B220+ or B:T Cell Ratio equivocal (+/– UVA) Tiered Strategy for Sensitization Testing Using the Enhanced Photo-LLNA SI UVA >25%  vs. SI NO

14. MB Research Laboratories In Vivo and In Vitro Solutions THANK YOU MB Research Laboratories 1765 Wentz Road, PO Box 178, Spinnerstown, PA 18968 Tel: 215-536-4110Fax: 215-536-1816Email: mbinfo@mbresearch.com