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New Photosensitization and Alternative Phototoxicity Methods

New Photosensitization and Alternative Phototoxicity Methods. George L. DeGeorge, Ph.D., DABT MB Research Laboratories. 3T3 NRU Phototoxicity Test. Seed 96 well plates with 3T3 cells. Plate A. Plate B. Dose 2 plates with 8 conc. of TA Incubate 37 º C, 5% CO 2 , 1 hr.

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New Photosensitization and Alternative Phototoxicity Methods

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

  2. 3T3 NRU Phototoxicity Test Seed 96 well plates with 3T3 cells Plate A Plate B Dose 2 plates with 8 conc. of TAIncubate 37ºC, 5% CO2, 1 hr Plate A: Expose to UVA 5 J/cm2/50 mins Plate B: Keep in the dark, 50 mins Return to 37ºC CO2 incubator for 24 hrs Add Neutral Red media; incubate 3 hrs; Rinse & Fix Read OD at 540 nm Determine IC50UVA and IC50Dark Calculate PIF ratio; if >6 then + phototoxin

  3. 3T3 NRU Phototoxicity Test • IC50: 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 = IC50 (-UV)/IC50(+UV)

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

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

  7. 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 Histological Cross Section of EpiDerm 200. Mag = 440X Transmission Electron Micrograph of Intercellular Lamellar Lipid Sheets: Broad-Narrow-Broad-Spacing (150K X) Courtesy of MatTek Corp.

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

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

  10. 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. In Ovo Phototoxicity Assay (IOPA)

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

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

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

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

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