mb research laboratories new photosensitization and alternative phototoxicity methods george l....
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MB Research Laboratories
New Photosensitization and Alternative Phototoxicity Methods
New Photosensitization and Alternative Phototoxicity Methods
George L. DeGeorge, Ph.D., DABT
MB Research Laboratories
MB Research Laboratories
Read OD at 540 nm
3T3 NRU Phototoxicity Test3T3 NRU Phototoxicity Test
Seed 96 well plates with 3T3 cells
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
Add Neutral Red media; incubate 3 hrs; Rinse & Fix
Return to 37ºC CO2 incubator for 24 hrs
Plate BPlate A
Determine IC50UVA and IC50Dark
Calculate PIF ratio; if >6 then + phototoxin
MB Research Laboratories
3T3 NRU Phototoxicity Test3T3 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)
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In Vitro/AlternativesIn 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
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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 SimulatorSolar Simulator
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3T3 NRU Phototoxicity Assay 3T3 NRU Phototoxicity Assay
•Evaluates photo-cytotoxicity
•Mouse Fibroblast Cell Lines
•OECD Guideline (TG 432)
•New Required Test; No Animal PT Test
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EpiDerm™: Human Skin EquivalentEpiDerm™: 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.
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The Air/Liquid Interface (ALI) Tissue Culture TechniqueThe Air/Liquid Interface (ALI) Tissue Culture Technique
Tissue Culture Well
Culture Insert
ALI Tissue
Medium
Membrane
Courtesy of MatTek Corp.
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EpiDerm Phototoxicity AssayEpiDerm 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
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In Ovo Phototoxicity Assay (IOPA)In Ovo Phototoxicity Assay (IOPA)In Ovo Phototoxicity Assay (IOPA)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.
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Photosensitization AssaysPhotosensitization Assays
• Photo-LLNA: Uses mice instead of GP• Validation by ECVAM and ICCVAM• EPA and OECD list as “Default preferred method”
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Negative
SI and SIUVA < 3 SI or SIUVA ≥ 3
Photoallergen
SIUVA / SI ≥ 1.25
Apply Test Substance to Mouse Ears (+/– UVA)
Inject Thymidine / BrdU Excise Nodes
Analyze Proliferating LNC (Calculate SIs)
Sensitizer
(+/– UVA)
Basic Photo-LLNABasic Photo-LLNA
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Irritating SensitizerIrritantNegative
SI and SIUVA < 3 SI or SIUVA ≥ 3
negative
negative
Ear Swelling >25%
>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-Ak+
Immunophenotyping %B220+ or B:T Cell Ratio
equivocal
(+/– UVA)
Tiered Strategy for Sensitization Testing Using the Enhanced Photo-LLNA
Tiered Strategy for Sensitization Testing Using the Enhanced Photo-LLNA
SIUVA >25% vs. SI
SIUVA >25% vs. SI
NO
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In Vivo and In Vitro SolutionsIn Vivo and In Vitro Solutions
THANK YOUTHANK YOU
MB Research Laboratories1765 Wentz Road, PO Box 178, Spinnerstown, PA 18968
Tel: 215-536-4110 Fax: 215-536-1816 Email: [email protected]