mb research laboratories new photosensitization and alternative phototoxicity methods george l....

MB Research Laboratories

New Photosensitization and Alternative Phototoxicity Methods

New Photosensitization and Alternative Phototoxicity Methods

George L. DeGeorge, Ph.D., DABT



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


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)


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


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


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


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.


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.


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


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.


Photosensitization AssaysPhotosensitization Assays

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


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


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


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]

mb research laboratories new photosensitization and alternative phototoxicity methods george l....

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