568. EVALUATION OF PLASMACYTOID DENDRITIC CELL-BASED ASSAY TO DETERMINE CHEMICAL ALLERGENICITY

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Ayehunie, S., Hayden, P., Kaluzhny, Y., Child, M., Snell, M., and Klausner, M. MatTek Corporation, Ashland, MA. Presented at 49th Annual SOT Meeting, Salt Lake City, Utah (2010) Summary:

Human dendritic cells have been used to evaluate the allergenicity potential of chemicals and develop alternatives to existing animal models utilized throughout industry to monitor products for contact sensitization. Development of such non-animal alternative assay systems for hazard assessment directly addresses REACH (Registration, Evaluation, and Authorization of Chemicals).

In this study, we investigated whether CD86 expression in plasmacytoid dendritic cells (pDC) can be used to identify contact allergens. Human DC were generated from CD34+ progenitor cells and the pDC fraction (CD123+/CD11c-) was harvested using FACS sorting.

The pDC were exposed to an expanded list of chemical allergens (n=49) or irritants (n=42). Concentrations of each chemical that resulted in >50 % viability as determined by FACS analysis of propidium iodide stained cells were used. Allergens were identified based on stimulation index (SI) calculated by the fold increase in CD86 expression.

A preliminary prediction model was developed: materials with SI ¡Ý1.5 in at least 50% of the pDC donors (n=2-5 donors) were labeled as allergens; materials with SI < 1.5 were labeled as non-allergens.

Of the 91 materials tested, historical data for 71 materials were available from mouse local lymph node assay (LLNA) and human studies; these data were used to analyze the sensitivity, specificity, and accuracy of the pDC based assay system versus the LLNA method and human response.

Evaluation of the pDC method resulted in sensitivity=95%, specificity=81%, accuracy=89%; for the same 71 materials, the LLNA gave sensitivity=85%, specificity=84%, and accuracy=85%. Thus, performance of the pDC was comparable to that of the LLNA (1). In conclusion, the pDC method appears to be a sensitive and specific predictor of allergenicity.

The assay is advantageous because high throughput screening of chemicals is possible, donor-to-donor variation can be monitored, the cells are of human origin, and the assay is considerably more cost effective than the LLNA or other in vivo tests.

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Keywords: Allergen presentation, Allergenicity, Allergens, Balsam of Peru, Birbeck granules, CD1a, CD34+progenitor cells, CD80, CD83, CD86, Contact allergenicity, Contact dermatitis, Dendritic cells (DC), HLA-DR, Hazard identification, Immunological Research, Inflammation skin, Langerhans cells (LC), Lymph node assay (LLNA), Myeloid DC, Non-allergens, Plasmacytoid DC (pDC), Reproducibility, Skin sensitization, Tween 20

Materials Tested: 5-Methyl 2,3 hexanedione, Aluminum chloride, Ammonium hexachloroplatinate (HCPt) , Bandowski¡¯s base, Benzalkonium chloride, Benzoyl chloride, Beryllium sulfate, Chloramine-T, Chlorobenzene, Chlorpromazine, Cinnamaldehyde, Cobalt chloride, Dimethyl sulfoxide, Dinitrochlorobenzene, Dinitrochlorobenzene sulfonic acid, Diphenylmethane diisocyanate, Ethanol, Ethylene glycol dimethacrylate, Fluorescein isothiocyanate, Glutaraldehyde, Glycerol, Hexane, Hydrocortisone, Hydroquinone, Isopropanol, Kanamycin, Lead acetate, Manganese chloride, Neomycin sulfate, Nickel chloride, Nickel sulfate, Nonanoic acid, Penicillin G, Phenol, Phenyl benzoate, Potassium dichromate, Potassium hydroxide, Propyl gallate, Propylene glycol, Pyridine, Salicylic acid, Sodium lauryl sulfate, Tartaric acid, Tetramethylthiuram disulfide, Tween 80, Xylene, Zinc sulfate, p-Phenylene diamine

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