SBIR Phase II Grant Application
Alternative Photosensitization Assay in the Mouse (Photo-LLNA)

Specific Aims

Until recently, modified guinea pig sensitization assays were used to test the photoallergenic potential of chemicals.  However, the EPA, OECD and other international regulatory authorities have disallowed or replaced these tests with the mouse Local Lymph Node Assay (LLNA). Currently, there exist no validated alternative photoallergy (photosensitization) assays.  The pharmaceutical, chemical, consumer product and cosmetics industries have increasing concerns about protecting consumers from UVR- and chemically-induced allergic contact dermatitis and urgently need rapid, cost-effective photoallergenicity assays.  To address this need, we have adapted the LLNA protocol to include UV irradiation and use of flow cytometry to measure lymphocyte proliferation and activation markers, which correlate with an allergenic response and improve the accuracy and predictivity of Photo-LLNA.  Phase I results and other preliminary data demonstrate the technical feasibility of the assay, and since our assay uses BrdU incorporation to measure lymphocyte proliferation, there is an added bonus of eliminating the need for radioisotopes, reducing hazardous waste.  To develop the assay to full commercial potential as the only available alternative photoallergy test, we will take concerted steps to optimize and validate the Photo-LLNA in our lab.  These Phase II specific aims are outlined below.

 

Specific Aim One:  Confirm the reproducibility and reliability of the basic Photo-LLNA to identify known photoallergens and the false positive irritant, sodium lauryl sulfate (SLS).

  • Determine the stimulation index, +/-UVA (SIUVA and SI), at various doses of key benchmark chemicals (chlorpromazine, bithionol and SLS), and identify positives using the criteria of SIUVA or SI > 3. 

  • Measure dermal irritation using a threshold of >25% increase in ear swelling to identify irritants.

  • Classify photoallergens by comparing SIUVA vs. SI at each concentration tested, using statistical tests and/or a threshold of an SIUVA/SI ratio of >1.25 as the criteria to identify positive chemicals.  

Specific Aim Two:  Include immunophenotypic analysis endpoints to characterize and classify an expanded series of known photoallergens and non-photoallergens.

  • Using an expanded set of photoallergens, non-sensitizers, irritants and vehicles, determine SI and ear swelling at each concentration, +/- UVA exposure,

  • Measure B220+ (B cell) and CD3+ (T cell) populations using flow cytometry, and calculate the B:T cell ratio which will be used to discriminate (photo)allergens from (photo)irritants.

  • Using flow cytometry, measure surface marker expression (%I-Ak and %CD69 positive) which correlate to the activation status of lymphocytes derived from the nodes of treated mice.

Specific Aim Three: Using the assistance of our statistical consultant, evaluate the changes in SI, ear swelling, B:T cell ratio and lymphocyte activation parameters to correctly classify test articles using our tiered decision tree.

  • Compare the methods of analysis: (1) 25% increase irradiated:unirradiated samples, and (2) ANOVA to correctly classify each tested chemical.

  • Based on our classification of each chemical, create contingency tables to compare our results with the known human and guinea pig properties available in the literature.

  • Using post hoc analysis of the data, perform a cost-benefit analysis on each endpoint to determine if it enhances the predictivity of the Photo-LLNA, and to determine if the number of animals per data set can be further reduced.

Specific Aim Four:  Establish the Photo-LLNA as a validated GLP-compliant assay and prepare for Phase III commercialization, marketing and acceptance of the basic and enhanced Photo-LLNA.

  • Develop all necessary data collection forms and procedures to be in compliance with GLP regulations.

  • Distribute optimized, validated Photo-LLNA protocols and SOPs for final expert consultants’ review.

  • Market the assay by distributing the protocol and copies of posters and manuscripts to a selected list of 100-200 sponsor companies (500-1000 companies in Phase III).

  • List the final protocol in our Capabilities Statement and Price List and on our LLNA website and our Alternative Test Methods webpage.

 
   

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