In chemico alternatives to contact allergen identification are being developed and in separate studies, monoclonal antibodies (mAb) to methylene diphenyl diisocyanate (MDI) bound proteins are being produced. To date in chemico reactivity experiments have been performed with 57 electrophilic chemicals that are skin sensitizers, irritants and non-skin sensitizers that have data available for the local lymph node assay (LLNA). The nucleophilic probes being used are nitrobenzenethiol (NBT) and pyridoxylamine (PDA). Reactivity of 29 chemicals to NBT was evaluated and correlation of rate constants to LLNA EC3 data was modeled. A contract was awarded for kinetic analysis of extremely rapidly reacting electrophilic allergens requiring a stopped-flow spectrophotometer, which is not available at NIOSH. Amine reactive chemicals (28) were evaluated with PDA for reactivity as a marker of their skin sensitization potency and their rate constants to LLNA EC3 data was also modeled. LLNA studies were performed for 6 benzoquinone derivatives as part of testing the predictive power of the reactivity assays. Kinetic data was used to identify appropriate doses and time points for simple endpoint assays with potential to estimate allergenic potency. These assays have been submitted to the Interagency Coordination Committee on the Validation of Alternative Methods (ICCVAM) for inter-laboratory validation testing and potential adoption. Current work is focused on experiments aimed at modifying the in chemico NBT and PDA reactivity assays to determine rate constants for prohaptens by incorporating cytochrome P450 (CYP) isoforms in the system. Several attempts to produce mAb to MDI bound protein have been made. One specific IgM has been produced, but a specific IgG producing hybridoma has not yet been isolated.
