In vitro techniques for hazard identification for potential sensitizers have been of increasing importance to the NTP. In FY21, BRT screened several additional chemicals to complete the data set for chemicals nominated by agency partners. The NTP, in collaboration with the EPA, CSPC, and other federal agencies, had compiled a list of 227 chemicals for in vitro testing for the potential to cause sensitization and hypersensitivity. These compounds were nominated based on the availability of in vivo data to explore the applicability domain for several in vitro approaches including the: 1) DPRA – Direct Peptide Reactivity Assay; 2) Keratinosens – keratinocyte activation; and the 3) h-CLAT – Human cell line activation test. These assays serve as the toolbox for the NTP in vitro testing battery that is being used to evaluate the potential for chemicals to cause sensitization. Data from the in vitro hypersensitivity assessment for the isothiozolinone chemicals were used by the EPA in their recently published risk assessment for this chemical class. The NTP has received 4 final reports from these in vitro studies which have been shared with interagency collaborators. An additional 5 reports are in the process of being finalized. A research collaboration agreement with a commercial vendor was established in FY21 to assess the utility of the GARDskin assay to predict the skin sensitization potential of mixtures and compounds which may be technically challenging to evaluate using other in vitro test methods. This test method uses gene expression profiling to discriminate skin sensitizers from non-sensitizers. A total of 31 chemicals from the agency partners studies were evaluated using the assay. The testing phase and data evaluation for these studies is complete and a report is being drafted. NTP is funding BRT participation in validation trials for the Electrophilic Allergen Screening Assay (EASA). This assay uses non-protein/peptide probes in a cell-free assessment to measure the ability of a test substance to “react” with biologically important molecules for the identification of electrophilic allergic contact dermatitis hazards. Study protocols were established in FY21, and the testing phase will begin in FY22.