In FY 20 we have accelerated the research program using in vitro approaches. A key in vitro project was the screening of a number of echinacea extracts and constituents derived from these extracts using a human whole blood culture system. Using his culture system, we examined cytokine release from human white blood cells following stimulation in vitro with various immune activators in the presence of the echinacea compounds. The data generated in these in vitro studies were instrumental in the selection of an echinacea extract to be used an in vivo immunotoxicity assessment of echinacea purpurea. Ongoing projects are investigating interindividual susceptibility factors and environmental risk factors using an in vitro human whole blood culture system. Endpoints include lymphocytotoxicity, cytokine release, and Natural Killer cell activity. The second project will use the same culture system and endpoints to investigate the applicability of the test system to investigating the immunotoxicity of PAC compounds. These data will be referenced against mouse in vivo data previously collected in the PAC-MAP mixtures studies and serve to anchor human relevance and strengthen the data that can be generated using the in vitro system. BRT is currently working on development of additional in vitro tools for this culture system that will facilitate interrogation of humoral antibody mediated immunity and T-cell driven cell-mediated immunity. This in vitro toolbox will be invaluable for the future of immunotoxicity testing and will position NTP at the cutting edge of testing in this field. Recently, in vitro techniques for hazard identification for potential sensitizers have been of great interest to the NTP. In FY19, BRT completed method transfer and optimization for a battery of tests for identification of chemicals with the potential to cause sensitization and hypersensitivity. The assays selected for the NTP Immunotoxicology contract included the following: 1) DPRA – Direct Peptide Reactivity Assay; 2) Keratinosens – keratinocyte activation; 3) h-CLAT – Human cell line activation assay, dendritic cells. These assays will now serve as the main assays in the NTP in vitro testing battery that will be used to evaluate the potential for chemicals to cause sensitization. The NTP, in collaboration with the EPA, CSPC, and other federal agencies, has compiled a list of 186 chemicals that are candidates for in vitro testing. 14 additional chemicals were identified by the NTP and an additional 27 pesticides were provided by an industry partner. Assessment of these 227 chemicals was completed in FY20. This project has led to the generation of 6 draft reports, 3 of which have now been closed. An additional 3 reports are in the process of being finalized. Development and evaluation of novel in vitro testing strategies is a major research initiative in the field of immunotoxicology. In FY20 we entered into a collaborative research agreement with an industry partner to eva...