Background: During the 2014 US Ebola crisis, an unprecedented amount of biohazardous waste was generated during patient care; healthcare facilities were overwhelmed by large volumes of waste. Few facilities had on-site waste treatment; therefore, waste was packaged and sent off site for incineration and disposal in Port Arthur, Texas, at a significant cost. Shipping this waste off site required the use of the US Department of Transportation (USDOT) Hazardous Materials (HMR 49 CFR) category A triple packaging for infectious substances. The most common treatment method for biohazardous waste in the United States is via commercial autoclaves. Because Category A waste packaging had not been tested to ensure effective treatment, we conducted autoclave efficacy studies to evaluate the various types of Category A packaging containing surrogate Ebola waste. If successful, this would potentially provide additional treatment options in the United States. Methods: Testing was conducted at commercial locations in 3 states: New York, Pennsylvania, and Florida. Various types of Category A packaging were obtained (Fig. 1). Waste loads were comprised of Ebola patient treatment material and included personal protective equipment, sharps containers, suction canisters, drapes, and associated items. Configured packaging was placed into autoclave bins to be processed. Each package tested included a biological indicator, a class 5 integrator. Where possible, thermocouples were added to record the thermoprofile of the waste. Initially, a modified cycle was tested (a prevacuum cycle followed by exposure to steam at 138°C for 60 minutes) and a postvacuum cycle. Cycle times were adjusted based upon initial results. Results: The initial New York autoclave was tested from a cold start (no vessel preheating) resulting in a failure to obtain efficacy (Fig. 1). Successful results were no growth in recovered biological indicators, acceptable color change in integrators, and reaching and holding temperatures >121°C for 30 minutes. After making modifications to treatment cycles, which included preheating of vessels, multiple prevacuum steps and a hold time of 60 minutes, successful results were achieved in testing conducted at the treatment plants in Pennsylvania and Florida. Conclusions: Commercial autoclaves can be effective in treating Category A biohazardous waste. Each autoclave considered for treatment requires a validation process using the types of packaging containing the waste, biological indicators, integrators, and thermocouples to present a complete assessment of the treatment process.Funding: Three companies provided support for this study: Approved Storage & Waste Hauling, Daniels International, and ProMed Solutions.Disclosures: Edward Krisiunas reports contracted research for Future Health Care Systems, Daniels Sharpsmart, and ProMed Solutions.