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To: Washington mJ From: Washington Field Re: 279A-WF-222936-USAMRIID, 1/11/2006 On the 6/26/2001 challenge, the concentrations of the Ba suspensions placed into the nebulizers were 1 x 10* cfu/ml, 1 x 10° cfu/ml, 1 x 10° cfu/ml, 1 x 10’ cfu/ml, 1 x.10° cfu/ml, and 1 x 10° cfu/ml. Each run exposed 10 mice at a time. The Aerosol Description Form noted that "AGIs contained 10 ml of PBS and 40 microliters of antifoam agent". In the 8/14/2001 challenge, the concentrations of the Ba suspensions placed into the nebulizers were 1 x 10’ cfu/ml, 1 x 10° cfu/ml, 1 x 10° cfu/ml, 1 x 10*° cfu/ml. It was noted on the Aerosol Exposure Sheet for run 4, "lots of foam". The Aerosol Description Form prepared b moted' that "AGIs were supplemented with_40 microliters 1:5 dilution antifoam + 10 ml PBS provided bf Bach of the 4 runs exposed 10 mice at a time. a omen ~J QO All of the aerosol technicians said that they would have noted on the challenge paperwork whether or not they had used antifoam during _an aerosol challenge, however, they would not have informed or the PIs of the use of antifoam unless specifically asked. If or the PIs read the log notes, they would have known whether or not the emulsion was used. Difficulty was encountered during aerosol challenges when a high concentration of the challenge agent was present in the nebulizer, or when the challenge agent possessed a high protein content, or when the collection material in the AGI contained a high protein concentration. Bubbling of the challenge agent in the nebulizer interfered with aerosolization, thus diminishing the effectiveness of the challenge. During aerosol challenges of substances with high protein concentrations, bubbling often occurred in the AGI and material was sucked into the vacuum tube attached to the AGI. Asa result, erroneous post-challenge concentrations were obtained. A lipid emulsion was used to prevent the bubbling. An antifoam emulsion was preferred; however, if antifoam was unavailable, olive oil was used as an alternative. The aerosol technicians preferred not to alter or add to the biological material provided by the investigator; however, successful completion of some aerosol challenges necessitated the addition of a lipid emulsion. If necessary, the antifoam emulsion was added to the nebulizer. During anthrax challenges, a clumpy, flocculent, b6 snow-like, milky material built up on the glass walls of the b7C nebulizer. [7 ___—satttributed this occurrence to the high concentration of the anthrax slurry placed in the nebulizer. Occasionally antifoam emulsion was added to the nebulizer to minimize bubbling and clumping of the anthrax. Approximately 40- 50 pL of antifoam were added to the nebulizer. The use of 22