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I ' - W .. . • . EFFECTS OF AGEING OF ART CIGARETTES ON EXTRACTABLE NICCTIImAU1D DELIVERY OF NICOTIIQE IN SMDKE 27 April 1989 gurflose: To determine if changes in the amount of extractable nicotine (EN), delivery of nicotine in mainstream smoke, or both occur when ART cigarettes experience accelerated ageing under selected conditions. g2t=d: Prepare 12,500 cigarettes of each of five aodels. Incubate each model in dedicated chambers where possible, desert (110° F, 15% RH), jungle (90° F, 854 RH) and in a freezer (-80° to -100°.C) for up to eight weeks. Samples will be withdrawn (500 cigarettes) from each_.condition.a= zero t:ma and at intervals of 2 days, 7 days, 2 weeks, 4 weeks, 6 weeks and 8 weeks. Submit 100 cigarettes from each condition and time for EN analysis and 100 cigarettes from each for analysis of nicotine in N!S smoke and other smoking data. Submit 50 cigarettes per collection to Project 1902 for microbial analysis. The remaining 250 cigarettes are f rozen at -80° C unt il needed for other (Project 6908) or repeat analyses. The five models are: 1. DL blend with no additions, = extracted; 2. the same blend, to which pre-eztraction humectants and AS have been added, = extractedt 3. the same blend, to which pre-eztraction humectants and AB have been added, and that has than been 2=ASJ2W 4. the same extracted bland to which 3% AH (dry weight basis) has been added; 5. the same extracted blend to which post-extraction casing and after-cut have been added (normal ARTT product formulatioa). A. Preparation of blend models and cigarettes It is important that all of these activities be completed within four days after initial blend formulation (see Figure 1) . IIe will assume that the DL strip blend will be assembled and that ART extraction will occur on 1 May 1989. About 500 lbs of DL strip blend Kill be prepared in the Semiworks. Fifty lbs will be set aside and cut for cigarette fabrication (Model 1). The remaining tobacco will receive pre-extraction hmoactants and AB and be cut. Fifty lbs will be set aside for cigarette fabrication (ltodel 2). The remaining tobacco will then be transported to the ART Pilot Plant for extraction. Following application of additional humactants and 718 at the Pilot Plant, the tobacco will be extracted by supercritical means, removing thereby ZA. 97% of extractable nicotine. The extracted blend will then be returned to the Semiworks. At the Semiworks, 50 lba of extracted blend will be tray-dried (Modal 3), 50 lbs will receive 3% (w/dw) AB and then be tray-dried (1[odel 4), and 50 lbs will receive post-extraction casing and be tray-dried (ltodel 5). Cigarettes (at least 12,500 of each model, n= packed) will be fabricated (Marlboro construction) and each of the five models separately boxed. Transport all cigarettes to T-8 by Thursday, 4 May. The time of placing cigarettes in the desert and jungle chambers (Monday, 8 May) is zero time.

DL Strip Blend (162) i 50= Order to 212 1.02 PG 1.0S G 1.52 AB Cut ( Tray-dry Cut 1_0>< PG 1.0Z G 1.5Z AB Tray-dry 500 [Model ? ..........., ...........~ Tray-dry Tray-dry 3xAB Tray-dry 50• Aiter-cut Figure 1. Preparation of models [Model .1 ) Afodel ,~

B. Incubation (I. Uydess and Project 1902) 1. Upon receipt of cigarettes (4 May), remove 500 of each model and place them in Mason jars and then in the freezer (T-520). All other cigarettes Nill remain at room temperature, in boxes, until the onset of incubation. 2. On Monday, 8 May, place 4000 cigarettes of each model (1-5) in Mason jars and then in the freezer (T-5). Five hundred of each model will be distributed for assays of the zero-time samples (see beloa). 3. Distribute 8000 cigarettes of each model among monitor trays (obtain from H. Mait), 2,000 cigarettes in each tray, divided into four groups of 500 each. The cigarettes should be so positioned as to encounter freely the circulating atmosphere of the chamber. Twenty monitor trays will be prepared in this manner. 3. Place two monitor trays from each of Models 1-5 in the OC desert chamber, locating those containing Models 3-5 in an area well-separated from all other cigarettes. It will be necessary to monitor the nicotine content of the atmosphere in that room from time to time (D. Watson). 4. Place two monitor trays from each of Mod.ls 1 and 2 in jungle cha.ber A and two monitor trays from each of Models 3, 4 and 5 in jungle chamber H(T- 8). Ensure that water of condensation will not contami•+ate the cigarettes placed in the jungle chambers. C. Collection and disposition of samples (I. Uydess and Project 1902) 1. Remove 500 cigarettes from each model at desert and jungle conditions at the folloxing intervals: 2 days, 7 days, 2 weeks, 4 v.eeks, 6 weeks and 8 weeks. Record the date and time of removal of samples from the chambers. (Assuming that zero time is 8 May, the succeeding samples should be withdrawn on 10 May, 15 May, 22 May, 5 June, 19 June and 3 July.) Place 200 from each collection in Mason jars and seal. Take these promptly to Cigarette Testing for equilibration (sequestered to minimize transfer of nicotine from other cigarettes). Project 1902 will reserve 50 cigarettes from each collection for +*mei3ate microbial analysis. Place the remaining 250 cigarettes from each collection into a suitable container (sealed) and freeze at -80° C (T-5). 2. At each collection time, remove 200 cigarettes of each model from the freezer and place them in a-30° C freezer (T-620) for one hr, then hold at room temperature for one hr. Transfer these thawed cigarettes to Cigarette Testing along with the experimental samples. ]m each sampling intezval, Ciga=atte sesting should rec.ive a total of fifteen conta+raz- of 200 c3qas+.ttes eaah. 3. When the cigarettes have been adequately conditioned (36 - 48 hr), transfer 100 (in a sealed container) to ARD for nicotine extraction and analysis (identify responsible person in CTD). D. Extraction and analysis of nicotine (ARD) It i& important that estimations of OV and extractable nicotine content be completed within 24 hr of receipt of samples. 1. Remove paper and filters from two groups of cigarettes (5-10 each) of each sample. Weigh the tobacco from each group. 2. Combine the paper and filters from each corresponding group of cigarettes. • T

3. Extract nicotine from each batch of tobacco (Method E86A) and, separately, from the corresponding paper and filters. Measure nicotine content by GC. Reserve mares of selected sapples for further (DSi) analysis by BCR (to be determined). 4. Using about 20 cigarettes from the same collection, measure OV. 5. Report OV and nicotine content of each batch as g= cent of dry tobacco weight and as milligrams per cigarette (data to B. Randy). s. Measurement of HS nicotine (Cigarette Testing) It is important that measurements of nicotine in smoke be completed within 24 hr after equilibration. To prevent transfer of nicotine from normal blends to the extracted cigarettes, moisture equ.tibratiom must take place in isolation from other cigarettes as far as is practicable. Smoke 100 cigarettes and assay nicotine by standard procedures. Report nicotine delivery 22K s;aaretY• and other smoking data (data to B. Sandy). !. Microbial analysis (Proj.ct 1902) On the day of collection (which will occur at the latest by 1000 hours), remove 50 cigarettes from each collection At X=dS= and divide into two groups of size large enough to assure acceptable results. Remove the paper and filters, maintaining aseptic conditions. Determine the populations in the tobacco of bacteria, yeast and mold by standard procedures. It is not necessary to measure differential endospore contents. G. Reporting, collation and interpretation of data Upon completion of assays of each collection, the data are to be reported to B. Handy (ARD), who will monitor progress and compile the results. Consulting with R. Jenkins and J. Tindall, B. 8andy, R. Carchman, D. Leyden and W. Sempflinq will collate and interpret the findings, and prepare a me®orandnm describing the results following completion. 8. Responsible persons ue understand that the following persons will assume responsibility for the execution of the components of this study. 1. Preparation of blend and cigarette fabrication: C. Ro.e 2: ART extraction: H. Alonso 3. Incubation and sample dispensation: r. tlpdss 4. Detetmination of MS nicotine: _. m;c*ham 5. Coordination of Analytical Research work and receptor of other data: 8. Randy 6. Microbial analysis: Z. Dldess T. Project 6908 participation: h. •+«#ield 8. Planninq: A. Carohsaa, II. Bemipfi3xq Richard Carchman

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