Tobacco Documents Online

__----.26 SF01 9001' Microbiological Associates 5221 RIVER ROAO • BETHESDA. MARYLANO 20016 TELEPHONE: (301) 654•3400 may 9. 1980 Dr. William U. Gardner Scientific Director The Council for Tobacco Research-U.S.A.,'Inc. 110 East 59th Street New York. NY 10022 Dear Bill, As usual, the following 3s our summary. 1. Eauipment a. Sham machine The Sham machine has been installed and works ve.ll. The new direct readiug meters for indicating air flows thtough the modules seem to be exactly what was required. The same meters are scheduled to be installed by P6I for the smoking machine with in the next 4 weoks. b. Modules - air closures The prototype air pressure closure devices installed on one ct-.annel of an ex posure rack seem to be a success. Closure of the modules is autocoatic, smooth and does not require technician assiscance. A request was made. to CTR that an official quote be provided by Pb1 for 30 of these air pressure closure devices. Doyle lfullinaY will attempt to provide a similar quote. c. Holders and inserts This is the. most critical equipment related problem. A scenario was prreentid which indicated a minimum of 3 months between order and receipt of A OIVISION OF '4hitta3+~csr . . . . . . . • . . . 100101_2 ko ui' i , EXHIBIT NO..?-3- S. NELSON ~:.:-~~ . CTR ~~IN '-4444

SF019002 1• ; the inserts. lforeover, we would suggest that the 300 new holders to be provided by PiI should be fabricated to accept these new inserts. These new holders equipped with inserts would then be at b:A for evaluation and testinb before the old holders are scheduled for modification to accept the inserts. Questions as to how the insert holder fits the mouse, types of plastics, types of filler materials for the inserts, durability of the inserts, etc. will be evaluated using the new 300 holderlinserts. These 300 holders will be fitted with the smaller insert (11/32 inch). The quote for making the inserts and mold has been requested and received by P61 from Wilks Plastic, Bockville, Md. The mold cost is $3000 and a typical inserts is 22C each. 1»Tilka makes the mold3 in house. The two different size inserts will be distinguished by a combination of small shape changes on the external edges of the insert and by color stamping. Thus it is our feeling that the ti$4000 to make the mold and inserts and 'L $9000 for the new holders should be authorized now and would seem to be a worthwhile "gamble" compared to the possibility of delaying initiation of the long term studies which may be a more expensive alternative. . Experimental a. CTR-101A. Chronic Exposure to 2RI cigarette emoke. A total of 12 tables were presented which describe the current status of this study. The salient features are: 1. A total of 684 animals remain on test for smoke exposure (this is on schedule). 2. Rates of machine associated deatha seeu to be decreasing, probably the result of improved equipment, increased experience and perhaps something related to the animals themselves. -2- 10010113 t..r f Pr'~e ~~HN 0''"f' 446°',.l~

S F01 y0023 3. The only smoke associated lesions observed are low levels of PA.`lA and congestion (also observed in the sham exposed animals). No malignant lung lesions have been observed in either the smoke or sham exposed animals up to approximately 68 weeks on test. 4. Some evidence of spontaneous neoplasiss have been observed in the shelf control animals, including une osteogenic sarcoma, one lymphosarcoma, one lymphocytic leukemia and one AAC. All were ob3erved after 64-68 weeks on test (74-7g weeks of age). 5. A variety of BaP lung carcinomas have been observed to date. The probability of dying of BaP induced lung cancer in BaP shelf control animals is 0.15 after 68 weeks on test (exactly as expected). 6. Exposure to 2R1 cigarette smoke did not influence the types, incidence or latency of BaY induced lung cancers compared sham exposed controls (p - 0.57). 7. The question of etiology of congestion was discussed and MA is to evaluate and provide some direction before the next CTR-Z1A meeting. ls. CTR-117-5. Acute toxicity with 2R1 cigarette smoke in male BC3F1/ Cum mice. The continuous exposure regimen of 17 runs, 8 puff/cigarette was initiated on 5/5/80 with 150 male BC3F1/Cum mice. Data were presented which indicated a high survival rate for the male mice, very similar to that for the females (Table 13). Fifteen to 30 mice in this experiment will be used in the dosimetry experiment (CTR-119) using 14C-dotriacontane. The remaining mice will continue to be exposed to this continuous exposure regimen for as long as possible (6 months or longer) before the long term chronic study using male mice is initiated in 1981 (CTR-124). c. CTR-117-4. SubacutE toxicity with 2R1 cigarette smoke in female BC3F1/Cum. The continuous exposure regimen of 17 runs, 8-10 puff/cigarette was initiated 1/7/90 using BC3FI/Cum female mice. Survival has continued to be high; -3- 10010114 CTFZ 11N 0-44"46t''`

SF019002 to date 151 mice remain on test. These mice will continue to be exposed until the dosimetry experiment (CTR-119) is performed, approximately the f irst week in June. A preliminary dosizerry experiment using this exposure regtmen is described below. d. CTR-119-1. Preliminary dosimetry experiment using the continuous 15/45 seconds smoke/air cycle on the Walton. A preliminary 14C-DTC-dosimetry experiment was carried out using the Walton so as to become familiar with the tissue sample oxidizer and to be able to compare the data in terms of previous experiments analyzed by tissue solubiliza- tion. Data were presented (Tables 14 and 15) for the labeled cigarettes which allowed calculation of the specif ic activity of the TPN (ti 38 14C-DCT/ug TP)i). These values were in the range of previous experiments. Tbe specific activity of the TPM did not change significantly with the nuvber of puffa/cigarette. Hice not previously exposed to amoke were used in this experiment and a _ large coefficient of variation was observed for TPlI deposition in the lung (Table 15). The experiment will be repeated with adapted animals so as to establish a better dose response, and to confirm the Ug TPN deposited in the lunge. Based on these preliminary data, the deposition would result in ti 500 ug TP2f/lung day. e. CTR-96F. Inhibition of DNA repair (with R. Rasaussen). An experiment using 2R1, 3A1 and 2A1 cigarettaa started in Septcmber 1979 to determine the effect of exposure to whole cigarette saake on the capacity to repair lung DNA damaged in an in vitro assay. A slightly lower dose of 2R1 sm;ke is given each day compared to the 3A1 and 2A1 cigarettes to reduce toxic effects of 2R1 smoke. An inhibition of DNA repair has been observed with 3A1 -4- 10010115 b./' T/ '`A I l ) 7 0,444G58

' Sf01y0U: and 2A1 :igarette smoke after % 11 weeks, while no inhibition has been observed with 2R1 cigarette smoke up to 'y 18 weeks. The equivalent dose of SPM from 2R1 cigarettes at which an effect was observed for 3A1 and 2A1 cigarettes was reached in February 1980, however, no inhibition of repair of damaged DtdA (unscheduled DNA synthesis) was observed. Approximately 30 mice remain in each group and these will continue to be exposed until rcheduling time on the smoking machine for the long term chronic studies forces termination (July-August). f. CTR-109. l-unosuppression (with H. Herscowitz). Several experiments have been performed recently with Dr. Herscovitz and the results will be summarized here. A detailed report will be prepared for the next CTR-HA Staff meeting in June. Briefly, it was reported by Herscowitz that short exposure (5-10 days) of BALB/c adce to low levels (4 cigarettes/day) of high nicotine cigarette smoke resulted in a 2 log decrease in the number plaque forming cells (PFC) found in the spleen. We have repeated the assay using both BC3FI/Cum and SALB/c mice and have not observed such a dec reasa. It was also reported that long term exposure ('- 1 year) to 2 high nicotine cigarettes/day resulted in a 2 log decrease in the PFC response. To insurd that the PFC assay was working, we developed the use of a cyclophosphamide positive control, ia which a dose dependent decrease in the PFC response was observed. BC3F1/Cum mice exposed to 2R1 cigarette smoke (as in CTR-1O1 A) were used to determiaed the PFC response after injection of sheep red blood cells. No depresAion in the PFC response was observed, while mice trea ted with cyclophosphamide were shown to have a decressed PFC response. The exp eriment is being repeated•so that sufficient numbers of animals and data point s will be availabe for cosplete analysis. -5- 10010116 CTI '. IMlI I '-/' 4II' I, ~u~..' M'

S F01 y002t g. CTR-111. ODC induction Aerosol generated TPA has been used to induce pulmonary ornithiae de- carooxylase (ODC). This system has been utilized to work out the exact assay conditions and experimental variables. An abstract describing this vork will be presented by Drs. Marshall Dinowitz, Kouri and Henry at the AACR meetings in San Diego this year (abntract previously submitted to CTR). It ha s been the ultimate aim of the work to determine whether cigarette smoke had an affect on ODC induction. BC3F1/Cum mice were exposed to 2R1 cigarette smoke (as in CTR-101A) and assayed for pulmonary ODC induction at various times after exposure. A detailed report of these on-going experiments will be available for the June CTR-MA meeting. Preliminary data suggest that: 1) ODC levels in sham control animals are imeaaed ti 1.5-2 times ov er the shelf control levels, 2) GDC levels in smoke exposed animals are incrtased 2-3 times over the sham coatrols atter 24, and 44, and 88 days exposure, 3) there may be a decrease in ODC levels to control values after 180 days exposure. h. Collaborative studies 1. Effect of cigarette s~aoke on oocytes (Dr. Don Mattison). '.Vo studies have been ongoing with Dr. lfattisons 1) the effect of cigarette smoke on the number of oacytes in BC3F1/Cum mice exposed a s in CTR-101A, and 2) the rate of atresia (natural depletion of oocytes) in four inbred strains of mice: BC3F1/Cum, C3H/Anf Cum (C3) C57B1/C1im (E) and DBA (D2). To date, exposure of SC3F1/Cum f emale mice to 2R1 cigarette smoke has not influenced the normal rate of oocy:e depletion. Almost complete depletion ia observed by ti 40 weeks of age in this strain. A short term, high dose smoke exposure study has been proposed for use with 3A1 and 2R1 smoke to attempt to completely address this question. Differences have been observed in the natural rate of depletion. C3 > B Y D2; BC3F1/Cua appear to be intersediate between the C3 and B. -6- 10010117 CTR HN ~`'..4'-4• 4470

S F01'a0i127 2. Effect of cigarette smoke on sister chromatid exchange (SCE) in BC3F1/Cum mice (Dr. K Benedict). BC3F1/Cum exposed to both ;A1 and 2R1 cigarette smoke have bean scheduled to be sent to Dr. Benedict for analjrsis of SCE within the next 6 months. Mice exposed to as high a dose et smoke as possible and as lona as possible will be utilized for this assay. 3. Determination of nicotine in blood or urine (Dr. A. Castro). As discussed previously, a collaborative study to determine nicotine in mouse plasma utilizing the radioimmune assay (RIA) for nicotine has been proposed. This will be given a higher priority than in the past so that two goals can be accomplished: 1) nicotine levels can be determined rolitinely in animals chronically exposed to c igarette smoke and 2) Lh-t RIA assay as developed by Dr. Castro and his associates san be vd:ia.:tcd hy another laboratory. Dr. Rockett provided a summary letter from Dr. Castro outli:ir.g a few of the technical reQuiretients. We vill be in contact with Dr. Castro shortly, with the main question concerning 50 ml of mouse plasma possibly required to construct a standard curve. 4. Additional short term assays: a) isolation of lung cells and Frotease inhibitiora (Dr. R. White) b) clinical chemistry and c) behavioral toxicology. Additional approaches to other short term tests as markers for smoke exposure have been discussed. Specific experimental approachea are'beiag explored with Drs. White 3nd/or Janoff to determine such things as 1) whether recovery of macrophages from mice could be performed routinely and 2) whether antiyrotease inhibitors could be evaluated in mice as has bepn.perfor'ed in rats. During this discussion, the areas of clinical chemistry and behavioral toxicology were brought up as being also very relevant to monitor long tein (lifetime) studies. MA will have a cl:r.ical chemistry laboratory operational in the next 6 months. A srPc+fied nunbcr of assays could be determined and the results analyzed before any long term cowittents to this area were made. Ekperimcntal details and more specific approaches will be available for the June CTR-!7A meeting. 3. Scherdulina of new lon term studies A tentative pert chart was presented for scheduling animals for "lifetime" smoke/sham ox posure for CTR-118 (BC3F1/Cua females) and C?R-124 (BC3F1/Cus males). Mice would b4 exposed to both 2R1 and 3A1 cigarette smoke ior 24-36 months. A -7- 10010118 CTR HN 0444 -"` 1

SF0190028 total of 960 mice per group was proposed. The first rtage of the female study is to start in June 1980 and will requ ire 4-6 months for all animals to be put on test. The first stage of the males is scheduled for % March 1981. The number of exposures would bEgi'n to dec rease in 1983, and decrease substantially in 1984. An estimate was made that '1 1 .1 million 2R1 cigarettes would be requir ed for all of the studies now on test and those proposed. Approximately 0.875 million 3A1 cigarettes would be required during this same time period. There are sufficient 2R1 cigarettes ia storage at PiA to meet these needs, however, the number of 3A1 cigarettes needed leaves little margin for error. A cigarette inventory will be performed by June I to verify the estimates and also for r•:neval of the storage contract 0020. CTR was to inquire about the availability of additional 3A1 cigarettes or tobacco from the University of Rentucky. In addition, a decision vill have to be made in the next 6 months or so as to how long the present cigarettes can continue to be stored since many of them were aanufactured in 1974-1976. An estimate should also be made witnin the next 6 months by CTR of how many cigarettes should be kept in storage since some re- arrangements in f reezer capacity are anticipated with the new lease arrangcments proposed tor 1981-1983. 4. Tentative three year budget. An estimated budget for 1981-1983 was presented to both Dr. W. Gardner, rlr. A. Yeaman and their respective staff inembers. As matters now stand, upon signing of the new leases for Bethesda Avenue, the computer proposal will be released immediately, as will the holders and inserts required for CTR-118 to -8- 10010119 i..r f R i f N 0` i, `"f. 4t`2

J • . s Fo'1 90029 are expected to remaia firm for another 60 days. In an attempt minimize the delay in scheduling for the new computer system development, ve propose to iaplement the previously submitted budget over a shorter period of tiae (ti July to December). It is estiaiated that the leases may be signed within the next 3-4 weeks. 5. Next scheduled meetinR The next scheduled meeting is June 18 in Bethesda. Call us if you have comments or questions. Sincerely, Richard F.. Kouri, Ph.D. Carol .]. Senry, Ph.D. Director Research Director, Dept. of I]cperimental Oncology CJH;kER:la cc : Dr. V. Lisaati Dr. Dr. Dr. Dr. Hr. ?tr. R. D. D. J. A. D. liockett Ford Stone Parker Yeaman Yentzer -9- 10010120 CTR HN 044473