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~ps ~ 4~1f Dermal Testing of*Smoke Condensate from Cigarette Containing Cocoa This test was designed to repeat the NCI Experimentl of a cocoa-containing cigarette. The tobacco blend was the'same . with the exception of the composition of the reconstituted tobacco whi.ch in this case contained more burley tobaccos than the NCI cigarette and was made by a paper process instead of the slurry process used for the NCI material. The cigarettes differed in -~,. that the NCI cigarette was non-filtered, while these cigarettes had a low-efficiency filter attached. Although the_ NCI study contained only the control cigarette and one containing 1% cocoa .(as did our original protocol), we also decided to add a 3% cocoa cigarette to the study. ~ =.. ; The tobacco blend data and•the cigarette specifications are -shown in Table 1._ Analytical smoking data are shown•in Table 2. ~The cigarettes were manufactured by Philip Morris Research and ~, Development and shipped to INBIFO, Cologne, W. Germany, for con- r~*'r':~ densate collection and dermal testing. Cigarettes were smoked on INBIFO machines.under standard smoking conditions and the smoke '" was collected in Elmenhorst cold traps The condensate was taken •up in acetone and concentrated to remove acetone and water by rotary .evaporators at 40°C under reduced pressure. The dry condensate was redissolved in acetone to a concentration of 25% w/v condensate and 2% water. Samples were further diluted with acetone to 18% and to 12%. Thus, 0.1 ml applications contained 12, 18, or 25 mg of dry condensate per 0.1 ml of solution. Female mice, LD-1 from Charles River (France), were assigned to 11 groups of 105 mice each. Two groups (0.1 and 0.3, INBIFO reports RL 49 (R)Al.and RL 50 (R)Al) received acetone only. The. other nine groups (1.0, 2.0, 3.0, 1.1, 2.1, 3.1, 1.3, 2.3,_3.3) received 60, 90, or 125 mg/wk of dry condensate in acetone from cigarettes containing 0, 1, or 3% cocoa powder. Painting was - done daily, 5 days per week, with 0.1 ml of the.appropriate solution. Applications were made for 75 weeks, and the animals were sacri- ficed during the 76th week. The animals were observed weekly for the appearance of lesions. A lesion was noted when it reached a diameter of 1 mm, but was not considered a tumor unless it reached a diameter of 2 mm and was elevated from the surrounding skin surface. At that time the lesion .was scored as a grossly visible tumor with time of appearance when it was first observed to be 1 mm in diameter Lesions which fai1ed to reach the 2 mm diameter or were not elevated were not scored. At'death or sacrifice all skins were stretched on cardboard and immersed immediately in Carnoy's solution for 4 hours. -`Visible lesions or opacities were taken, immediately dehydrated in 'alcohol, ;•and then embedded in paraffin for sectioning. Sections were stained with hematoxylin and eosin for histologic examination. a ~

M i RL 49 and in Tables 7-10 and 15-18 of Report RL 50. Papillomas All lesions visible by the above procedure were examined and classified as shown in Tables 10-16 and 24-30 of Report and acanthomas (1.05 - 1.10 in above tables) were considered to be vremaliRnant tumors, and carcinomas (1.11 - 1.12) were l classed as ma ignancies. These are totaled in Tables 39-41 of Report RL 49 and in Tables 24-26 of Report RL 50. Total 'numbers .of mice with skin tumors in each group are shown in Table 42 of --' Report RL 49 and in Table 27 of Report RL 50. Table 43 of Report„M~,•:`, 'with tumors for each group relative to the initial numbers of RL 49 and Table 28 of Report RL 50 shbw the incidence of mice mice. -. - Reports RL 55 and 56 cor4tain life-tables with histologically corifirmed tumors for all groups. These results are combined in added to tobacco has no effect on the~tumori enicit of ci - ` 1% cocoa sample is an anomalous result and that cocoa when 3%.cocoa. Therefore, we'conclude'that the1ow dose group for This is also true for the'90 and 125 mg/week data for 0, 1, and difference among the data. Further study shows this to be due to the low dose result for the 1% cocoa sample. If the 0% and 3% cocoa data are'compared,.the}i are not-different by x2. - = :',Chi squared (x2) analysis (Table 4) shows an overall Table 3 of this report. ~ ~ arette smoke condensate*on mouse'skin. ' Reference: 1., Toward Less Hazardous Cigarettes. Draft Report No..3: The Third Set of Experimental Cigarettes. National Cancer Institute Smoking and Health Program; 1976 August.

I ' r 4 TABLE 1 TOBACCO BLEND *Reconstituted Tobacco Flue-Cured Stems Burley Stems .Flue-Cured Scrap Burley Scrap Hot Water Solubles CIGARETTE SPECIFICATIONS Flue Cured Burley Oriental Reconstituted* Length ' .Tobacco Rod Filter Circumference :Paper Filter Tipping Paper Dilution Code X6DOEE, 0%Cocoa X6DOEF, 1% Cocoa* . X6DOEG, 3% Cocoa* 63mm 22mm 36.5% 23.7% 12.4% 27.4% 85mm -25mm Ecusta 351 (30-40sec Greiner Porosity) - (0.5-0.7% Phosphate 8.4/40,000 Cellulose Acetate Cork, 25mm 0 ..w .a At ..-I~ :r ,,,~, ,, `3

CIGARETTE SMOKE DATA* -~'TPM Nicotine Water FTC Tar Puff Count .X6DOEE 0% Cocoa mg/cigt - 35.8 sec duration, 58 .sec interval, 28 mm butt X6DOEF - X6DOEG 1% Cocoa 3°lCocoa mg/cigt mg/cigt

4 TABLE 3 TUMOR INCIDENCE, HISTOLOGICALLY CONFIRMED FROM LIFE TABLES Cocoa*Added 60 I -0% _ . ., .w 6.9 20.0 r' 26.4 r - 18.2 9.5 11.9. 3% 9.3 16.7 . 18.2

1 TABLE 4 ~..r.. ., _ CHI SQUARED ANALYSIS OF DATA :: ° Overall Cocoa , 0 vs.:'1 .O vs. 3 1 vs. 3