Tobacco Documents Online

-2- 1. OBJECTIVES The objective of this experiment was to com.,are the effect of several alternative modes of tobacco manufacture on the tumorigenic activity of the condensates produced by ciKarettes containing these materials. A subsidiary objective was co compare these data with an earlier JANUS experiment (B3). Unpainted mad acetone painted control Kroups of animals were also included in this ~etimenc. 2. DESIGN OF EXPEEIMENT Five modes of tobacco manufacture were used, as well as the 'conventional' method used to produce the control ciSLTette. The experimental treatments ware numbered as follows:- B9-1 Contro I cigarette. B9-2 Extracted tobacco. B9-3 Extracted tobacco. Extract returned. B9-4 Reconstituted sheet type 'A'. B9-5 Reconstituted sheet type tB'. B9-6 Reconstltuted sheet type 'C'. B9-7 Repeat of experiment B3. 39-901 Unpainted controls. B9-902 Acetone painted controls. Two condensate levels (40 ~ and 60 ~) were used for Erear~enna Bg-1 to B9-6. Only one dose level (40 mE) was used for B9-7. The condensate was applied three tiros each week th~ou&hout the perlod of the experiment in 0.3 ml of solution. The solvent used was acetone, so that B9-902 was painted three times per week with 0.3 ml of acetone. Thus the axperlment consisted of 15 Eroups of animals, each group BAT Co LTD - MINNESOTA TOBACCO LITIGATION m

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-3- containing 147 animals in the original design, a total of 2205 animals. These animals were unmated female albino mice of Cax-~or~h origin. ~hen submitted to the experiment they were 8 ~o 9 weeks old and weighed on average 20 K- All animals in the experiment were shaved with the same shearing rhythm. The de~ailsof animal husbandry and condensate production are to be found in (3). 3. RESULTS OF EXPERIMENT The practice at Battelle, Frankfurt, is to examine each anJmml weekly. Skin les~ons within the painted area are recorded as tumours if they have a diameter of 8c least 2 --- and protrude above the skin surface or have a firmer consistency than the surrounding skin. If at any subsequent examination a tumour is judged no have disappeared a regression is recorded. For the purpose of tabulating the incidence of tumours, the time of appearance of a tumour is taken as the time of appearance of che papilloma, recorded as described in the previous paragraph. The condition of malignancy is not diagnosed until autopsy; the time of onset of malignancy is zaken as the time of death of the animal carrying ~he tumour. Tumours are regarded as malignant if nhey produce metastases, or if they infiltrate the dermis and penetrate the tunlca muscularls~ or if they inf£1trate the dermis and penetrate the basal membrane of the epithelium. Occasionally tumours show infilnration of the dermis, but it cannot be clearly established that penenranion of uhe basal membrance had occurred. Such tumours were recorded as showing the first siena o£ malignancy but are now classified as benign (i.e. class 6 for animals and class & for lesions will be 0, see below). BAT Co LTD - MINNESOTA TOBACCO LITIGATION o

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-4- The results of the experlment are shown in Tables I to 15. These tables show the incidence of tumour-bearin8 animals in the 15 experimental Eroups of animals. The animals in an experimental Eroup may be allocated Co the followinE seven classes:- 1. Animals having no leslon. 2. Animals havinE lesions all of which permanently regressed. 3. Animals havinE lesions all of which are uncharacterised. 4. Animals havinE lesions, some of which permanently regressed, the r~mainder being uncharacterlsed. 5. Animals having at least one confirmed tumour. 6. Animals havinE at least one confirmed turnout showing the first siEns of maliEnancy, but no maliEnant tumour. 7. Animals havinE at least one malignant turnout. An animal must fall into one and only one oF the classes I to 5. Classes 6 and 7 are sub-divisions of class 5. The lesions borne by an animal in an experimental Eroup may be allocated to one of the followinE five classes:- i. "Lesions which permanently regress. 2. Lesions which are uncharacterised. 3. Lesions which are benisn. Lesions which show the first slgns of malignancy. 5. Lesions which are maliEnant. A lesion must fall into one and only one of these five classes. Zn order to analyse the incidence of tumour--bearins animals, Che time of appearance of tumour-bearLns animals in the experiment is tabulated as in Tables I to 15. The first column shows the duration BAT Co LTD - MINNESOTA TOBACCO LITIGATION ,",o

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-5- of the experiment divided into 4-week periods. The rimes recorded in the dace for each animal e.g. rime of appearance of tumour, eime of death, etc., are recorded in weeks. The second col,mm is defined as the number of animals aZive and tumourless at the beginning of the time period. The third column is defined as the number of animals becoming Cumour-bearlnK during the 4-week period. Column 4 is defined as the number of animals dying without becoming turnout-bearing during the A-week period. The fifth colu~-n is defined as the ~uanbar of animals alive without a malignant cumour at the beginning of the A-week period. The sixth column is defined as the number of animals which have no malignant tumour at the beginnlnK of the time period and which produce at leash one malignant tumour during the A-week period. Since the onset of malignancy is defined as the date of deaCh of an animal bearing a malignant turnout, colum~ six may be equivalentl7 defined as the number of animals dying with a malignan~ tumour during the 4-week time period. Similarly column 5 may be equivalently defined as the tonal number of animals alive at the beginning of the A-week period. The seventh colunm is defined as the number of animals dying without a maliEnanC Cumour during the A-weak period. Thus if the rows of che cables are numbered I to i then for colunms 2, 3, & and 5, 6, 7 Ni÷I = Bi - D1i - D2i where N. is the number of animals at risk et the beginning of time period i, DIi is the number of animals becoming tumour-bearing during time period i, and D2i is Che number of anLmals dying during cime period i wlChouC becoming cumour-bearlns. N1 is the number of anlmals enterln$ the experiment at the beginning of week 1. BAT Co LTD - MINNESOTA TOBACCO LITIGATION k.m

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-6- The ani~nals in an exPer£mental group enter the tables according to their occupancy of the seven classes described above. Thus animals from classes 3, A and 5 will enter column 3 of the cable; an£mals from classes 1 and 2 w£11 enter column 4 of the cable; animals in class 7 will enter column 6 of the table. Columns 3, 4, 6 and 7 are totalled. These cables £orm the basis of the statistical mnalysls. Subsidiary tables of the numbers of animals in the seven classes and of the numbers of lesions in the five classes, are shown in Tables 16 Co 30. Table 31 is a summary of Tables 16 and 17, and Table 32 is a su~=ary of Tables 18 co 30. It will be noted thaC Tables I to 15 frequently show chat the numbers of aninmls in the experimental groups were greater than those stated at the beg£nning of section 2. These extra animals are called "REPLACZHENT AHIHALS" and wez'e taken into the exper;,nent to replace animals dying during the first A weeks of treatment. The number of animals used in the 15 experimental Kroups were as £ollows:- B~l B~2 B~3 B~5 B~6 B9-7 Unpainted control Acetone control 40 mS 60 mg 1 1 2 1 0 O 0 5 0 10 1 3 11 1 1 A total of 37 replacement an£mals were used, mak£ng an overall total of 2242 animals used in the experiment. BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢...m

INDEX HAS INDICATED GAP IN BATES RANGE HERE