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FINAL REPORT P. LORILLARD COMPANY, INC. C ,1 Contract C-131 R October 14, 1964 It was the purpose of this study to determine the carcinogenicity for the skin of mice of certain tobacco smoke condensates and of one of their fractions. The amounts of smoke condensate painted on the skin were kept on an equal weight-per-animal basis in these experiments. Thus, in the case of the tars yielded by a filter cigarette, the smoke condensate applied to the mice was derived from approximately twice as much tobacco as in the case of a smoke condensate originating from a nonfilter cigarette. The results obtained express the relative carcinogenicity of each material painted onto mice in terms of car cinogenic activity per g of tar studied, and not in terms of the amount of tobacco smoked to prepare the test material. This is an important difference of this project from certain published studies (Bolk, Moore, Dowd and Clark, J. A. M. A. 181:668-672, 1963). The materials studied were: 1) smoke condensate derived from Pall Mall cigarettes, termed PM-100, 2) smoke condensate derived from Kent cigarettes with the original Kent filter, manufactured before 1962, termed herein K-75, 3) smoke condensate derived from Kent cigarettes with a filter, manufactured since 1962, termed K-12, 4) smoke condensate derived from Winston filter cigarettes, termed W-35 and 5) a fraction of whole smoke condensate (carbon tetrachloride fraction, or Fraction B). The latter was used at two dosage levels corresponding roughly to the amounts of Fraction B previously studied by Wynder in mice (herein called B-2) and to five times as much (called B-10). The influence of the solvent -{acetone) alone was studied in a control group.

Methods The mice used were female Millerton mice (Millerton Research Farm, Rudd Pond Road, Millerton, N. Y. ) weighing from 24 to 30 g at the start of the experiment. In contrast to our routine procedure, the animals used in this long-term experiment were not dipped into an insecticide mixture, nor were they given tetracycline in the drinking water after arrival, because it was intended to avoid the contact of the mice with any substance which might potentially have some carcinogenic potency. The animals were distributed into five groups of 200 each and two additional groups of 50 each in such a way that the weight distribution of the mice was the same in each of the seven groups. All mice were individually numbered. They were placed in a separate air-conditioned and light-controlled animal room in plastic disposable cages, ten mice per cage, with San-I-Cel as bedding material, Purina Chow for food and tap water ad libitum. Body weights of all animals were determined once a month. The triangular dorsal area between the iliac crests and the root of the tail was shaved by means of electric clippers, and this area was subsequently painted with a No. 5 camel's hair brush. The mice were shaved repeatedly as necessary to maintain a smooth painting area. Throughout the entire study, the same attendant performed this operation, except for vacation periods when he was relieved by a man whom he had personally trained. The amounts of material painted onto the backs of the mice were measured each day by weighing the material before and after painting; the average amounts of solution applied per mouse per week, thus obtained, are shown in Table I. The materials applied were coded (see Table II), and our personnel handling the animals did not know the key to this code. A painting schedule is presented in Table III.

3 TABLE I Materials studied and amounts applied (computed as outlined in text, averaged for entire duration of experiment) expressed in mg of acetone solution. 011964'71 ature and Source Average Amount of Acetone Concentration of Number of of Material Solution Applied in mg/Mouse/ Acetone Solution in Animals Painted Week g/g ~Smoke condensate derived I from Pall Mall cigarettes (PM-100) 206 50 % 200 Smoke condensate derived from Kent cigarettes before 1962 (K-75) 202 50 % 200 Smoke condensate derived from Kent cigarettes since 1962 (K-12) 208 50 % 200 Tar derived from Winston cigarettes (W -35) 195 50 % 200 Fraction B in twice its con- centration in crude tar (B-2) 233 2% 200 Fraction B in ten times its concentration in crude tar (B-10) 263 10 % 50 Acetone (Control Group) ~ 100 % 50 == Not determined

4 TABLE II MATERIAL PAINTED CODE B-2 H K-12 J K-75 K PM-100 L W-35 M B-10 N

5 TABLE III ~ MATERIAL PAINTED ANIMAL NUMBERS Monday H 1-200 J 171-270 K 341-440 L 511-610 M 681-780 N 851-870, 891-900 Tuesday H 1-100 J 171-370 K 441-540 L 611-710 M 781-880 N 861-870, 901-910 961-970 Wednesday H 101-200 J 271-370 K 341-540 L 511-610 M 681-780 N 851-860, 891-910 Thursday H 1-100 J 171-270 K 341-440 L 511,710 M 781-880 N 861 -870, 891 -900 961-970 Friday H 101-200 J 271-370 O 10+ K 441-540 /r L 611-710 ~ M 681-880 ~ N 851-860 901-910 ~ , 961-970 W * See Table 11 for code

6 At each painting, the mice were observed for appearance of papillomas; when the technician noted a lesion at least 1 mm in diameter, this was shown to a supervisor for verification and was recorded as a papilloma. Such mice were kept until the progressive growth of the lesion had been definitely established, and then the animals were killed and discarded except for a few, the lesions of which were sectioned and studied microscopically. Painting was started on January 7, 1963 with three weekly paint- ings. In this early phase of the experiment, some animals died from nicotine toxicity. On January 31, all animals that had thus died were replaced by animals of comparable weights. There were only 36 replace- ments; this replacement procedure did, therefore, not affect the outcome of the study. Painting of the animals receiving the high concentration of Fraction B(B-10) was discontinued after 56 weeks; the experiment involving the lower concentration of Fraction B (B-2) was terminated after 82 weeks of painting. In both cases, the cumulative percentage of papillomas exceeded 90% at termination. Painting of the remaining groups was discontinued at the end of 88 weeks. All remaining mice were sacrificed when painting was terminated. All materials listed in Table I were prepared in the Lorillard Research Laboratories and were shipped to us in acetone solution on dry ice. These materials were kept in the freezer under nitrogen. Bottles were vigorously shaken before use. Small aliquots were placed into test tubes for painting, and these tubes were kept in ice water during use. Results The numbers of animals with papillomas have been expressed in percent of the number of survivors, and these results are presented in Figure 1. Table IV gives the numerical data at the termination of each of the seven groups. Figures 2 and 3 show photographs of some typical papillomas.

7 By our criteria, namely by detecting the time of appearance of the first papilloma and by observing the rate of subsequent papilloma formation, the smoke condensate from one of the filter cigarettes (Winston) was equally carcinogenic as an equal amount of condensate from the un- filtered cigarette (Pall Mall), or even slightly more carcinogenic; but the smoke condensates obtained from the Kent cigarettes with the older filter (K-?5) as well as from Kent cigarettes with the newer filter (K-12) were both less carcinogenic than those from Winston and Pall Mall cigarettes (68% and 70% papillomas from the Kents after 88 weeks versus 78% and 84% for the same period for the two other brands). These differences, however, are small and their statistical significance is difficult to ascertain. A much more striking observation was the markedly accelerated formation of papillomas caused by Fraction B. With all whole smoke condensates, the first 5% of papillomas occurred after 34 to 38 weeks of painting, and a 50% level was reached between 70 to 78 weeks. In contrast, 5% of papillomas were already observed after 19 weeks with the high con- centration of the B-Fraction (B-10), and 50% of papillomas occurred after 42 and 62 weeks in the B-10 and B-2 groups, respectively. Monthly changes in body weights of the animals are shown in Table V.

-8- TABLE IV DATA AT TERMINATION OF PAINTING Material Applied Number of Weeks No. Ani mals Papilloma s After Beginning of Il Painting I At Start At Ter mination No. - Animals P A ercent nimals PM-100 88 200 132 103 78.0% K-75 88 200 123 84 68.2% K-12 88 200 107 75 70.0% W-35 88 200 139 117 84. 1% B-2 82 200 156 151 96.8% B-10 56 50 50 47 94.076 CONTROL 80 50 30 1 3.376

011964'77 - 9 - T AB LE V AVERAGE MONTHLY BODY WEIGHTS DATE WEIGHED PAPS -I H J K L M N CONTROL 1-2-63 27.7 27.4 28.1 27.7 27.6 27.6 2-4-63 29.8 28.5 29.5 29.2 29.0 28.8 3-4-63 31.6 30.6 30.2 29.6 31.5 31.6 30.3 4-9-63 31.3 30.3 30.8 31.0 30.8 31.0 30.1 5-2-63 33.2 32.9 32.2 31.7 32.9 32.0 32.2 6-4-63 32.5 31.4 31.1 31.2 32.3 32.5 31.8 7-2-63 31.6 30.8 30.5 30.6 31.5 32.1 31.7 8-1-63 33.4 34.4 32.0 32.0 31.6 31.8 32.3 9-3-63 34.5. 33.5 32.7 32.2 32.4 33.0 33.4 Paps 36.2 32.0 35.0 33.3 33.6 32.8 10-3-63 36. 0 34.7 33.8 33.6 35.2 34.7 35.0 Paps 36.5 34.2 36.3 34.3 34.7 34.2 11-5-63 34.4 33.4 32.7 33.4 33.3 33.2 34.3 Paps 35.2 33.9 32.9 36.7 33.8 33.0 ~ Where two lines appear after one date, the first one indicates the means for mice without papilloma, and the second line, marked Paps, indicates the means for all mice with Paps.

o11ss4'78 - 10 - TABLE V Cont'd. DATE WEIGHED PAPS* H J K L M N CONTROL 12-6-63 35.6 34.4 33.8 33.6 34.1 33.6 35.3 Paps 34.6 34.2 34.8 33.1 34.6 33. 3 1-2-64 36.4 34.7 33.8 33. 5 34.1 33.9 35.5 Paps 36.0 34. 5 33.6 34.2 34.1 33.6 2-13-64 36.6 35.7 35.2 36. 5 35.4 35. 3 Paps 36.5 35.9 35.3 35.8 36.5 3-4-64 34.7 33.5 33. 5 32.4 33. 8 34. 2 Paps 31.7 34.8 34.0 33.6 34.1 4-2-64 36.1 34.6 34.0 32.7 34.4 35.3 Paps 36.5 35.3 35.0 34.3 34.8 5-6-64 32.2 34.6 34. 3 31.2 34. 3 35.3 Paps 36.6 35.7 36.5 34.6 34.5 6-4-64 37.0 35.6 34.2 33.7 34.2 35.9 Paps 38.2 35.4 35.0 33.7 34.9 7-7-64 37.1 34.4 35.4 34.4 34.0 36.7 Paps 37.5 35.3 35.5 34.6 35.0 8-10-64 33.8 35.4 35.3 34.3 35.0 36.2 Paps 37.2 35.5 35.4 34.6 34.4 9-8-64 35.4 35.5 35.1 34.9 37.0 Paps 35.1 35.1 34.3 37.7 9-21 -64 36. 1 35.9 35.0 35. 0 37.4 Paps 35.3 36.2 34.5 34.0

From the photographs of papillomas taken from representative animals after approximately 48 weeks of painting, it may be seen that these lesions have the typical appearance of tobacco tar-induced papillomas. No attempt was made to study their histological evolution from benign hyperplasia to cancer, but histological studies showed typical squamous cell carcinomas in a number of instances. Dis cus s ion It may be concluded that at high dosages, the condensates obtained from smoke of cigar-ettes with and without filter tips are capable of caus- ing papillomas and cancers in the skin of mice. This is in accordance with the earlier literature quoted extensively in our previous study on skin paint- ing of mice with tobacco smoke condensates derived from pipe, cigar and cigarette tobaccos (J. Nat. Cancer Inst. 31:1445-1459). The differences of carcinogenicity of equal amounts of such tars are minor. Nevertheless, there are tars produced by smoking through such filters as the Kent micronite filter which appear to have reduced carcinogenicity (as compared to unfiltered smoke) when applied to the skin of mice. This was not true in the case of another type of filter studied here which yielded tar of carcino- genicity equal to that of unfiltered smoke. The carcinogenicity of the condensates from smoke of the Kent cigarettes would then be considerably lower than that of the two other cigarettes when compared on a per-cigarette instead of on a per-weight basis. The most rapid development of skin papillomas was obtained with Fraction B. This is the carbon tetrachloride-eluted subfraction of the neutral tar and amounts to 1.7% of the crude tar. Wynder and Wright (Cancer 10:255-271, 1961) had noted marked biological activity in this fraction. Since at high dosage this B fraction causes much earlier appear- ance of papillomas, it may be considered to contain a highly active co- carcinogen and primarily appears to act as a promoter.

- 12 - Since the total tumor incidence obtained, 95% and 98%, is also higher than that seen with the tars themselves, it must be assumed that adequate amounts of initiators (polycyclic hydrocarbons) or other carcinogens are also present. The controls in this experiment showed a small incidence of papillomas (3. 3%); this suggests a slight contamination of acetone with some chemical carcinogen. This is minimal and insignificant. There was no significant difference in the mean body weights between animals with and without papillomas, receiving the same material. These biological tests strongly suggest the usefulness of an intensive search for cocarcinogens (promoters) in Fraction B. Elimina- tion of such substances might well reduce carcinogenic activity of tobacco tars down to insignificance.

100 80 S60 K-75 0 20 PM-100 / J, W-35 I ~ I ~ CONTROL 20 30 40 50 60 70 WEEKS OF PAI NTI NG 80

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