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AMERiCARI HEALTH FOUisIDATiON NAYLOR DANA INSTITUTE FOR DISEASE PREVENTION Dana Road, Valhalla, New York 10595 Telephone: 914-592-2600 American Hlsallh Foundation 320 East 43rd Stteet New York, New York 10017 Telephone:,212-953-1900 OFFICERS AND BOARD OFT9USTEES David J. Mahoney, Chairman Dr. Jimmy H. Bell Chairman and Chief Executive Otficer Lorl l l ard Norton Simon, Inc. i.~ William J. Levhta,Past'Chairman IKes$arcii Center Founder-Chairman levitt dSona, Inc. t'. 420 English Street Ernat L. Wynder, M.D., President p.Q.BOx 21688 John D. Twiname, Managing Ditecton Green abOIO y N. C. 2 7 4 2 0 Eric M. Javits, Secretary ;~ Senior Partner Javits & Javlts TRUSTEES: . JohnJ. Burns, Ph.D. Vice President for,Research HoNmann-La Roctie„Inc. Thomas L. Chrystle Chairman Merrill Lynch White Weld Capital Markets Group Theodore Coaper, M.D. Dean Cornell University Medical'College Mrs. Charles A. Dana D. Ronald Daniel Managing Director, McKinsey & Companyj,lnc. Ralph Landau, Sc.D: Chairman & Chief Executive Officer Haicon International,Jnc. Hildegarde E.,Mahoney Bruce S. Nevins President Perrier, Great Waters of France, Inc. Joseph Robbie Managing Partner Miami Dolphins, Ltd. Hon. Donald'H. Rumsfeld President d Chief~Executive Officer G.D. Searle & Gompany John W. Simmons Presitlenf8 Chief Executive Officer Morton-Norwich Products, Inc. Mrs. Jennifer Jones Simon Thomas A. Watson ADM. E. R. Zumwalt, Jr., USN (Ret.) President 8 Chief~Execut)vs Officer American MedicallBuildings, Inc. David L. Davies Executive Vice President John H. Weiaburger, Ph.D. Vice President for Research Naylor Dana Institute Charles B. Arnold, M.Di. M,P.H. Vice President for Research Health Maintenance Institute Dear Dr. Bell: I have enclosed the data from the samples which you have forwarded to Dr. Hoffmann. We have found that our current methods of collect- ing the distillate from tobacco provides us with very consistent mutagenicity results. Because of the potential for "hot spots" in qur initial distilling apparatus, we employed a modified unit using a Hewlett-Packard gas-chromatograph as illustrated in figure 1. U.sing this unit it was found that under our standard conditions (flow rate = 50 ml per minute using He carrier gas) almost complete.transfer of distille , mater- ial at a specific temperature occurred afte 1.0 hour. Our initial studies using this apparatus involved heating at temperaturesof 200°, 250°, and 300°'. No mutagenic activity was observed in S. typhimurium TA 98 at 200° in either the whole distillate of its basic subfraction. At 250°, where most of the largest amount of di- stillate is collected, only marginal mutagenic activity was observed. Only at 300° have we found mutagenic activity of at least 4 x con- trol levels (DMSO) for the whole distillate. 2

NAYLOR DANA INSTITUTE for DISEASE PREVENTION 2 It is our current practice, therefore, to di- still tobacco at 2500 for 1.0 hour, then to replace the traps and continue the distilla- tion at 300°C for an additional hour. The mutagenicity assays on your fractions A, B, and C are outlined in Table 1. There is little doubt that your chemical procedures are effective in concentrating the mutage- nic activity. Table 2 outlines the results of our stepwise temperature distillation procedure using 200°, 250°, and 300°C. Tncluded are the results of mutagenicity assays of the neutral, basic, and acidic fractions of these spe-cific distillates. All solutions for bioassay are prepared using Aldrich "Gold Label" dimethylsulphoxide. If we can be of any furthe r assistance in this study, please do not hesitate to call on us. Miss Lorraine Tulley or Miss Eliza- beth-Ann Fow are available to assist you in establishingidentical mutagenicity assay pro- cedures, if you feel this is required. Sincerely, Edmond J.LaVoie, Ph.D. Head Section of Metabolic Biochemistry Div. of Environmental Carcinogenesis encl. EJL/bs cc. Dr. F.J. Schultz

r Table 1. HIS+RE'VEFtANTS PLATE SAMPLE DOSE .PLATE (ug) PLATE PLATE I II f. X ~jr ~ 1000 7).Y- 80 75 y8- y 0 500 Go 7S - 65 lsS 4Z s s:,- ,. A 250 YG.~- 55 38 100 c Z 49 35 3~'sL 3~ C?/. 50 ~ 33 18 25 17 2 5 1000 yy 3~ 9 8 81 rz 500 !1 Z- 113 ill B 250 1yZ. 148 136 100 11 `l 100 128 50 ~3 84 102 13/ sv 33 3Y 25 58 53 1000 lbp,s- 114 87 /~8- 1/9 ?6 500 /7ys-153 206 ;tr? t7$'./Y7 C 250 ~ Sz 131 173 /'31 /*Q~~ 100 8y•>91 88 a' ? 6r 'S2 5 0 S (- 50 62 33 ' 30 25 3!y 31 32 Strain . TA . 98; Spont.: 18 Control DMSO: 19

Table AVERAGE (HIS+ B2EVERTANTS/PLATE)'' TEMPERATURE (° C) PLATE ( l,tg ) TRAP I TRAP I I NEUTRALS 1 BASES 1 ACIDS 1 1000 50 50 31 77 43 500 46 48 36 69 36 200 250 48 44 40 .36 30 100 50 32' 33 39 26 100U 34 43 48' 33• 25 2'50 500 34 46 40 25. 28 250 40 48 42 23 32 100 50 51 45 1$ 37 1000 60 46 52 90 46 500 67 45 48 108 42 300 250 60 41 49 92 38 100 42 39 45 100 35 Strain TA.98; Spont:20 Control DMSO: 20 1Fractions of combined materials of Trap I and II.

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