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TITLE: ANNUAL REPORT ACTIVITY NO. 189 No. Collection, Separation, and Elucidation of the Components of Cigarette Smoke and Cigarette Smoke Condensate Part I. Chemical Characterization of Experimental Cigarette Smokes Part II. Inhalation Bioassay Monitoring and Support DOE (ERDA/AEC) - NIf€(NCI) - 40-485-74 Person-in-Charge: M. R. Guerin (25~') Principal Investigator: R. A. Jenkins (I00'i~o`) Support Personnel: 310 man-hours per week Oak Ridge National Laboratory* P. 0. Box X Oak Ridge, Tennessee 37830 June 30, 1978 *Operated by Union Carbide Corporation for the U.S. Department of Energy. ~, uCFt•4414a PAGE (13 a.731

DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE PUBLIC HEALTN SERVICE NATIONAL I&ISTITUTES OF NEALFIi PROJECT OBJECTIVE AND PROGRESS REPORT SECTION I - FILL IN TtIIS INFORMATION WITH EVERY SUBA4ITTAL PROJECT (ChecFk one) ,~ Interagency Agreement C Research Contracts C P.L. 480 Program CONTRACT, AGREEMENT OR P.L. 480 NUMBER DOE(ERDA/AEC)-NTW(NCI) 40-485-74 NAME AND ADDRESS OF AGEId.Y RECEIVING NIH SUPPORT Oak Ridge National Laboratory P. 0. Box X Oak Ridge, TN 37830 INVESTIGATOR INFORMATION (Co4plete for new agreementa and contracts or for subsequent changes - otherwise leave blank) (Not applicable to P.L. 480 progracs) PROJECT C:APIE (Last, Ylrst.' I19ddle Initial) SOCIAL utCURITY NO. i I ;•' E fxrs. perviceetce -~ -- - in whode hra.) - OIRECTOR - (umt t to 1) Oueri n, Mi chael R. 330-34-1228 25 . Jenkins, Roger A. 373-50-5944 100 OTHER - SENIOR Michael P Maskarinec 181-42-6229 75 SCIENTISTS . , 2. (If applicable) 3. Caton, John E. 183-32-8183 25 INSTITUTIONS* IdAJOk CuI(iPOPdEFdT 7;ac'r}oo1 of Medicine. t DEPARTMENT LUbdLUislGn of i,aur Gomponeni Llstec OF HIGHER Dentistry, etc.1 Department of Chemistryr6iology, etc.; EDUCATICN (Contracts only) *if contractor is an institution of higher education, designate major component and department in which project director Is assigned. SUMMARY OF OBJECTIVES AND WORK SCOPE (100 words or less. Must be completed and submitted for each new project. For project renewals, c.=plete only if changed significantly from prior reporting period.) This is a renewal proposal. . NIH-1688 (Prescribed by NIH Manual Issuances 1165 and 6000-3.50.60) Rev. 9-72

SECTION II - MUST BE COMPLETED AND SUBMITTED AT END OF EACH PROJECT PERIOD SALIENT RESULTS TO DATE (200 words or less) Comparison of tar, nicotine, C0, C02 and HCN deliveries of three dog inhalation exposure devices has been made. The smokes of seventy-six brands of foreign and domestic commercial cigarettes have been analyzed for deliveries of selected con- stituents. Based on the concentrations of only a few selected constituents•in ciga- rette smoke condensate, a mathematical model for predicting results of long term mouse skin painting tumorigenicity bioassay has been developed. Chronic cigarette smoke inhalation studies have been monitored to estimate factors which control animal smoke dose and dose variabi,iity. Radiolabelled tracer studies to determine clearance of nicotine and its metabolites from dogs have been performed. A reliable, simple method for the quantitative determination of urinary nicotine and cotinine has been developed. Construction of an experimental assembly has indicated that the concept of a particulate/flow sensor system as a non-invasive dosimeter is feasible for dog inhalation exposures. w ...~.,~ ARTICLES + List the last three papers (do not include abstracts or reviews) published or accepted PUBLICATIONS, for publication based on work done on the project during prior reporting period. If none, write "none". List reports filed with the Federal Clearinghouse for Scientific Information REPORTS, ETC. reporting the work done during the same period. If none, write "none". AUTHOR(3) TITLE OF ARTICLE NAME OF JOURNAL DATE OR PERIODICAL PUBLISHED R, t~, M. P. Maskarinec A Novel Method for the Iso•la- , Narvey, and J. E. Cato tion and Quantitative Analysis Analytical of Nicotine and Cotinine in Toxicology In press i Fluids d M W H G i t R Influence of Tobacco Type of Recent Advances in . an . . r es . i G Smoke Composition Tobacco Sciences 1977 uer n 3, 121 1977 S. G. Zeldes and A. D. Trapping and Determination of I mnalytical Horton Labile Compounds in the Gas Chemistr y 1978 Phase of Cigarette Smoke , 50, 779 1978)- INVENTION REPORTS MADE DURING PRIOR REPORTING PERIOD INVENTORIS NAME (Last first, initial) TITLE OF INVENTION (100 spaces or less) DATE OF REPORT NONE > NIH-1688 (Back) RfY. g-7$ •

i TABLE OF CONTENTS Page INTRODUCTI®N-------------------------------------------------------- 1 PART I. ANALYSIS OF CIGARETTE SMOKE AND SMOKE CONDENSATE INTRODUCTION------------------------------------------------------ 3 I. Chemical Characterization of Tobacco Smoke from Beagle Dog In- halation Exposure Systems----------- -------------------------- - 9 II. Final Data - Series IV Cigarette Smoke and Condensate Chemical Analyses------------------------------------------------------ 21 III. Chemical Analysis of Smokes of Foreign and Domestic Commercial Cigarettes---------------------------------------------------- 33 IV. SWRI Baboon Study Smoking Extremes Experiment- -------------- 85 V. Statistical Modeling of Histopathological Probabilities------- 93 PART II. BIOASSAY MONITORING AND SUPPORT INTRODUCTION------------------------------------------------------ 99 I. Monitoring of the Chronic Inhalation Exposures--- --------- 107 II. Instrumental Approaches to Bioassay Monitoring---------------- 289 III. Trapping and Determination of Organic Gas Phase Constituents of Cigarette Smoke-------------------------------------------- 301 IV. Determination of Nitric Oxide and Nitrogen Dioxide in Cigarette Smoke by Chemiluminescent Analysis---------------------------- 307 PART III. DOSIMETRY AND BIOIMPACT INTRODUCTION------------------------------------------------------- 311 I. BRL-ORNL Collaborative Smoke Particulate Deposition Experiment 313 II. L4C Tracer Studies to Develop Sampling Protocols for Quantita- tive Nicotine Dosimetry Following Smoke Exposure-------------- 315 III. Isolation and Quantitative Analysis of Nicotine and Cotinine in Physiological Fluids---------- 7 --- ------------------------- 323 IV. Physiological Fluids Studies: Mutagenicity and Profiling----- 331

1 INTRODUCTION Since 1968, Oak Ridge National Laboratory has been acting as an analytical chemistry facility for the National Cancer Institute's Smoking and Health Program. As NCI has pursued the goal of identifying the charac= teristics of a less hazardous cigarette, smoke and smoke condensates have been bioassayed in order to assess their biological impact. It has been the primary mission of ORNL's NCI project to provide for the quantitative chemical definition of the material being bioassayed. During the mouse skin painting bioassays associated with the Series I-IV of experimental cigarettes, this role has consisted mainly of the routine determination of a large number of NCI-specified constituents in the condensate being tested for mouse skin carcinogenesis. As NCI has shifted toward the more complex and sophisticated inhalation bioassays employing various animal models, ORNL's role has been to provide definition of the inhalation exposure atmospheres both under tightly controlled conditions at ORNL and at the actual sites where the inhalation studies are conducted. Because of ORNL's expertise in smoke chemistry, S&HP management has called upon our facility to respond rapidly to a wide variety of special requests. These special requests have involved such diverse studies as troubleshooting inhalation exposure equipment, evaluating the efficacy of benzo(a)pyrene antibodies in cigarette filters, and analysis of the smoke of commercial cigarettes from foreign countries. This latter function has become increasingly important, as NCI seeks to understand the world-wide epidemiology of tobacco-smoke-associated cancer. In addition, ORNL has begun, a small but significant program to develop methodologies and assess the utility of certain biological tests and bio- chemical parameters in determining the biological impact of cigarette smoke on experimental animals.

2 The work described here refers to progress made since approximately May 1, 1977, in the areas of the analytical chemistry of cigarette smoke and smoke condensate, bioassay monitoring and support, and tobacco smoke dosimetry and biochemical impact on experimental animals.

3 PART I. ANALYSIS OF CIGARETTE SMOKE AND SMOKE CONDENSATE INTRODUCTION Efforts during the past year have been characterized by a shift in emphasis away form the performance of a large number of protocol analyses on smoke condensate generated by other NCI contractors and a shift toward development of procedures for meaningful sampling and analysis of inhalation exposure atmospheres generated by smoke exposure systems at ORNL. In addi- tion, a proportionately greater fraction of our work has been in direct response to special requests by S&HP management. Most of this effort has directed toward the chemical characterization of smokes of foreign and domestic commercial cigarettes. In accomplishing this work, 3565 "deter- minations" which yielded 1543 "analyses" (a final, reported result) were performed. Analyses performed on samples returned from working site visits are not in these figures. Because of the "sensitivity" of smoke chemistry results reported by ORNL, repeated analyses of reference cigarettes plays an important role in our quality assurance program. Table I-lA lists data for the periodic measurement over the last two years of Kentucky Reference Cigarette smoke particulate phase constituents. The data generated in recent months are consistent with previous results. Overall reproducibility is better than ± 5 percent; thus, the smoking and analytical procedures are performing very reliably. Periodic data for the determination of Kentucky Reference Cigarette smoke gas phase and whole smoke constituents are shown in Tables • I-lB and I-1C. The recent data are again consistent with previous results, although the overall reproducibility is slightly less than for particulate phase constituents. However, the better than ± 10 percent relative standard deviations for the two year overall data are quite acceptable and are an

4 TABLE I-1A Periodic Measurements Kentucky Reference Cigarette (1R1) Deliveries Particulate Phase Constituents Date _ TPMa (_ mO Water (mg) Nicotine (mg) Tar (mg) 5/20/76 44.57 5.22 2.66 36.69 8/12/76 44.14 4.94 2.72 36.48 9/20/76 46.90 - - - 9/20/76 45.80 - - - 9/20/76 45.50 - - - 12/02/76 43.18 5.04 2.50 35.64 8/08/77 44.05 5.60 2.51 35.94 8/24/77 46.79 5.85 2.68 38.26 10/11/77 45.67 4.58 2.52 38.57 12/19/77 43.19 4.84 2.79 35.56 1/04/78 44.82 4.33 2.66 37.83 1/18/78 43.93 2.71 2.50 37.72 2/02/78 44.68 4.32 2.57 37.79 3/02/78 44.30 4.15 2.52 37.63 3/15/78 43.46 3.28 2.62 37.56 4/21/78 43.20 3.31 2.30 37.59 5/16/78 43.30 3.72 2.60 36.98 5/25/78 43.75 3.11 2.76 37.88 Average 44.51 4.40 2.59 37.21 Std. Dev. 1.19 0.86 0.13 0.94 Rel. Std. Dev. 2.68 19.46 4.85 2.54 aTotal particulate matter

5 TABLE I-1B Periodic Measurements Kentucky Reference Cigarette (1R1) Deliveries Whole Smoke and Gas Phase Constituents CO CO HCNa NOX Date (n~1 ) m1~ Date (u97 Date ( as hl0 ) _ _ ~sg , 5/26/76 18.11 37.17 9/15/76 434.9 11/02/76 270.7 7/26/76 16.57 35.11 11/24/76 415.1 11/24/76 264.4 9/27/76 16.20 34.00 11/30/76 433.9 11/23/76 307.8 9/27/76 15.60 34.50 12/09/76 447.6 11/24/76 255.1 9/28/76 15.90 32.30 1/07/77 417.3 12/08/76 294.4 8/08/77 19.10 41.78 1/17/77 393.8 12/29/76 245.5 8/24/77 18.79 38.64 2/24/77 428.8 1/06/77 280.7 10/11/77 17.98 33.63 2/14/77 452.3 3/14/77 268.9 12/19/77 17.40 38.65 9/28/77 396.0 6/29/77 266.3 ~ 1/04/78 19.49 41.61 8/19/77 403.3 7/08/77 272.3 1/18/78 18.62 39.80 9/08/77 415.8 9/27/77 281.0 2/02/78 18.72 40.52 10/26/77 384.5 2/08/78 288.4 3/02/78 18.13 40.71 2/10/77 385.4 3/12/78 241.5 3/15/78 19.21 41.78 3/29/78 398.9 4/21/78 17.13 38.80 Average 17.80 37.93 414.8 272.1 Std. Dev. 1.26 3.26 22.2 18.8 Rel. Std. Dev. 7.11 8.60 5.36 6.90 aWhole smoke, other constituents are gas phase.

6 TABLE I-1C Periodic Measurements Kentucky Reference Cigarette (1R1) Deliveries Organic Gas Phase Constituents Acetaldehyde Date (uO Acrolein tu9 } Isoprene (u9} Formaldehyde (uO 12/06/76 1060 _ 110 575 12/13/76 1027 108 525 12/29/76 1037 109 565 1/24/77 1002 114 4/12/77 1055 109 576 10/07/76 119 9/27/76 116 10/04/76 117 9/16/77 114 9/22/77 125 4/19/77 30.8 4/20/77 4/20/77 28.5 4/20/77 5/06/77 36.7 5/16/77 34.7 6/06/77 28.2 6/14/77 28.2 6/14/77 28.9 6/20/77 29.2 12/12/77 121 31.4 1/30/78 30.7 4/03/78 1020 122 580 Average 1034 116 564 30.7 Std. Dev. 21.9 5.5 22.6 2,9 Rel. Std. Dev. 2.11 4.77 4.0 9.41