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THE SUBJECTIVE AND ELECTROPHYSIOLOGICAL EFFECTS OF SMOKING CIGARETTES WITH CONSTANT TAR BUT VARYING NICOTINE LEVELS (PT)

-PROTOCOL P 0500/3203 930827 ,~c. "4.C'a PAGE 1 A clinical test is planned to investigate the subjective effects of smoking cigarettes with constant tar but varying nicotine levels. Electrophysiological responses will also be measured using the pattern reversal-evoked potential (PREP). The subjective responses to the cigarettes will be measured by means of a descriptive ballot. Using the ballot, the subjects will evaluate such factors as flavor, satisfaction, acceptability, and concentration. post-smoking EEG responses to a reversing (i.e., shifting) check- erboard pattern. Previous work (Gullotta and Shultz, 1982; Gullotta and Hayes, 1984) has indicated that a component of the PREP, the P1, is sensitive to nicotine as delivered in cigarette The PREP is used as a noninvasive technique involving the attach- ment of electrodes to the scalp and the recording of pre- and

PROTOCOL P 0500/3203 930827 PAGE 2 2 INTRODUCTION The sponsor of this study is attempting to optimize the ratio be- tween positive factors (e.g., taste quality, consumer satisfaction) and possible negative factors (e.g., toxicity). Considerable progress has been made in reducing the value of the denominator of this ratio. For example, the introduction of cigarette filters has substantially lessened the quantity of com- pounds with possible biological activity. Unfortunately, reducing the yalue of the denominator often has the negative effect of con- comitantly reducing taste quality and consumer acceptability. To improve overall quality, then, it would be necessary to find ~ methods to breakthis loop. Progress in this area has been dif- r---_ __.-`.--~-- - ficult for two reasons. First, until recently (Grubbs et al., 1991), it has not been possible to systematically and selectively modify tobacco constituents - a necessary prerequisite for con- ducting valid studies. Second, our ability to measure the results of this modifications has not progressed beyond the use of rela- tively primitive and notoriously unreliable descriptive ballots. n Recent advances in both tobacco technology and subjective measure- ment techniques now make it possible to conduct studies on cigarette taste and consumer acceptability in„a-scientifically sound manner. With respect to advances ~n tobacco technology, it has been demonstrated that tobacco qonjitituents can be selectively removed by using supercritical extraction methods. The subjective attributes of these constituents can then be systematically evailuated by comparing cigarettes made with extracted tobacco to cigarettes made from extracted tobacco blended in various propor- tions with normal tobacco. With respect to advances in subjective measurement techniques, studies conducted in our laboratory over the last several years have shown that the pattern-reversal evoked potential (PREP) can

•PROTOCOL P 0500/3203``930827 PAGE 3 ~. be used to obtain information about;cigarette taste and accept- waveformand that these changes correlate with subjective assess- ,t`~q.i".` ;. . :._ . , .. . :. produces p'redictable and'highly reproducible changes in the PREP abilityi'Specifically, we have demonstrated that cigarette smoking Cigarettes prepared from tobaccothat has been supercritically ex- ~tracted~.of~'.alkaloids indicate that.these compounds are significant contributors.to taste and satisfaction: The major tobacco alkaloid tributorthe subjective characteristic of cigarettes. Yet, is,'of oourse, nicotine, and it'is believed to be a prime con- surprisingly little is known aboutthe sensory characteristics of -. _: .. . ,........r nicotine.'.That is, few reports (Gullotta, unpublished report) ex- _.~? ;ist:where nicotine, and only nicotine,.has. been varied and its sensory:characteristics' evaluated. A scientifically sound inves.-_,} productawithout significant loss of acceptibility. amount.ofthis pharmacologically active substance in a consumer characteristic of cigarettes will allow the minimization of the ~. . ','i "y - . .. ,. . . . . . . . igation.,of the contribution of .nicotine on the subjective Thepurpose of the planned.investigation is to systematically in- have -beenprepared fromtobacco,,that-has been supercritically ex-. ': ' 'fi ` . . .. . . . . , . , - acceptability. The study will be conducted using cigarettes that -vestigate''-`the contribution of.nicotine to cigarette flavor and tracted`of;nicotine,blended in various proportions with normal. 'tobacco. Flavor and acceptability will be measured using the PREP, togetherwith traditional descriptive ballots. l1l,k'f A.p~OC, G..tZ SC. 04 M- YY ht g'Zb cY. ~ dca.0. Y ~ eC kt~fiC a.ti he risx associatea with tne participation in tni.s stuay is negii- , ;gible. The.outcome of this study will be of scientific merit in the"field of understanding the contribution of nicotine to cigarette taste,,flavor, acceptability, consumer satisfaction and • ability ..to, concentrate. The aquired knowledge will offer health benefits by facilitating the optimization' of the ratio between positive and possible negative factors of cigarettes. Therefore, N.

PROTOCOL P 0500/3203 930827 PAGE 4 we considered the performance of this study as very well jus- tified.

PROTOCOL P 0500/3203 930827 PAGE 5 3 DESIGN AND PROCEDURES --------------------- --------------------- 3.1 Research 3.1.1 Inclusion and exclusion criteria For the study, 30 healthy male volunteers with normal or corrected to normal vision will serve as subjects. All subjects will be recruited locally and will be paid for their participation. For inclusion in the study, the subjects must fulfill the following criteria: I (1) between 20 and`40 years of age (2) smoke at least 10 nonmenthol cigarettes per day for at least 2 years (3) be in good general health as determined by prior physical ex- amination including blood chemistry (4) agree to sign informed consent form __.(5)-'agree to sign a non-disclosure form (6) can be reached by telephone Participation in the study will not be permitted if the subjects meet any of the following exclusion criteria: (1) vision that is not normal or not corrected to normal (2) inability to completely understand informed consent (3) long-term medication (4) history of respiratory illness (5) history of neurological disorders (6) history of drug or alcohol abuse (7) concurrent participation in other clinical studies (8) staff of the institute and their relatives

PROTOCOL P 0500/3203 930827 PAGE 6 On testing days, subjects will be required to report to the in- stitute 2 hours prior to the beginning of the test. They will be required to abstain from smoking or ingesting any form of nicotine for 2 hours prior to testing. They will be required to abstain from ingesting caffeine in any form for 2 hours before the tests. Additionally, the subjects will be required to abstain from using alcohol, illegal drugs, drugs requiring a doctor's prescription or other medications that might produce drowsiness, excitation, or affect mental alertness for at least 8 hours prior to testing. Medications that might otherwise affect vision at times of testing are also prohibited. Subjects will be discontinued and replaced if they ask to withdraw from the study, meet any of the exclusion criteria, or have ad- verse reactions during the test session. Subjects will also be discontinued if they, on more than two occasions, fail to comply with any aspect of the experimental requirements. All records identifying subjects by name, will be locked in a secure location and will only be made available to the clinical study director, the attending physician and the experimenters. _.._ ._.._..~........_..._...._.,,.....~,._..._....,,....... s-v~ri3-l-be'~sYOpe~''Ty"disposed` of when no longer need'ed: All o~ records, such as data stored in computer data bases and hard copies will be coded. 3.1.2 Informed Consent (see Appendix 1) Subjects will read a detailed explanation of all the pertinent facts of the experiment. This will include:

PROTOCOL P 0500/3203 930827 PAGE 7 (1) data confidentiality, (2) what tobaccos are in the cigarettes, (3) what, if anything, has been added to the tobacco, (4) how nicotine was removed from the tobacco, (5) nicotine and tar deliveries, (6) what the PREP is and how it is recorded, (7) cleanliness and sterilization procedures. After the subjects have read the material, the attending physician will go over the important facts verbally to ascertain whether the subjects fully understood the information. Subjects will then be asked to sign an informed consent form agreeing to participate in the study. 3.1.3 Nondi$closure agreement ~ (see Appendix 2) 3.2 Ethical Principles The studies will adhere to the ethical guidelines for non- therapeutic biomedical research involving human subjects as set forth in the World Medical Association Declaration of Helsinki/Tokyo/Venice/Hongkong (1964, 1975, 1983, 1989) and those set forth by the council for International Organization of Medical Sciences (CIOMS) in the International Ethical Guidelines for Biomedical Research Involving Human Subjects (First Draft, January 1992).

•PROTOCOL P 0500/3203 930827 PAGE 8 3.3 Subiect Remuneration Subjects will be paid 50,-DM for the first test session. This amount will be increased by 10,-DM for each of the following ses- sions (max. 10). The subjects will be payed irrespective of whether the tests were successfully completed or not. The only ex- ceptions would be that subjects would not be paid for a test session if they failed to comply with the conditions specified in 3.1.1. 3.4 Subiect Insurance All subjects will be insured according to the legal requirements for the conducting of clinical studies (S40 AMG). 3.5 Cigarette Specifications (see also APPENDIX 3) -1 11 The cigarettes have been prepared using a American tobacco blend. A single lot of tobacco was divided into portions, one portion of which was extracted of nicotine using CO2 supercritical fluid ex- traction procedures (Grubbs et al., 1991). The extracteo, tobacco ~ was combined in varying proportions with unextracted tobcco. This resulted in cigarettes with constant tar (approx. 17 mg) but vary- ing levels of nicotine (0.12 to 1.56 mg/cig.). These tar and nicotine levels are well within the range of those found in cigarettes commercially available in Germany (see appendix 4). Nine cigarette types were produced with the following tar and nicotine levels:

PROTOCOL P 0500/3203 270893 PAGE 9 CIGARETTE CODE TAR NICOTINE (mg/cig.) (mg/cig.) AGH 17.0 0.12 AGI 16.8 0.19 AGJ 16.8 0.41 AGK 17.2 0.66 AGL 16.5 0.83 AGM 16.6 0.98 AGN 16.9 1.21 AGO 17.0 1.40 AGP 16.9 1.56 TABLE 1 TAR AND NICOTINE DELIVERIES OF TEST CIGARETTES Remarks: data provided by the client

PROTOCOL P 0500/3203 930827 PAGE 10 3.6 Toxicology As the test cigarettes correspond to commercially available cigarettes, no additional toxicological risks are associated with their smoking. Detailed information on the composition of the test cigarettes, as it is given in appendix 3, was deposited at the BGA Institut fur Arzneimittel and is registered under the number ... (see APPENDIX 5). 3.7 Safety of Experimental Cigarettes The cigarettes are made from normal American blend tobacco without any additional ingredients. Even erroneous mixtures of tobaccos will lead to cigarettes with compositions of ingredients within the normal range. The CO2 extraction process does not leave any residues and is well established and widely used also in food processing. The subjects will be continuously observed during and immediately after smoking. If adverse effects (e.g., perspiration, vertigo, nausea) are noticed, the test session will be halted and the study physician will be summoned immediately. 3.8 Treatment of Adverse Effects For adverse effects, treatment will be mainly symptomatic. For cases of emergency, oxygen supply, atropine, pressors, vasocon- strictors, and anticonvulsants will be held ready. In case of strong adverse effects the subject will remain under medical treatment and care until the effects will have disappeared.

.. . ~. . PROTOCOL P 0500/3203 930827 PAGE 11 3.9 Study Design A completely randomized factorial design will be employed where (on different occasions) each subject will smoke each of the cigarette types. Order of smoking will be randomized for each sub- ject using established randomization procedures. The study will be conducted double blind where neither the subjects nor the inves- tigators are informed as to which test cigarette is being smoked in a given test session. 3.10 Study Chronology Prior to initial testing, subjects will be trained in using the descriptive ballot and the controlled smoking procedure (7 puffs per cigarette, see Appendix 6). This will be accomplished by smok- ing 2 cigarettes which greatly differ in nicotine delivery. For each test session, subjects will be scheduled by telephone at least 24 h in advance. They will first be asked whether they have any illnesses or are taking any medications which could preclude testing. These illnesses and medications would include, but not be limited to, influenza, colds, visual disturbances, medicines that affect concentration or vision. When it is concluded that testing is possible, a date and time will be set and the subjects will be told to report to the in- stitute 2 h prior to testing to ensure that they refrain from nicotine and caffeine use. Once subjects report for testing, they will be asked about com- pliance to the deprivation requirements and once again asked about health or medications that would preclude testing on that day. Subjects will then be prepared for testing.

PROTOCOL P 0500/3203 930827 PAGE 12 3.11 Descriptive Ballot After smoking each test cigarette, subjects will be required to fill out a ballot, evaluating the cigarette on 7 variables (see sample ballot in Appendix 7). Each variable will be rated on a scale between 1 and 7. Four of the variables relate to cigarette <flavop while the other 3 evaluate satisfaction, acceptability, and concentration. 3,12. Pattern Reversal-Evoked Potentials 3.12.1 Instrumentation A 30-cm (diagonal measurement) video monitor (AAmazing Technologies Corp.) will be used to display the checkerboard pat- tern which will be used as the stimulus for PREP recording. The checkerboard pattern will be generated using a Neuroscan software program (Neurosoft, Inc.) designed for that purpose. PREPs will be. recorded using Grass Instruments A.C. amplifiers (model 12A5). Low frequency filters will be set at 1.0 Hz and high'-ji fi?'e"quency filters will be set at 1.0 KHz. Analogue to digital con- version will be accomplished using a NeurosGan. .12-Bit A/D converter (Neurosoft Corp.) at a digitization rate qf 1280 Hz and at a sample duration of 200 ms. The digitized PREP waveforms will be processed by a personal com- puter using a Neuroscan (Version 1.0) data acquisition system (Neurosoft Corp.). At the end of an experimental session, data will be transferped to optical disk (Storage Dimensions, Inc.) for off-line processing.

•f PROTOCOL P 0500/3203 930827 PAGE 13 3.12.2 PREP acquisition 9-mm gold disc electrodes will be positioned over scalp locations O1, 021 0Z, FP, and CZ (Jasper, 1958) and affixed with collodion. A 6th electrode will be affixed to the forehead and serve as ground. After the electrodes have been affixed, they will be ' a_. filled with a conducting solution (i.e., electrolyte). Electrode impedances will then be checked and the scalp under the electrodes abraded slightly, if necessary, to achieve impedances under 5 Ka. O1, 02 and OZ, and FP will serve as recording electrodes, while CZ will.serve as the reference electrode. The subjects will sit in a 40-m3 testing chamber, under dim il- lumination, 1.0 m in front of a video monitor. Subjects will be asked to focus on and count changes in color of a dot which will appear in the center of the screen at the beginning of the record- ing session. Once the session begins, a black and white checkerboard pattern with 10 checks down and 10 checks across will be displayed by the monitor. The dimension of each check of the display will be 2.3 cm2. The black and white checks will reverse positions at a rate of 2/s. Each reversal will trigger the data acquisition system to sample a 200 ms epoch of the EEG. One hundred and fifty epochs will be acquired in this fashion and will constitute the presmoking baseline. _ Subjects will then be required to smoke one of the cigarettes using a controlled smoking procedure (see APPENDIX 4) where number of puffs, interpuff interval and manner of puffing are specified. Immediately after extinguishing the cigarette, the subjects will once again focus on the video monitor and PREPs will be recorded in an identical manner as they were prior to smoking. Following the postsmoking PREP recording, subjects will rate the cigarettes using the descriptive ballot. The electrodes will then be removed from the subjects scalp using an acetone free collodion remover (Maridon Corporation) and the subjects will be free to

PROTOCOL P 0500/3203 930827 PAGE 14 l/~~'~-~~•~' - leave the laboratory. This chronology of events will occur until each of the subjects has smoked, on different occasions, all of the cigarette types used in the investigation. PREPs collected during a given test session will initially be stored on virtual disk. After that, the PREPs will be transferred to optical laser disk for long-term'storage. Upon retrieval from storage,, the waveforms will be inspected for (band pass, high pass = 1.0 Hz, low pass = 40 Hz). Following fil- artefacts (e.g., muscle co`ntaEJ5&tI'6^_n) and digitally filtered tering waveform baseline corrections will be performed and the responses will be averaged. Averaged responses will be txans.f.erred4 tp.aaa=J-_di-sk for waveform measurement. N2(130-150 ms.). Latency values will bederived for each peak by for each averaged waveform. The 4 peaks, together with their nor- mal latency ranges, are: Po(40-60 ms), N1(60-80 ms), P1(90-110ms), Four peak latencies and 3,peak to peak amplitudes will be obtained determining maximal amplitudes within the specified latency ranges for the peaks. Each peak to peak amplitude value will be derived by measuring the amplitude from the peak of one component to the peak of the adjacent component. The measured values will be evaluated statistically. Ballot: Single factor (cigarette)-repeated measures ANOVAs (Keppel, 1973) will be performed for each of the seven descriptors

PROTOCOL P 0500/3203 930827 PAGE 15 on the ballot. p-values of 0.05 or lower will be considered as statistically significant. Post-hoc tests (Duncan's Multiple Range) (Keppel, 1973) will be performed where appropriate. PREP: Each latency and amplitude measure will be analyzed by a separate 2-factor repeated measures analysis of variance (ANOVA). The within subject factors will be condition (pre- vs postsmoking) and treatment (cigarette), and the treatment x condition interac- tion. Factors will be considered statistically significant if they achieve a p-value of 0.05 or lower. Greenhouse-Geisser-corrected values will be used where appropriate to account for the non- independence of measures (Keppel, 1973). The ANOVA will be followed by post-hoc (Duncan's Multiple Range) tests where ap- propriate.

PROTOCOL P 0500/3203 930827 PAGE 16 4 SAFETY CONSIDERATIONS The testing chamber will be equipped with a window so that the subjects will be under continuous visual observation at all times during testing. In addition, the chamber will be equipped with a passive audio communications system so that the subjects will be in voice contact with the experimenters throughout the testing. An on-site physician will be called immediately if the subject re- quests medical assistance or if the experimenters suspect that such assistance is required. In addition, the subjects will be re- quired to be checked by the attending physician prior to leaving the Institute at the end of a test session. For good hygienic conditions, all electrodes and other implements that have come in contact with the subjects will be cleaned after use. Cleaning will consist of washing the electrodes and imple- ments in soap and water, followed by soaking in a disinfectant solution. The experimenters will wash their hands with disinfec- tant soap and water after each experiment. To insure electrical safety, the EEG equippment is certified by the Technischer Oberwachungsverein Rheinland, Koln (TOV).

PROTOCOL P 0500/3203 270893 PAGE 17 5 STUDY ORGANIZATION AND RESPONSIBILITIES 5.1 Study Organization The study shall be performed at INBIFO Institut fRr biologische Forschung, FuggerstraRe 3, D-51149 Koln. Following units at INBIFO are involved in this study: Sensory Physiology (SP) and Information Systems (IS). The performance of the methods is in the responsibility of the respective team manager. Date of assays: ... Name and Address of the Clinical Study Director: 5.2 Responsibilities Attending Physician: ............. ........................... Date Dr.med. Dr.rer.nat. K. von Holt Physician and Physicist (Arzt und Diplomphysiker) Sensory Physiology: ............. ........................... Date Dr. F.P. Gullotta Ph.D., Experimental Psychologist

PROTOCOL P 0500/3203 270893 PAGE 18 Information Systems: ............. .......................... Date Dr.rer.nat. W. Gomm Mathematician (Diplommathematiker) General Manager; Dr.rer.nat.•W.•Reininghaus• Date ............. Physicist (Diplomphysiker)

PROTOCOL P 0500/3203 270893 PAGE 19 6 STORAGE OF MATERIALS AND RECORDS Records will be stored in our archives for at least 30 years after delivery of the final report to the client (SOP QA 10). Samples of the cigarettes will be stored in our archives as long as their quality under state-of-the-art storage conditions allows further evaluation, but not longer than 30 years (SOP QA 11). All records and samples can be claimed by the client.

PROTOCOL P 0500/3203 270893 7 REFERENCES Grubbs, H.J., Prasad, R., Howell, T.M., Process for removal of basic material, U.S. Patent 5, 018, 540 (1991) Gullotta, F.P., Hayes, C.S., unpublished report (1984) Gullotta, F.P., Shultz, C.J., unpublished report (1982) PAGE 20 Jasper, H.H., The ten-twenty electrode system of the International Federation, Electroencephalography and Clinical Neurophysiology 10: 371-375 (1958) Keppel, J., Design and analysis: A researchers handbook, Englewood Cliffs, N.J.: Prentice-Hall, Inc., 1973 END OF PROTOCOL