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INTRODUCTION, OVERVIEW, AND CONCLUSIONS DEVEIAPMENT AND ORGANIZATION OF THE REPORT This report from the Surgeon General's Advisory Committee on the Health Consequences of Using Smokeless Tobacco represents the first comprehensive assessment of the biomedical and behavioral literature describing experimental and human evidence on the health consequences of using smokeless'tobacco. The content of this report is the work of numerous erperts within the Department of Health and Human Services as well as distinguished scientists outside the organization. Each chapter of the report was prepared based on manuscripts written by scientists who are recognized for their understanding of the specific content areas. Manuscripts were subjected to extensive peer review by a large number of experts in the specific areas of interest. The report includes a "Preface" that presents the essence of the entire re- port and an "Introductioa, Overview, and Conclusions." The body of the report consists of the following four chapters: • Chapter 1-Prevalence and Trends of Smokeless Tobacco Use in the United States • Chapter 2-Carcinogenesis Associated With Smokeless Tobacco Use Chapter 3--Noacancerous Oral Health Effects Associated With Smokeless Tobacco Use • Chapter 4-Nicotine Exposure: Pharmacokinetics, Addiction, and Other Physiologic Effects HISTORICAL PERSPECTIVE The use of smokeless tobacco is a worldwide practice with numerous vari- ations in the nature of the product used as well as in the customs associated with its use. In the United States, smokeless tobacco consists of chewing tobacco and snuff. The predominant mode of use of these nonsmoked tobaccos is oral, although they may be placed in or inhaled into the nasal cavity. Tobacco sniffing, however, has been and remains a rare practice in the United States. Smokeless tobacco was used in the United States in the early 1600's when snuff made its way to the Jamestown Colony in Virginia through the efforts of John Rolfe in 1611 (1). Evidence of tobacco chewing, however, was not found until a century later in 1704 (2). The use of tobacco, including smokeless tobacco, has been controversial since its introduction. In the past, tobacco use was considered by some as beneficial. As early as 3500 B.C., there are indications that tobacco was an article of established value to the inhabitants of Mexico and Peru. It 2581258014 XV

appears that people who frequently lacked sufficient food alleviated their hunger pains by chewing tobacco (3). Smokeless tobacco was also thought to have several medicinal uses. Among Native Americans, for example, chewing tobacco was used to alleviate toothaches, disinfect cuts, and relieve the effects of snake, spider, aad insect bites (4). Moreover, during the 19th and early 20th centuries in America, dental snuff was advertised to relieve toothache pain; to cure neuralgia, bleeding gums, and scurvy; and to pres-erve and whiten teeth and prevent decay (1). _ On the other hand, the history of tobacco use has had numerous adversaries, including the following (1): • In 1590 in Japan, tobacco was prohibited. Users lost their property and were jailed. • King James VI of Scotland in the early 1600's was a strong antismoking advocate who increased taxes on tobacco 4,000 percent in an attempt to reduce the quantity imported to England. • In 1633, the Sultan Murad IV of Turkey made any use of tobacco a capital offense, punishable by death from hanging, beheading, or starvation. He maintained that tobacco caused infertility and reduced the fighting capabilities of his soldiers. • The Russian Czar Michael Fedorovich, the first Romanov (1613-1645), prohibited the sale of tobacco, stating that users would be subjected to physical punishment and that persistent users would be killed. • A Chinese law in 1683 threatened that anyone possessing tobacco would be beheaded. • During the mid-1600's, Pope Urban VIII banned the use of snuff in churches, and Pope Innocent X attacked its use by priests in the Catholic Church. • Other religious groups also banned snuff use: John Wesley, the founder of Methodism, attacked its use in Ireland; the Mormons, Seventh-Day Adventists, Parsees and Sikhs of;India, Buddhist monks of Korea, members of the Tsai Li sect of China, and some Ethiopian Christian sects forbade the use of tobacco. • Frederick the Great, King of Prussia, prevented his mother, the Dowager Queen of Prussia, from using snuff at his coronation in 1790. • Louis XV, ruler of France from 1723-1774, banned snuff use from the Court of France. Scientific observations concerning the health effects of smokeless tobacco use were first noted in 1761 by John Hill, a London physician and botanist who reported five cases of polypuses, a"swelling in the nostril that was hard, black and adherent with the symptoms of an open cancer" (5). He concluded that nasal cancer could develop as a consequence of tobacco snuff use (sniffing). 2501250015 a xvi

Evidence that suggested a possible association between smokeless tobacco use and oral conditions in North America and Europe was not reported until 1915 when Abbe identified several tobacco chewers among a series of oral cancer patients and commented that smokeless tobacco use may be a risk factor for this cancer (6). In the late 1930's, Ahblom observed in Sweden that more patients with buccal, gingival, and "mandibular" cancers than with other cancers reported the use of snuff or chewing tobacco (7). In the United States, case reports of oral cancer among users of snuff or ,chewing tobacco appeared in the early 1940's (8). The first epidemiologic study of smokeless-tobacco was not conducted until the early 1950's (9). Since that time, several scientists have described a pattern of increased risk of oral cancer among smokeless tobacco users. Investigations of other possible health effects of smokeless tobacco use (e.g., noncancerous oral effects, addiction, and other physiologic consequences) are more recent subjects of scientific inquiry that have been undertaken pri- marily in the past two decades. A brief review of the health consequences of smokeless tobacco was presented in the 1979 Surgeon General's report on smoking and health (10). Since that review, the results of additional studies addressing the role of smokeless tobacco in health have become available and thus provide the basis of this current comprehensive review. REVIEW METHODS For the purpose of evaluating the scientific evidence to be included in this report, the Advisory Committee called upon the same criteria to determine causality as have been used for a number of Surgeon General's reports on smok- ing for the past two decades. The following criteria were used as the primary guidelines for assessing whether any associations between smokeless tobacco use and each of the disease areas or health condition's under examination were likely to be causal in aature: • Consistency of the association-similar observations by multiple investigators in different locations and situations, at different times, and using different methods of study. • Strength of the association-high ratio of disease rate for the population exposed to the suspected risk factor compared to the population unexposed to the risk factor. • Specificity of the association-associations with the exposure exist for a specific or limited set of diseases, and associations with the disease exist for a specific or limited set of exposures. • Temporal relationship of the association-exposure tq the suspected etiologic factor precedes the disease. • Coherence of the association-epidemiologic observations are conso- nant with all else that is known about the disease. 2501250016 XVil

In addition to these criteria, the general principles employed by the Inter- national Agency for Research on Cancer (IARC)* in evaluating the carcinogenic risk of chemicals or complex mixtures (table 1) were used as needed to supple- ment the primary causation criteria (11). OVERVIEW The use of smokeless tobacco products in the United States was widespread until the end of the 19th century. With the advent of antispitting laws, loss of social acceptability, and increased popularity of cigarette smoking, its use declined rapidly in this century. However, recent national data indicate a resurgence in smokeless tobacco habits with more than 12 million persons - estimated as users of some form of smokeless tobacco in 1985. An upward trend in use is emerging, particularly among young males. Given the evidence that smokeless tobacco is regaining popularity, seri- ous questions have been raised about its adverse health effects. Most notably, this behavior has been linked to cancer, specifically, oral cancer. Analytic epidemiologic studies now indicate that the use of oral snuff increases the risk of oral cancer several fold and that among long-term snuff dippers the excess risk of cancers of the cheek and gum may reach nearly fiftyfold. This conclusion is consistent with the judgment of a recent working group of the IARC, which assessed the carcinogenic risk associated with tobacco habits other than smoking (11). The conclusion that smokeless tobacco causes cancer results from several lines of evidence: the presence of high levels of carcinogens in smokeless tobacco; the metabolic conversion of products of smokeless tobacco into geno- toxic agents; the consistency of the oral cancer-smokeless tobacco association across epidemiologic investigations conducted in diverse locations; the trend in increasing oral cancer risk with duration of exposure; the strength of the association with oral cancer; and the occurrence of the highest risks for cancers at the anatomic sites where the tobaccoo exposures are the greatest. In addition, a number of clinical observations and studies show an asso- ciation between smokeless tobacco use and some noncancerous and precancerous oral health conditions. The development of a portion of oral leukoplakias in both teenage and adult users can be attributed to the use of smokeless tobacco. The risk of developing these leukoplakic lesions increases with increased ex- posure, and a number of studies now suggest that some snuff-iaduced leukoplakias can undergo transformation to dysplasia and further to carcinoma. The evidence concerning the adverse health effects of smokeless tobacco use on other oral soft and hard tissues is only suggestive at this time. *The IARC was established in 1965 by the World Health Assembly as an indepen- dently financed organization within the framework of the World Iieaith Orga- nization. It conducts a program of research concentrating particularly on the epidemiology of cancer and the study of potential carcinogens in the human environment. 2501259017 t , xviii

The magnitude of blood nicotine levels resulting from smokeless tobacco use has been shown to be similar to that from cigarette smoking. Therefore, the nicotine-related health consequences of smoking would also be expected to result from smokeless tobacco use. Given the nicotine content of smokeless tobacco, the users ability to sustain elevated blood levels of nicotine, and the well-established data implicating nicotine as an addictive substance, it is reasonable to expect that smokeless tobacco is capable of producing nicotine addiction in users. There is also some suggestive evidence that nicotine may play a contribu- tory or supportive role in the development of coronary artery and periphera]i vascular disease, hypertension, peptic ulcer disease, and fetal mortality and morbidity. . The conclusions in this report on the relationship between smokeless to- bacco use and cancer, noncancerous and precancerous oral conditions, and addiction and dependence are substantially in agreement with those published at a recent National Institutes of Health (NIH) Consensus Development'Confer- ence on the Health Implications of Smokeless Tobacco Use (12). CONCLUSIONS Prevalence and Trends of Smokeless Tobacco Use in the United States 1. Recent national data indicate that over 12 million persons used some form of smokeless tobacco (chewing tobacco and snuff) in 1985 and that approxi- mately 6 million used smokeless tobacco weekly or more often. Use is increasing, particularly among young males. 2. The highest rates of use are seen among teenage and young adult males. A recent national survey indicates that 16 percent of males between 12 and 25 years of age have used some form of smokeless tobacco within the past year and that from one-third to one-half of these used smokeless tobacco at least once a week. Use by females of all ages is consistently le'ss than that of males; about 2 percent have used smokeless tobacco in the last year. 3. State and local studies corroborate the national survey findings. The prevalence of smokeless tobacco use by youth and young adults varies widely by region, but use is not limited to a single region. In several parts of the country, as many as 25 to 35 percent of adolescent males have indicated current use of smokeless tobacco. Carcinogenesis Associated With Smokeless Tobacco Use 1. The scientific evidence is strong that the use of smokeless tobacco can cause cancer in humans. The association between smokeless tobacco use and cancer is strongest for cancers of the oral cavity. 2. Oral cancer has been shown to occur several times more frequently among snuff dippers than among nontobacco users, and the excess risk of cancers of the cheek and gum may reach nearly fiftyfold among long-term snuff users. 2501258018 XLX

3. Some investigations suggest that the use of chewing tobacco also may increase the risk of oral cancer. 4. Evidence for an association between smokeless tobacco use and cancers outside of the oral cavity in humans is sparse. Some investigations suggest that smokeless tobacco users may face increased risks of tumors of the upper aerodigestive tract, but results are currently inconclusive. 5. Experimental investigations have revealed potent carcinogens in snuff and chewing tobacco. These include nitrosamines, polycyclic aromatic hydro- carbons, and radiation-emitting polonium. The tobacco-specific nitrosamines N-aitrosoaornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone, have been detected in smokeless tobacco at levels 100 times higher than the regulated levels of other nitrosamines found in bacon, beer, and other foods. Animals exposed to these tobacco-specific nitrosamines, at levels approximating those thought to be accumulated during a human life- time by daily smokeless tobacco users, have developed an excess of a variety of tumors. The nitrosamines can be metabolized by target tissues to compounds that can modify cellular genetic material. 6. Bioassays exposing animals to smokeless tobacco, however, have generally shown little or no increased tumor production, although some bioassays suggest that snuff may cause oral tumors when tested in animals that are infected with herpes simplex virus. Noncancerous and Precancerous Oral Health Effects Associated With Smokeless Tobacco Use 1. Smokeless tobacco use is responsible for the development of a portion of oral leukoplakias in both teenage and adult users. The degree to which the use of smokeless tobacco affects the oral hard and soft tissues is variable depending on the site of action, type of smokeless tobacco pro- duct used, frequency and duration of use, predisposing factors, cofactors (such as smoking or concomitant gingival disease), and other factors not yet determined. 2. Dose response effects have been noted by a number of investigators. Longer use of smokeless tobacco results in a higher prevalence of leukoplakic lesions. Oral leukoplakias are commonly found at the site of tobacco placement. 3. Some snuff-induced oral leukoplakic lesions have been noted upon continued smokeless tobacco use to undergo transformation to a dysplastic state. A portion of these dysplastic lesions can further develop into carcinomas of either a verrucous or squamous cell variety. 4. Recent studies of the effects of smokeless tobacco use on gingival and periodontal tissues have resulted in equivocal findings. While gingival recession is a common outcome from use, gingivitis may or may not occur. Because longitudinal data are not available, the role of smokeless tobacco in the development and progression of gingivitis or periodontis has not been confirmed. 2501258019 XX

5. The evidence concerning the effects of smokeless tobacco use on the salivary glands is inconclusive. 6. Negative health effects on the teeth from smokeless tobacco use are suspected but unconfirmed. Present evidence, albeit sparse, suggests that the combination of smokeless tobacco use in individuals with existing gingivitis may increase the prevalence of dental caries compared to nonusers without concomitant gingivitis. Reports of tooth abrasion or staining have not been substantiated through controlled studies; only case reports are available. Nicotine Exposure: Pharmacokinetics, Addiction, and Other Physiologic Effects 1. The use of smokeless tobacco products can lead to nicotine dependence or addiction. 2. An examination of the pharmacokinetics of nicotine (i.e., nicotine absorp- tion, distribution, and elimination) resulting from smoking and smokeless tobacco use indicates that the magnitude of nicotine exposure is similar for both. 3. Despite the complexities of tobacco smoke self-administration, systematic analysis has confirmed that the resulting addiction is similar to that produced and maintained by other addictive drugs in both humans and animals. Animals can learn to discriminate nicotine from other substances because of its effects on the central nervous system. These effects are related to the dose and rate of administration, as is also the case with other drugs of abuse. 4. It has been shown that nicotine functions as a reinforcer under a variety of conditions. It has been confirmed that nicotine can function in all of the capacities that characterize a drug with a liability to widespread abuse. Additionally, as is the case with most other drugs of abuse, nicotine produces effects in the +iser that are considered desirable to the user. These effects are caused by the nicotine and not simply by the vehicle of delivery (tobacco or tobacco smoke). 5. Nicotine is. similar on all critical measures to prototypic drugs of abuse such as morphine and cocaine. The methods and criteria used to establish these similarities are identical to those used for other drugs suspected of having the potential to produce abuse and physiologic dependence. Specifically, nicotine is psychoactive, producing transient dose-related changes in mood and feeling.. It is a euphoriant that produces dose-related increases in scores on standard measures of euphoria. It is a reinforcer (or reward) in both human and animal intravenous self-administration paradigms, functioning as do other drugs of abuse. Additionally, nicotine through smoking produces the same effects, and it causes neuroadaptation leading to tolerance and physiologic dependence. Taken together, these results confirm the hypothesis that the role of nicotine in the compulsive use of tobacco is the same as the role of morphine in the compulsive use of opium derivatives or of-cocaine in the compulsive use of coca derivatives. 2501250020 XXL

6. The evidence that smokeless tobacco is addicting includes the pharma- cologic role of nicotine dose in regulating tobacco intake; the common- alities between nicotine and other prototypic dependence-producing sub- stances; the abuse liability and dependence potentia]l of nicotine; and the direct, albeit limited at present, evidence that orally delivered nicotine retains the characteristics of an addictive drug. 7. Several other characteristics of tobacco products in general,'including smokeless tobacco, may function to enhance further the number of persons who are afflicted by nicotine dependence: nicotine-delivering products are widely available and relatively inexpensive; and the self-administra- tion of such products is legal, relatively well tolerated by society, and produces minimal disruption to cognitive and behavioral performance. Nicotine produces a variety of individual-specific therapeutic actions such as mood and performance enhancement; and the brief effects Qf nico- tine ensure that conditioning occurs, because the behavior is associated with numerous concomitant environmental stimuli. 8. All commonly marketed-and consumed smokeless tobacco products contain substantial quantities of nicotine. The nicotine is delivered to the central nervous system in addicting quantities when used in the fashion that each form is commonly used (or as recommended in smokeless tobacco marketing campaigns). 9. Since the exposure to nicotine from smokeless tobacco is similar in magnitude to nicotine exposure from cigarette smoking, the health consequences of smoking that are caused by nicotine also would be expected to be hazards of smokeless tobacco use. Areas of particular concern in which nicotine may play a contributory or supportive role in the pathogenesis of disease include coronary artery and peripheral vascular disease, hypertension, peptic ulcer disease, and fetal mortality and morbidity. REFERENCES 1. Christen, A.G., Swanson, B.Z., Glover, E.D., and Henderson, A.H. Smokeless tobacco: The folklore and social history of snuffing, sneezing, dipping, and chewing. J. Am. Dent. Assoc. 105: 821-829, 1982. 2. Gottsegen, J.J. Tobacco. A Study of Its Consumption in the United States. New York, Pitman, 1940, p. 3. 3. Voges, E. The pleasures of tobacco-How it all began and the whole story. Tobacco J. Int. 1: 80-82, 1984. 4. Axton, W.F. Tobacco and Kentucky. Lexington, University Press of Kentucky, 1975, pp. 8, 25, 58-59. 5. Redmond, D.E. Tobacco and cancer: The first clinical report, 1761. N. Engl. J. Med. 282: 18-23, 1970. 6. Abbe, R. Cancer of the mouth. New York Medical Journal 102: 1-2, 1915. 2501258021 Xxll

7. Ahblom, H.E. Predisposing factors for epitheliomas of the oral cavity, larynx, pharynx, and esophagus. Acta Radiol. 18: 163-185, 1937 (in Swedish). 8. Friedell, H.L., and Rosenthal, L.M. The etiologic role of chewing tobacco in cancer of the mouth. JAMA 116: 2130-2135, 1941. 9. Moore, G.E., Bissinger, L.L., and Proehl, E.C. Tobacco and intraoral cancer. Surg. Forum 3: 685-688, 1952. - 10. U.S. Public Health Service. Smoking and Health. A Report of the Surgeon General. Department of Health, Education, and Welfare, Public Health Service, Office of the Assistant Secretary for Health, Office on Smoking and Health (DHEW Publication No. PHS 79-50066). Washington, D.C., U.S. Govt. Printing Office, 1979, pp. 13-38 to 13-41. 11. International Agency for Research on Cancer. Monographs on the evaluation of the carcinogeaic risk of chemicals to humans. Tobacco habits other than smoking; betel-quid and areca-nut chewing and some related nitro- samines. IARC Monogr. 37: 291, 1985. 12. National Institutes of Health. Consensus Development Conference Statement on the Health Implications of Smokeless Tobacco Use, January 13-15, 1986. xxiii

Table 1 General Principles in Evaluating Carcinogenic Risk of Chemicals or Complex Mixtures (International Agency for Research on Cancer) . Evidence for carcinogenicity in experi:tental animals: --Qualitative aspects: (a) Experimental parameters under which chemical was tested. (b) Consistency with which chemical shown to be carcinogenic. (c) Spectrum of neoplastic response. (d) Stage of tumor formation in which chemical involved. (e) Role of modifying factors. -Hormonal carcinogenesis. -Complex mixtures. -Quantitative aspects; increasing incidence of neoplasms with increasing exposure. • Evidence for activity in short-term tests: -Use of valid test system. -Sufficiently wide dose range and duration of exposure to the agent and appropriate metabolic system employed in test. -Use of appropriate controls. --Specification of the purity of the compound, and in the case of complex mixtures, source and representativeness of sample tested. • Evidence of carcinogenicity in humans: -For studies showing positive association: (a) Existence of no identifiable positive bias. (b) Possibility of positive confounding considered. (c) Association unlikely to be due to chance alone. (d) Association is strong. N (e) Existence of dose-response relationship. Ca9 a -For studies showing no association: (a) Existence of no identifiable negative bias. (b) Possibility of negative confounding considered. (c) Possible effects of misclassification of exposure or outcome have been weighed. xxiv

CHAPTER 1 PREVALENCE AND ZRENDS OF SMOKELESS TOBACCO USE IN THE UNITED STATES

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1 PRODUCT CHARACTERISTICS . . . . . . . . . . . . . . . . . . . . . . . . 1-1 TRENDS IN PRODUCTION AND SALES . . . . . . . . . . . . . . . . , , . 1-1 Categories of Products . . . . . . . . . . . . . . . . . . . .~ . ; , 1-1 Temporal Trends . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-2 ZRENDS IN SELF-REPORTED USE: SURVEY DATA . . . . . . . . . . . . . . . 1-2 National Survey Data . . . . . . . . . . . . . . . . . . . . . . . . 1-2 State and Local Survey Data . . . . . . . . . . . . . . . . . . . . . 1-5 CONCLUS IONS . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . 1-8 RESEARCH NEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . . 1, 1-8 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-9

INTRODUCTION This chapter defines the various forms of smokeless tobacco that are used in the United States and examines the data that pertain to trends in prevalence and patterns of use. Trends in smokeless tobacco production and sales and self-reported use are considered. Methodological considerations are discussed and research needs are identified. Tobacco was used by pre-Columbian American Indians in smokeless forms as well as smoked (1). Cultivated by American colonists, tobacco became a major commodity in trade with Europe. Until the end of the 19th century, the use of smokeless tobacco products was widespread in the United States. Its use declined rapidly in this century with the advent of antispitting laws, loss of social acceptability, and increased popularity of cigarette smoking (1,2). Use was primarily confined to rural and agricultural areas and to occupational settings where smoking was not allowed, such as mining and some industries (3,4). In the Southeastern United States, especially in rural areas, oral use of dry snuff remained popular among women (5,6). PRODUCT CHARACTIItISTICS Today, smokeless tobacco is produced in two general forms: chewing tobacco and snuff (7-10). Chewing tobacco is chewed or held in the cheek or lower lip. Three primary types of chewing tobacco are marketed: looseleaf, plug, and twist. Snuff has a much finer consistency than chewing tobacco and is held in place in the mouth without chewing. It is marketed in both dry and moist forms. Although smokeless tobacco is not subject to combustion and is usually used orally in the United States, products differ with regard to several factors, including type of tobacco plant used, parts of the tobacco plant used, method of curing, moisture content, and additives. For example, looseleaf chewing tobacco is made from air-cured, cigar-type leaves from tobacco that is grown in Pennsylvania and Wisconsin. In contrast, dry snuff is made primarily from fire-cured dark tobacco that is grown in Kentucky and Tennessee. Plug tobacco and snuff come in dry and moist forms. t~iany smoke- less tobacco products are sweetened with sugar or molasses. Many are flavored; licorice is a common additive for chewing tobacco while mint and wintergreen often are used to flavor snuff. Table 1 describes the types of smokeless tobacco and how they are used and packaged (7-10). TRENDS IN PRODUCTION AND SALES United States Department of Agriculture (USDA) records on the annual production and sales of smokeless tobacco serve as indicators of the popula- tion's consumption. Changes in consumption can be inferred from changes in production and sales. Because sales figures closely resemble those for production, only production will be reported. Categories of Products The USDA reports production and sales by product category (i.e., chewing tobacco and snuff). The definitions of categories changed in 1981. Prior to

f 1981, total figures for chewing tobacco were derived by summing data for the subcategories of plug, twist, looseleaf, and fine-cut; snuff was a separate category. However, fine-cut tobacco is used in moist snuff. To reflect this fact, after 1981 USDA shifted fine-cut from the category of chewing tobacco to moist snuff. To observe and clarify temporal trends for the purposes of this review, the data presented in figure 1 reflect a uniform category sys- tem across years. In these records, fine-cut tobacco is counted consistently as snuff (11-17). Temporal Trends Figure 1 depicts temporal trends in the quantities of smokeless tobacco that were manufactured in the United States from 1961 to 1984. Between 1944 and 1968, total smokeless tobacco production declined 38.4 percent from 150.2 to 92.5 million pounds. Subsequent increases in production reached 135.6 million pounds in 1985. Between 1970 and 1985, total snuff production increased 56 percent from 31.3 to 48.7 million pounds. This increase was due to changes in the production of moist snuff; the manufacture of dry snuff declined (3). The difference in trends in the production of moist and dry snuff is shown in figure 1 for the years 1981 through 1985. Separate production data are not available for the two types of snuff prior to 1981. Between 1970 and 1981, however, the production of fine-cut tobacco, used in the manufacture of some moist snuff, increased threefold from 4.8 to 15.2 million pounds. Between 1970 and 1985, the production of chewing tobacco increased 36 percent from 63.9 to 86.9 million pounds. This increase was due to the.pro- duction of looseleaf tobacco, which increased 87.3 percent from 39.5 to 74.0 million pounds. The production of plug and twist tobacco declined during this period. TRENDS IN SELF-REPOR7.'ED USE: SURVEY DATA National Survey Data National data from 1964 to 1985 are available from eight different national probability surveys and a national survey of college students. The majority of the data pertain to persons over the age of 17. The principal characteristics of these surveys are shown in table 2. Office on Smoking and Health Surveys Early data on the use of chewing tobacco and snuff are available from the 1964, 1966, 1970, and 1975 Adult Use of Tobacco Surveys that were conducted by the National Clearinghouse for Smoking and Health, currently the Office on Smoking and Health (OSH) (18,19,20). National probability samples of 5,700 to 12,000 individuals over the age of 21 from randomly selected households were interviewed by telephone regarding the use of tobacco products. Between 1964 and 1975, the prevalence of smokeless tobacco use remained fairly stable. Results are summarized in table 3. Three patterns in these data may be noted: 250125502?

• Less than 5 percent of the population reported using smokeless tobacco. • Nationally, use was higher among males than females. • Among males, the prevalence of use of chewing tobacco was higher than that for snuff. National Health Interview Survey In 1970, the National Center for Health Statistics included a question on current use of snuff and chewing tobacco in its National Health Interview Survey (NHIS) (21). One respondent per household provided information on all household members age 17 and older. Data were collected on approximately 77,000 persons in 37,000 households. Estimates indicate that 1.4 percent of males used snuff and 3.8 percent used chewing tobacco (table 4). Simmons Market Research Bureau, Inc. National probability data that were collected annually from 1980 through 1985 for the Simmons Study of Media and Markets provide estimates of the prevalence of snuff use among adults who were 18 years of age or older. Sam- ple size ranged from 15,000 to 19,000. Data are summarized in table 5 for the years 1980 to 1985. The prevalence rate for "current use" of snuff was 2.4 percent of males in 1980, and 0.8 percent for females. Rates for males peaked at 4.2 percent in 1982 and were 3.2 percent in 1985. Since 1982, the highest rates of use have consistently been observed in the age group 18 to 24 years old. Comparatively higher rates of use were also observed in the age groups 25 to 34 years old and over age 65 (22). The Simmons National College Study reports data from a probability sample of full-time students 18 years or older who were attending baccalaureate-grant- ing colleges and universities in the coterminous United States. In 1983, 2,011 students were sampled, and 2,373 students were sampled in 1985. Five to 7 per- cent of males indicated use of snuff compared to 0.2 percent of females (table 6). The prevalence rate among male students exceeded that of the general*adult male population (tables 5 and 6). In 1985, prevalence among college males was twice that of other adult males, while the rate for college women was less than one- third that among the general adult female population. The combined prevalence for male and female college students (3.5 percent) was very similar to that for 18 to 24 year olds in the general population (2.8 percent) (tables 5 and 6) (23). Current Population Survey In the fall of 1985, the Census Bureau collected health information on approximately 120,000 persons in 58,000 households in its Current Population Survey (CPS) (24). OSH sponsored a supplement to this survey, which included a question on current use of snuff and chewing tobacco. One respondent per household provided information on all members age 16 and older. Provisional estimates of smokeless tobacco use indicate that 1.9 percent of males used snuff and 3.9 percent used chewing tobacco (table 4). 2501250028

National Institute on Drug Abuse Household Survey The recently completed 1985 National Household Survey on Drug Use pro- vides the national probability data on current use and correlates of use of smokeless tobacco by youth. It is the eighth in a series of national proba- bility surveys conducted among household residents in the coterminous United States by the National Institute on Drug Abuse (NIDA). Data are collected on the use and adverse consequences that are associated with 11 drugs or drug classes. The 1985 survey oversampled for blacks and Hispanics and younger age groups. The total sample consists of approximately 8,000 face-to-face interviews. The data presented here are based on a preliminary analysis of 4,564 interviews. Provisional estimates are presented in tables 7 through 9. Sixteen percent of males under the age of 21 reported using chewing tobacco or snuff within the last year, in contrast to 11 percent of older males (table 7). The decline in older age groups is seen more clearly when narrower age categories are used (table 8). An estimate of the prevalence of weekly use may be obtained by combining the use frequency categories of "most days a week" and "1 or 2.days a week" (table 9). Use at least once a week peaks in the 18- to 25-year-old age groups at 8 percent. As in previous surveys, the use among females was consistently much lower than among males. Responses suggest slightly higher rates of use among women 40 years of age and older than among younger women (table 8) (25). Discussion of National Survey Data Despite varying methodologies among the national surveys (table 2), sufficient commonalities permit meaningful comparisons. The 1970 and 1975 OSH surveys and the 1980 to 1985 Simmons Study of Media and Markets indicate that the use of snuff by adult males remained constant within a range of 3 to 4 percent. Use by adult females also remained constant at about 1 percent. During this same 15-year period, the population over the age of 18 increased 32 percent from 133.5 million to 175.8 million (26). The production of al1l forms of smokeless tobacco increased 42 percent from 95.2 to 135.6 mi•llion pounds, and the production of fine-cut/moist snuff tripled. This may indicate the emergence of a new population of users. The 1970 NHIS and the 1985 CPS both relied on the use of proxy respon- dents. Estimates of smokeless tobacco use are likely to be lower than the actual population prevalence because respondents may not always be aware of smokeless tobacco use by other members of the household. In fact, in 1970, the NHIS estimated that 1.4 percent of males used snuff and 3.8 percent used chewing tobacco. In the same year, the OSH Adult Survey, which did not use proxy respondents, provided corresponding estimates of 3 and 6 percent. Similarly, the CPS estimates that 1.9 percent of males used snuff in 1985, while the Simmons Study of Media and Markets estimates 3.2 percent. However, comparisons between the 1970 NHIS and the 1985 CPS for the purpose of examining trends are appropriate. They suggest little change in the overall*rate of adult male use of smokeless tobacco but indicate a marked change in the age distribution of users (table 4). In 1970, the use of smoke- less tobacco was most common among older men; in 1985, the prevalence in the younger age groups had greatly increased. 2501250029

Both the Simmons Study of Media and Markets and the NIDA survey show the highest rates of use among young adults ages 18 to 24. The Simmons National College Study indicates that male college students are as likely to use snuff as are other 18 to 24 year olds. The Simmons data also show a slight elevation in prevalence among persons over the age of 65, which reflects the age distri- bution of traditional users of smokeless tobacco. If the NIDA prevalence estimates are applied to current population fig- ures (26), there are at present over 12 million persons in the United States ages 12 and older who have used some form of smokeless tobacco in the past year. Three million are under the age of 21, and 1.7 million of these are males 12 to 17 years old. An estimated 6 million persons use smokeless to- bacco at least weekly. Of these, 0.5 million are males ages 12 to 17; 1.3 million are males ages 18 to 25; and approximately 780,000 are females. The 1980 to 1985 Simmons Study of Media and Markets estimated that 2 to 4 million persons over the age of 18 were users of snuff. Of these, 0.6 to 1.2 million were between the ages of 18 and 24. Table 10 summarizess data on the prevalence of smokeless tobacco use by region from three national surveys conducted in 1985. Among these adult samples, use was highest in the South and lowest in the Northeast, with the West and North Central/Midwest falling in between. These surveys provide self-report data only; no direct validation attempts were made. Because no strong social sanctions regarding smokeless tobacco use exist for adults, systematic misrepresentation by them is unlikely. However, under the conditions of a personal interview, as used in the NIDA study, adolescents would be more likely to underreport than overreport their use of smokeless tobacco. In addition, the preliminary estimates from the NIDA sur- vey have not been adjusted for oversampling of blacks and Hispanics. In this sample, blacks and Hispanics reported less smokeless tobacco use than whites, and their overrepresentation would result in underestimates of national prevalence. State and Local Survev Data State and local surveys provide much of the information after 1980 on the use of smokeless tobacco. Since most of these surveys were conducted in schools, often motivated by apparent increases in students' use of smokeless tobacco products, there may be a selection bias. However, the large and growing number of reports and the wide geographic coverage support the con- clusion that smokeless tobacco use is not a localized phenomenon. Indeed, the consistency of such data suggests that smokeless tobacco has become a product that is used by large numbers of teenage and young adult males. Adult Use Several reports provide a tentative profile of local usage patterns of smokeless tobacco among adults. In 1979, tobacco use information was collected from 4,282 men between the ages of 21 and 84 in 10 geographic areas as part of the National Bladder Cancer Study, a population-based case control study (27). The overall prevalence for having "ever used snuff for 6 months 2501258030 1-5

or more" among the control subjects (randomly selected from the general popu- lation) was 5 percent; for chewing tobacco, the corresponding figure was 12 percent. A breakdown by age indicated much more use of smokeless products by older men than younger men (table 11). Glover and his colleagues conducted a random sample telephone survey of 280 persons in Pitt County, North Carolina (28). A user was defined as a person who answered "yes" to the question,` "Do you dip or chew tobacco?" Forty percent of males and 9 percent of females answered positively. High rates of use are probably not a new phenomenon since there is a tradition of smokeless tobacco use among both sexes in this area, and tobacco is a major agricultural product. Gritz, Ksir, and McCarthy surveyed a sample of 214 students at the University of Wyoming (29). In their sample, 27.1 percent of males and 4.1 percent of females reported "current use," with the criterion for "current use" unspecified. The vast majority of users (84 percent) used moist snuff. Glover and his colleagues reported a survey of 5,894 students in physical education classes at 72 colleges and universities from 8 States (Oregon, Arizona, Colorado, Oklahoma, Minnesota, Ohio, South Carolina, and Connecticut) (30). Twenty-two percent of the males who were surveyed reported using smoke- less tobacco compared to 2 percent of the females. Combined rates of use for both sexes ranged from 15 percent in Oklahoma to 8 percent in Connecticut. The majority of the users reported using less than one can or pouch per week. Adolescent Use Studies of school age youth conducted since 1980 are summarized in table 12 (31-45). Five different criteria for classifying use have been selected for data display: daily use, weekly use, monthly use, current use (no frequency specified), and ever used. Recent regional data on the use of smokeless tobacco have been collected by a number of National Cancer Institute grantees in the course of their ongoing research on tobacco use by youth (46). Through collaboration, these investigators have achieved more standardization in data collection than in previous studies, which makes comparisons among the different locales more meaningful. Although there were some differences in methodology, all of the studies addressed one or both of the following research questions: I. What percentages of males and females have ever used smokeless tobacco? 2. What percentages of males and females have used smokeless tobacco in the last 7 days? Adolescent males may be subject to pressures that simultaneously dis- courage and encourage smokeless tobacco use. Underreporting of use may result from the presence of teachers and the setting in which the survey is admin- istered. Overreporting may result from peer pressure to be seen as a smokeless tobacco user. Accurate reporting may be facilitated by collecting breath or saliva samples when surveys are completed. Respondents who believe that their self-reports can be objectively verified via biochemical testing tend Z501258031

. ~ }..~ '..7. .. , . . t' . to provide more accurate responses (47-49). Biochemical validation was used in 14 of the 17 subsamples reported in table 13. Most studies do not distinguish between snuff and chewing tobacco. In reports where the two have been separated, both substances were found to be in use (34,42,43). Rates of smokeless tobacco use were consistently higher among males than females. This difference is especially marked when more precise classifica- tions for regular use are employed. While substantial numbers of adolescent . females report having tried smokeless tobacco at least once, very few use it on a regular basis (33-35,37,39,46). The use of smokeless tobacco by youth was generally higher in rural than urban areas, in small communities, and in areas where there is a tradition of smokeless tobacco use (34,37,46). However, high rates of use have also been reported in large metropolitan areas as well (37,40,46). 1 Table 14 summarizes data on smokeless tobacco use by ethnic groups col- lected by investigators using standardized questions (46). To date, little information has been available on smokeless tobacco use by nonwhites, and some early research suggested that minority•youth were not taking up the practice (42). In these studies, however, Hispanic youth showed rates of smokeless tobacco use comparable to whites, and Native American rates were consistently higher. In most locales, use was less common among Asians and blacks. Nationally, black college students are less likely to use snuff than are white college students (table 6). Prevalence estimates for smokeless to- bacco use by black adults, however, have equaled or exceeded those of whites (tables 5 and 11). The likelihood of using smokeless tobacco appears to increase with age as well as over time (32-35,37,42,46). Only one study has collected both cross-sectional and longitudinal data. Hunter and her colleagues assessed tobacco use by children in Bogalusa, Louisiana, in 1976-77 and again in 1981- 82 (42). The use of both snuff and chewing tobacco increased over time within age categories, within age cohorts, and across age categories (table 12). A decrease in use was observed in the oldest age category, 16-17 years old, but has not been seen in other locales (tables 12 and 13). The decrease may reflect age-related changes in normative behavior particular to that area or a cohort effect. Peer and family members are found consistently to be important influences on smokeless tobacco use by children and adolescents. Young users of smokeless tobacco have more friends who also use smokeless tobacco (34-36,39,45) and may themselves identify friends' encouragement as a reason for use (35,44). Users of smokeless tobacco are also more likely to have family members who themselves use smokeless tobacco (34,36,45) and encounter less parental disapproval for the practice (31,34). In a special National Program Inspection study prepared by the Office of the Inspector General of the Department of Health and Human Services, young current and former users of smokeless tobacco were interviewed in depth (50). Two hundred ninety students in junior and senior high schools from 16 States 1-7

U volunteered to participate. All had used smokeless tobacco on a weekly or daily basis. While this study was not designed to provide prevalence estimates, it provides useful information about the attitudes and practices of some adolescent smokeless tobacco users. Over 90 percent of these respondents used snuff exclusively, and over 55 percent indicated that they would have strong cravings if they tried to quit. On the average, this group reported first trying snuff at age 10 and beginning regular use by age 12. Fifty percent cited pressure from friends as their primary reason for initiating use, but continued use was most often attributed to enjoyment of taste (64 percent) and habit strength ("being hooked," 37 per- cent). Over 85 percent thought that dipping and chewing can be harmful to health, but less than 55 percent considered regular use to present a moderate or severe risk. CONCLUSIONS 1. Recent national data indicate that over 12 million persons used some form of smokeless tobacco (chewing tobacco and snuff) in 1985 and that approxi- mately 6 million used smokeless tobacco weekly or more often. Use is increasing, particularly among young males. 2. The highest rates of use are seen among teenage and young adult males. A.recent national survey indicates that 16 percent of sales between 12 and 25 years of age have used soae form of smokeless tobacco within the past year and that from one-third to one-half of these used smokeless tobacco at least once a week. Use by females of all ages is consistently less than that of males; about 2 percent have used smokeless tobacco in the last year. 3. State and local studies corroborate the national survey findings. The prevalence of smokeless tobacco use by youth and young adults varies widely by region, but use is not limited to a single region. In'several parts of the country, as many as 25 to 35 percent of adolescent males have indicated current use of smokeless tobacco. RESEARCH NEEDS More systematic and detailed national and local surveys on smokeless tobacco should be conducted.* National probability sample surveys need to be supplemented with surveys of suspected "hot spots" to detect the extent of high-risk areas in the country and the prevalence of use in these areas. Standardized methods are essential to facilitate appropriate comparisons among data. The current state of assessment is similar to the early days of research on cigarette smoking before standardized formats for assessment of prevalence and quantification of dosage became available. Accurate and repro- *The 1986 OSH Adult Use of Tobacco Survey will address many of the items listed below. 2501258033 1-8

ducible dosage measurement for smokeless tobacco products is needed. Standar- dization may prove more difficult than for cigarette smoking because of the multiplicity of product forms. Specific items that require standardization include the following: • Collection of data separately for snuff and chewing tobacco. • Definition of "user" needs to be classified according to the fre- quency of use. To date, little attention has been given to finer distinctions of use, including quantity used, the appropriate unit of measurement, and time that the product is allowed to remain in the mouth. • Description of use. Data need to be gathered on patterns of use as well as the relationship of use to cigarette smoking. • Reporting of age of initiation and duration of use. • Definition of quit attempts and a quitter. • Natural history of smokeless tobacco use and its relationship to other substance use, including other forms of tobacco, particularly cigarettes. • Surveys need to be of adequate sizes to permit stratification of the samples by relevant variables such as gender, age, ethnicity, socioeco- nomic status, cigarette smoking status, and various behavioral factors such as attitudes and knowledge, peer pressure, and academic status. REF EREIJCES 1. Christen, A.G., Swanson, B.B., Glover, E.D., and Henderson, A.H. Smokeless tobacco: The folklore and social history of snuffing,,sneezing, dipping, and chewing. J. Am. Dent. Assoc. 105: 821-829, 1982. 2. Schuman, L.M. Patterns of smoking behavior. In: M.E. Jarvick, J.W. Cullen, E.R. Gritz, T.M. Vogt, and L.J. West. (eds.). Research on smoking behavior (NIDA Research Monograph 17). U.S. Government Printing Office, (Stock No. 017-024-00694-7), 1977. 3. Shelton, A. Smokeless sales continue to climb. Tobacco Reports, p. 42, August 1982. 4. Maxwell, J.C., Jr. Chewing, p. 31, September 1980. snuff is growth segment. Tobacco Reports, 5. Winn, D.M. Tobacco chewing and snuff dipping: An association with human cancer. In: I.K. O'Neil, R.C. Borstel, C.T. Miller, J. Long, and H. Bartsch. (eds.). N-Nitroso Compounds Occurrence, Biological Effects and Relevancy to Human Cancers. IARC Scientific Publication No. 57, Oxford University Press, 1985. 25a1258o34

6. Rosenfeld, L., and Callaway, J. Snuff dipper's cancer. Am. J. Surg. 106: 840-844, 1963. 7. Agricultural Market'Service. Tobacco in the United States. U.S. Department of Agriculture (Miscellaneous Publication No. 867), 1979. 8. Davis, D.L. Smokeless tobacco products and their production in the United States. Presented at the NIH Consensus Development Conference on the Health Implications of Smokeless Tobacco Use, Bethesda, Maryland, January 13-15, 1986, and personal correspondence. 9. Bantle, L.F. Smokeless tobacco-a trend to watch. Tabak J. Int. 4: 344-346, 1980. - 10. Rizio, D. Smokeless tobacco. Tabak J. Int. 2: 183-184, 1984. 11. U.S. Department of Agriculture, Economic Research Service. Tobacco: Outlook and Situation Report. Washington, D.C., 1985. 12. U.S. Department of Agriculture, Agricultural Marketing Service. Annual report on tobacco statistics, 1973. (Statistical Bulletin No. 528). Washington, D.C., 1974. 13. U.S. Department of Agriculture, Agricultural Marketing Service. Annual report on tobacco statistics, 1976. (Statistical Bulletin No. 570). Washington, D.C., 1977. 14. U.S. Department of Agriculture, Agricultural Marketing Service. Annual report on tobacco statistics, 1981. (Statistical Bulletin No. 685). Washington, D.C., 1982. 15. U.S. Department of Agriculture, Agricultural Marketing Service. Tobacco stocks, as of January 1, 1983. Washington, D.C., Marketing Service, 1983. 16. U.S. Department of Agriculture, Agricultural Marketing Service. Tobacco stocks, as of January 1, 1984. Washington, D.C., 1984. 17. U.S. Department of Agriculture, Agricultural Marketing Service. Tobacco stocks, as of January 1, 1986. Washington, D.C., 1986. 18. National Clearinghouse for Smoking and Health. Use of tobacco: Practices, attitudes, knowledge, and beliefs. U.S. Fall 1964 and Spring 1966, July 1967. 19. U.S. Department of Health, Education, and Welfare. Smoking and health- a report to the Surgeon General. Office on Smoking and Health (DHEW Publicatipn No. PHS 79-50066), 1979. 20. National Clearinghouse for Smoking and Health. Adult use of tobacco, 1975. U.S. Department of Health, Education, and Welfare, Public Health Service, 1976. 2501258035

21. National Center for Health Statistics. National Health Interview Survey, 1970 (unpublished). 22. Simmons Market Research Bureau, Inc. Study of Media and Markets, 1980- 1985. 23. Simmons Market Research Bureau, Inc. Simmons National College Study, 1983 and 1985. 24. Office on Smoking and Health. Current Population Survey, 1985 (unpublished). 25. Rouse, B.A. National prevalence of smokeless tobacco use. Presented at the NIH Consensus Development Conference on the Health Implications of Smokeless Tobacco Use, Bethesda, Maryland, January 13-15, 1986. 26. U.S. Department of Commerce, Bureau of the Census. (CP Series P25, No. 922), 1982. 27. Hartge, P., Hoover, R., and Rantor, A. Bladder cancer risk and pipes, cigars, and smokeless tobacco. Cancer 55: 901-906, 1985. 28. Glover, E.D., O'Brien, K., and Holbert, D. Prevalence of smokeless tobacco use in Pitt County, North Carolina. Int. J. Addict. (in press). 29. Gritz, E.R., Ksir, C., and McCarthy, W.J. Smokeless tobacco use in the United States: Past and future trends. Ann. Behav. Med. 7: 24-27, 1985. 30. Glover, E.D., Johnson, R., Laflin, M., and Christen, A. Smokeless tob.ac- co trends in the United States. World Smoking and Health (in press). 31. Marty, P.J., McDermott, R.J., Young, M., & Guyton, R. Prevalence and psychosocial correlates of dipping and chewing in a group of rural high school students. Health Educ. (in press). 32. Marty, P.J., McDermott, R.J., and Williams, T. Patterns of smokeless tobacco use in a population of high school students. Am. J. Public Health 76: 190-192, 1986. 33. Newman, I.M., and Duryea, E.J. Adolescent cigarette smoking and tobacco chewing in Nebraska. Nebr. Med. J. 243-244, 1981. 34. Bonaguro, J.A., Pugy, M., and Bonaguro, E.W. Multivariate analysis of smokeless tobacco use by adolescents in grades four through twelve. Health Educ. (in press). 35. Lichtenstein, E., Severson, H.H., Friedman, L.S., and Ary, D.V. Chewing tobacco use by adolescents: Prevalence and relation to cigarette smoking. Addict. Behav. (in press). 36. Severson, H., Lichtenstein, E., and Gallison, C. A pinch or a pouch in- stead of a puff? Implications of chewing tobacco for addictive processes. Bulletin of Psychologists in Addictive Behaviors 4: 85-92, 1985. 2501258036

37. Jones, R.B. Smokeless tobacco: A challenge for the 80's. Journal of the Wisconsin Dental Association 10: 717-721, 1985. 38. Chassin, L., Presson, C.C., and Sherman, S.J. Stepping backward in order to step forward: An acquisition oriented approach to primary prevention. J. Consult. Clin. Psychol. (in press). 40. Greer, R.O., and Poulson, T.C. Oral tissue alterations associated with the use of smokeless tobacco by teenagers, I. Clinical findings. Oral Surg. 56: 275-284, 1983. Behav. (in press). 39. Chassin, L., Presson, C.C., Sherman, S.J., McLaughlin, L., and Gioia, D. Psychosocial correlates of adolescent smokeless tobacco use. Addict. 41. Poulson, T.C., Lindenmuth, J.E., and Greer, R.O. A comparison of the use of smokeless tobacco in rural and urban teenagers. CA 34: 248-261, 1984. 42. Hunter, S.M., Croft, J.B., Burke, G.L., Parker, F.C., Webber, L.S., and Bereason, G.S. Longitudinal patterns of cigarette smoking and smokeless tobacco use in youth. Am. J. Public Health 76: 193-195, 1986. 43. Guggenheimer, J., Zullo, T.G., Krupee, D.C., and Verbin, R.S. Changing trends of tobacco use in a teenage population in western Pennsylvania. Am. J. Public Health 76: 196-197, 1986. 44. Schaefer, S.D., Henderson, A.H., Glover, E.D., and Christen, A.G. Patterns of use and incidence of smokeless tobacco consumption in school-age children. Arch. Otolaryngol. (in press). 45. Young, M., and Williamson, D. Correlates of use and expected use of smokeless tobacco among kindergarten children. Psychological Reports 56: 63-66, 1985. 46. Boyd, G.M., et al. Use of smokeless tobacco among children and adoles- cents in the United States. Prev. Med. (in press). 47. Evans, R.I., Hansen, W.G., and Mittelmark, M.B. Increasing the validity of self-reports of smoking behavior in children. J. Appl. Psychol. 62: 521-523, 1977. 48. Murray, D.M., O'Connell, C.M., Schmiel, L.A., and Perry, C.P. The validity of smoking self-reports by adolescents: A reexamination of the logic and pipeline procedure. Addict. Behav. (in press). 49. Bauman, K.E., and Dent, C.W. Influence of an objective measure on self-reports of behavior. J. Appl. Psychol. 67: 623-638, 1982. 50. Office of the Inspector General. Youth use of smokeless tobacco: More than a pinch of trouble. U.S. Department of Health and Human Services, January 1986. 1-12

Table 1 Characteristics of Smokeless Tobacco Products Product CHEWING TOBACCO: Looseleaf Plug Twis t BE08SZ10sZ Description How Us ed Packaging* Made from air-cured, cigar leaf A piece of tobacco, 3/4 to 1 tobaccos of Pennsylvania and inch, in diameter is tucked Wisconsin. Consists of stripped and between the gum and jaw, processed tobacco leaves. The leaves usually to the back of the are stemmed, cut, or granulated and mouth. are loosely packed to form small strips of shredded tobacco. Most brands are sweetened and flavored with licorice. Made from enriched tobac4o leaves Chewed or held in the cheek or (Burley and bright tobacco and lower lip. May be held in the cigar tobacco) or fragments wrapped mouth for several hours. in fine tobacco and pressed into bricks. May be firm (less than 15 percent moisture) or moist (15 percent or greater moisture). Most plug tobacco is sweetened and flavored with licorice. ing and sweeteners. Handmade of dark, air-cured leaf tobacco treated with a tarlike tobacco leaf extract and twisted into strands that are dried. Majority is sold without flavor- rProduct weight (includes moisturz). Pouch, typically 3 ounces. A few brands market a 1.5 ounce pouch. A compressed brick or flat block wrapped inside natural tobacco leaves. Packaged in clear plastic. Packages range from 7 to 13 ounces. Also sold by the piece. Similar to plug. A pliable but dry rope. So ld by the piece, packaged in plastic bags. No standard weight. Sold in small (approximately 1-2 ounces) and larger sizes based on the number of leaves in the twist.

Table 1 (continued) Characteristics of Smokeless Tobacco Products 'roduct NUFF: Mois t Dry Description How Used Packaging Made from air-cured and fire-cured A small amount ("pinch") is Cans and plastic tobacco. Consists of tobacco stems placed between the lip or cheek containers, and leaves that are processed into and gum and is typically held typically 1.2 fine particles or strips. Some for 30 minutes or longer per ounces. products are flavored. Has a pinch. moisture content of up to 50 percent. Most dry snuff is made from fire- cured tobaccos of Kentucky and Tennessee. After initial curing the tobacco is fermented further and processed into a dry powdered form. Products vary in strength and flavor- ing. Generally has a moisture content of less than 10 percent. Same as moist snuff. May also Metal cans or be sniffed. glass containers, vary from 1.15 to 7 ounces per container. cEOeszIasZ i

ldute c National Prevalence of Smokeless Tobacco Use: Data Sources Survey Type Date Respondents Number of Respondents/ Households Products Office on Smoking and Health Personal Interview 1964 Adults > 21 5,794 Snuff and Chewing Tobacco Separately Office on Smoking and Health Personal Interview 1966 Adults > 21 5,770 Snuff and Chewing Tobacco Separately Office on Smoking and Health Telephone 1970 Adults > 21 5,200 Snuff and Chewing Tobacco Separately Office on Smoking and Health Telephone 1975 Adults > 21 12,000 Snuff and Chewing Tobacco Separately National Health Personal 1970 Persons > 17 77,000/ Snuff and Chewing Interview Survey Supplement (National Cen- Interview Including Proxy ~ 37,000 Tobacco Separately ter for }lealth Statistics) Simmons Study of Questionnaire 1980 Adults > 18 15,000- Snuff Only Media Markets 1981 19,000 Simmons Market 1982 Research Bureau, 1983 Inc. 1984 1985 Questions "Have you ever used-- at all regularly?" "Do you use--now?" "Have you ever used-- at all regularly?" "Do you use--now?" "Have you ever used-- at all regularly?" "Do you use--now?" "Have you ever used-- at all regularly?" "Do you use--now?" Does presently use any other form of smokeless tobacco, such as snuff or chew- ing tobacco? 1980 to 1983 "Do you use it yourself--snuff (smokeless tobacco)?" 1984 to 1985 "Do you yourself use any of the following tobacco pro- ducts?" Snuff (ST) listed as an option. Qh08SZ t OSZ

Table 2 (continued) irvey Type Date Respondents Number of Respondents/ Household Products inmons National Questionnaire 1983 College 2,011- Snuff Only ,11ege Study, Students 2,373 mmony Market 1985 > 18 search Bureau, tc. irrent Popula- Personal 1985 Persons 120,000/ Snuff and' on Survey Interview >16 58,000 Tobacco ipplement-- ,nsus Bureau ,r Office on ioking and alth DA Household Including Proxy ersonal 985 ersons > 12 ,000 Chewing Separately nuff and irvey Interview Chewing Tobacco Combined Questions "Please mark which of the items listed be- low you yourself use." Snuff (smokeless tob- acco) listed as an option. Does presently use any other form of to- bacco, such as snuff or chewing tobacco? What other forms of tobacco does presently use? "On the average, in the past 12 months, how often have you used chewing tobacco or snuff or other smokeless tobacco?" l f1oSSZl 0Sz

Table 3 Use of Smokeless Tobacco in the United States by Individuals Over 21 Years of Age* Percenta e of Users Males Fema les Use Category 1964 1966 1970 1975 1964 1966 1970 11975 Now use snuff 2.0 3.1 2.9 2.5 2.0 2.1 1.4 1.3 Used to use snuff 3.6 3.9 4.2 4.0 0.9 1.0 1 1 1 1 Have ever used snuff** 5.7 7.2 7.1 6.4 2.9 3.1 . 2.6 . 2.4 Now use chewing tobacco 5.1 7.1 5.6 4.9 0.5 0.4 0.6 0.6 Used to use chewing tobacco 12.0 13.2 19.1 16.1 1.0 1.1 1.8 1.2 Have ever used chewing tobacco** 17.2 20.5 24.7 21.0 1.5 1.5 2.4 1.8 *"Use" not further defined with respect to frequency. **Includes those who used to use, but did not state if they used it currently. Source: National Clearinghouse on Smoking and Health.

~i~qx-i = Table 4 Prevalence of the Use of Snuff and Chewing Tobacco Among Males by Age:* 1970 NHIS and 1985 CPS Surveys 1970 HIS 1985 CPS Percentage Percentage Product A e of Us ers A e of Us ers Snuff 17-19 0.3 16-19 2.9 20-29 0.6 20-29 2.7 30-39 0.7 30-39 1.8 40-49 1.2 40-49 1.5 50+ 2.7 50+ 1.4 Total 1.4 Total 1.9 Chewing 17-19 1.2 16-19 3.0 Tobacco 20-29 1.9 20-29 4.2 30-39 2.8 30-39 3.7 40-49 3.0 40-49 3.3 50+ 6.5 50+ 4.2 Total ~ 3.8 ~ Total 3.9 *"Use" not further defined with respect to frequency. Sources: National Center for Health Statistics, National Health Interview Survey, 1970 (unpublished). Office on Smok- ing and Health, Current Population Survey, 1985 (un- published).

Tab 1e 5 National Prevalence of Current Use of Snuff by Gender, Age, and Race for 1980 Through 19851 Sam le 1980 1981 1982 1983 1984 1985 Total 1.6 2.2 2.6 2.3 1.9 1.9 Gender Males 2.4 3.7 4.2 3.8 3.0 3.2 Females 0.8 0.8 1.1 0.9 1.0 0.7 Age 18-24 1.4 2.6 4.3 3.5 3.2 2.8 25-34 2.5 2.8 3.1 3.0 2.0 2.1 35-44 1.0* 1.3 1.6 1.8 1.5 1.0 45-54 1.3* 1.3 1.4* 1.0* 1.1* 1.5 55-64 1.2* 1.7 1.7 2.3 1.1* 1.3 > 65 1.6* 2.8 2.6 1.4 2.5 2.4 Race I Black 2.3* 1.6* 3.0 2.9 2.9 2.4 White 1.5 2.2 2.6 2.3 1.9 1.9 Other 1.9* 1.4* 1.1* NA Q.4* 1.2 lAdults def ined *Number Source: defined as individuals over 18 years of age. with respect to frequency. of cases too small for reliable estimates. Use not further Simmons Market Research Bureau, Inc. Study of Media and Ma-kets 1980-1985. Percenta e of Users 1-19

Table 6 Prevalence of Snuff Use Among College Students 18 Years of Age or Older by Gender and Yearl Percentage of Users Sample 1983 1985 Total 2.7 3.5 Gender Males 5.4 6.7 Females 0.1* 0.2* Race B lack 1.5* 1.4* White 5.1 3.6 Other 4.9* 4.3* 'Current use; frequency of use not specified. *Projection relatively unstable because of small sample. Source: Simmons Market Research Bureau, Inc. Simmons National College Study, 1983 and 1985.

Table 7 National Prevalence of Smokeless Tobaqco Use by Adult Status and Sex NIDA Sample, 19851 Pe-centaste of Users Males Use Category Used in past year Used formerly Never used I < 20 Years 16 4 79 > 21 Years 11 7 82 Females < 20 Years 2 2 96 > 21 Years 2 2 96 1P-eliminary estimates not adjusted for oversampling of blacks and Hispanics Source: National Institute on Drug Abuse 1985 National Household Survey on Drug Abuse. Preliminary results presented at the NIH Consensus Development Conference on the Health Implications of Smokeless Tobacco Use, Janua-y 1986.

Table 8 Recency of Smokeless Tobacco Use by Sex and Age Groupl Percentage of Users by Age Groups Use Category Used in past year Used formerly Never used 12-17 1 18-25 1 26-39 1 40 Males{Females{ MaleslFemalesl MalesiFemalesi Males 16 4 80 1 2 97 16 7 77 I 1 98 8 8 84 , + Females 3 2 95 1Preliminary estimates not adjusted for oversampling of blacks and Hispanics. Source: National Institute on Drug Abuse 1985 National Household Survey on Drug Abuse. Preliminary results presented at the NIH Consensus Development Conference on the Health Implications of Smokeless Tobacco Use, January 1986.

Tab le 9 Frequency of Smokeless Tobacco Use in Past Year1 Percentage of Users A e Grou s for Males Past Year Use of Males and Females Smokeless Tobacco 12-17 18-25 26-39 40+ A e 12 and Above Most days/week 3 7 5 4 2 ' I or 2 days/week 2 1 1 ~ 1 1 1 or more days/week 5 8 6 5 3 3-51 days/year 5 5 3 3 2 1-2 days/year 6 3 2 1 2 Not in past year 4 7 5 8 3 Have tried 20 23 15 ~ 16 ~ 10 Never 80 77 ~ 85 84 90 1Preliminary estimates not adjusted for oversampling of blacks and Hispanics. Source: National Institute on Drug Abuse 1985 National Household Su_'vey on Drug Abuse. Preliminary results presented at the NIH Consensus De- velopment Conference on the Health Implications of Smokeless Tobacco Use, January 1986.

Table 10 Prevalence of Smokeless Tobacco Use by Census Region, 1985 Percenta e Re ortin Use Prevalence Category Northeast North Central South West CPS Chewing tobacco 1.6 3.7 7.0 3.9 Snuff 1.2 2.3 3.1 1.6 S immons Snuff 1.5 1.3 2.9 1.3 NIDA* (Snuff and/or chewing tobacco) Weekly use or more often 1.0 2.0 5.0 4.0 Any use in past year 1 4.0 6.0 8.0 ~ t 9.0 *Preliminary estimates not adjusted for age and race. Sources: Office on Smoking and Health, Current Population Survey, 1985 (unpub- lished). Simmons Market Research Bureau, Inc., Study of Media and Markets, 1980-1985. National Institute on Drug Abuse, 1985 Household Survey on Drug Abuse. Preliminary results presented at the KIH Con- sensus Development Conference on the Health Implications of Smokeless Tobacco Use, January 1986.

Table 11 Prevalence of Snuff and Chewing Tobacco Use by Adult Males in 10 Geographic Areas Percenta e Re ortin Ever Used Sam le n Snuff Chewin Tobacco All men 4,282 5 .12 Age 21-44 240 0 2 45-64 1,653 3 6 65-84 2,389 7 16 Area of Residence Atlanta 186 8 23 Connecticut 654 4 12 Detroit 355 8 20 Iowa 552 12 14 New Jersey 1,288 2 10 New Mexico 129 7 20 New Orleans 115 1 6 San Francisco 542 2 8 Seattle 255 10 6 Utah 206 5 7 Race White 3,892 5 11 Nonwhite 390 5 18 Source: National Bladder Cancer Study. Hartge, P., Hoover, R., and Kantor, A. Bladder cancer risk and pipes, cigars, and smokeless tobacco. Cancer, 55: 901-906, 1985. Research supported by the National Cancer Institute, the Food and Drug Administration, and the Environmental Protection Agency.

Table 12 Prevalence of Use of Smokeless Tobacco Among Youth by Gender and Grade: Regional and State Level Surveys Reported Since 19801 Location (reference) Grade(s) Males Females Total n DAILY USE Arkansas (31) 10-12 26.0 179 Arkansas (32) 10-12 15.0 901 Nebraska (33) 7-12 2.5 0.0 2,612 Ohio (34) 4-12 Chewing Tobacco 11.4 0.2 1,004 Snuff 19.7 0.4 1,004 Oregon (35) 7 8.8 0.7 443 9 18.5 0.0 249 10 23.1 2.4 130 Oregon (36) 7 4.6 710 8 5.8 139 9 9.7 432 10 10.6 255 Wisconsin (37) 7 3.0 0.0 8 6.0 0.0 9 3.0 0.0 10 8.0 0.0 11 11.0 0.0 12 Total 15.0 0.0 WEERL Y USE (Or More Often) Nebraska (33) 7-12 4.8 ! 0.0 2,616 Wisconsin (37) 7 12.0 8 18.0 9 15.0 10 24.0 11 25.0 12 37.0 Total 1.0 25,000 lUnless otherwise indicated, figures represent the usage of chewing tobacco and/or snuff. Multiple entries have been made for studies that provide for more than one classification criterion. 2501258051 1-26

Table 12 (continued) Location (reference) Grade(s) Males ~ ~ Females I Total ~ n MONTHLY USE (Or More Often) Arizona (38) 8-12 18.4 -- -- 1,080 Midwestern State (39) 10-12 33.0 0.0 -- , 323 Nebraska (33) 7-12 7.1 0.0 -- I 2,616 CURRENT USE (Frequency Not Specified) Arkansas (31) 10-12 31.8 2.2 -- 179 Arkansas (32) 10 I -- -- 13.8 326 11 -- -- 20.6 ~ 330 12 -- -- 23.7 245 Tota1J 36.7 ( 2.2 I -- i 901 Colorado (40) 10-12 i 21.6 i 0.6 i -- 1 1,119 Colorado (41) 10-12 I 26.0 ~ 0.0 i -- ~ 445 Louisiana (42)* h 1976-1977 ! ! Chewing Tobacco 8-9 11.0 -- -- ' 10-1L ( 17.0 ! 12-13 25.0 -- -- 14-15 24.0 ~ ! 16-17 15.0 ~ -- -- Snuff 8-9 4.0 ! 10-11 7.0 1 -- ~ -- ~ 12-13 ~ 5.0 ~ -- ~ -- ~ 14-15 ~ 11.0 ~ ! 16-17 ~ 5.0 ~ -- -- ~ Total l -- -- i -- 2,880 ~ 1981-1982 I I ( ~ Chewing Tobacco 8-9 24.0 ! ~ -- ~ -- ~ 10-11 32.0 ! I 1 12-13 39.0 - - - = 14-15 43.0 _ _ t!1 ! -- ( -- *Age listed rathe-

Table 12 (continued) Location (reference) Grade(s) i Males - F ( Females , f Total i n Total -- -- ,981 1,981 Snuff 8-9 21.0 -- -- 10y11 26.0 -- -- 1•2-13 32.0 -- -- 14-15 30.0 -- -- 16-17 14.0 -- -- ~ Total -- -- -- 1,981 Pennsylvania (43) 7-12 30.0 0.0 -- 538 Texas (44) 7-12 19.0 0.0 -- 5,392 Wyoming (29) 7-9 24.5 1.2 -- ~ 2,408 EVER USED Arkansas (45) K I -- -- 21.4 ~ 112 Ohio (34) Chewing tobacco 4-12 ( 58.0 I 12.0 I -- ( Totall -- 1,007 Snuff 4-12 ~ 64.0 ~ 24.0 ~ -- ~ Total( -- ~ -- ~ 1,007 0regon (35) 7 I 63.4 19.9 I -- ( y.5 4y .5 9 72.7 16.4 -- ~ 249 10 76.7 ~ 23.8 -- ~ 133 ' Wisconsin (37) 7 32.0 I -- -- -- ~ 8 ~ 45.0 l -- -- ~ -- 9 ~ 47.0 ' -- -- - 10 50.0 -- ~ ~ _ -_ - 11 ~ 47.0 -- ~ -- f -- 12 ~ 48.0 ~ -- -- ~ Total) -- I -- I 11.0 ~ 25,000 1-28

Tab le 13 Prevalence of Use of Smokeless Tobacco Among Youth by Gender and G-ade: Local Surveys Using Standardized Questions Sample Grade USED IN LAST 7 DAYS I Males Females Percentage n 1 Percentage n California Suburban/Rural 6 4.7 (469) 0.7 (407) 7 14.8 (574) 1.4 (557) 8 9.2 (487) 1.6 (499) Minnesota Suburban/Urban 9 18.1 (2,015) 2.4 (2,146) Montana Urban 4 9.4 (477) 2.0 (403) 5 11.9 (429) 1.5 (392) 6 13.9 (446) 3.2 (402) New York Urban 4 ~ 3.9 (306) ~ 0.3 (298) 5 2.9 (272) ~ 0.4 (275) 6 ~ 10.7 (252) ~ 0.4 (243) ~ New York New York City 6 1.1 (1,488) i 0.9 (1,494) New York I ( Suburban 7 t 3.0 (2,016) i 0.0 (1,811) ~ Oregon ( Suburban/Ru-a1 6 6.0 (602) ~ 0.9 (542) 7 l 9.1 (627) I 0.8 (613) 8 I 13.6 (663) ~ 1.0 (608) r1i 9 17 3 (572) ~ 0 5 (567) C11 . . ~ 10 22.2 (514) ~ 2.3 (471) 11 ~ 22.7 (440) 0.5 (431) N regon tJ1 Da a LJ1 .9 Suburban/Urban 6 1.9 (571) 0.4 (525) 7 4.6 (570) 1.4 (575) 8 6.8 (514) 0.8 (533) 9 14.8 (588) 1.2 (575) 1-29

,:- Table 13 (continued) Males ~ Females S amp le Grade ( Percentage n , Percentage n Southeastern United States 6 9.8 (305) 1.3 (228) 10 SMSA's 7 12.1 (346) 0.6 (325) 8 10.4 (279) 1.6 (313) Vermont Rural 5 9.3 (288) 0.3 (317) 6 14.9 (328) 1.0 (289) Ve*mont Urban 4 2.8 (216) 0.0 (199) 5 4.8 (207) 1.0 (201) 6 5.4 (204) 0.0 (193) Washington Rural 4 4.4 (45) 0.0 (47) 5 6.4 (141) 1.3 (156) 6 8.8 (968) 2.1 (964) 7 13.1 (521) 4.1 (514) 8 14.8 (316) 5.2 (325) ~ Washington 1 Rural 10 C 23.7 (215) 0.4 (233) ~ ~ ~ ~ EVER USED ~ ~ California Suburban/Rural 6 I I 32.6 (473) ~ 7.8 (411) ~ 7 56.2 (578) ~ 19.6 (567) 8 ~ 56.7 (492) i 20.0 (504) ~ i California ! I Los Angeles 7 24.9 (273) 6.7 (310) ~ ` SHARP ( ~ ~ California ~ ~ tV ~ Los Angeles 7 ~ 25.3 (479) ~ 7.7 (480) Q SMART 8 ~ 31.9 (429) ~ 8.1 (418) ~ ~ LJ'i ( ~ California ~ Q C11 Los Angeles 8 32.0 (1 240) 6.9 474) (1 U9 , ~ , TVSP

Table 13 (continued) Males Females Sample Grade Percentage n ( Percentage n Minnesota Suburban/Urban 9 62.1 (2,001) 22.9 (2,133) Montana Urban 4 41.0 (480) 17.5 (401) 5 56.9 (431) 19.3 (394) 6 68.2 (443) 24.6 (402) New York Urban 4 23.1 (307) 3.4 (298) 5 33.5 (272) 5.1 (275) 6 47.8 (255) 7.0 (243) New York New York City 6 6.7 (1,488) 3.0 (1,494) New York Suburban 7 25.3 (2,016) 4.1 (1,811) Orego n Suburban/Rural 6 48.3 (607) 16.2 (551) 7 57.9 (639) 19.8 (630) 8 64.5 (677) 23.8 (617) 9 70.4 (577) 26.7 (576) 10 74.7 (522) 31.1 (485) 11 77.5 (445) 34.2 (436) Oregon Suburban/Urban 6 32.4 (568) 8.7 (523) 7 44.9 (568) 16.8 (572) 8 54.1 (512) 17.2 (535) 9 61.3 (589) 24.7 (575) Southeastern United States 6 7.6 309) 1.4 229) t`J GJ'1 Q ~ 10 SMSA's 7 49.0 (353) 13.5 (325) ~ 8 51.4 (280) 15.6 (314) G~'I C7o Ve*_tnont Rural 5 8.8 289) .2 317) Q tJ'I M 6 54.8 (332) 7.2 (290) 1-31

Table 13 (continued) Males Females Sample Grade Pe.rcentage n Percentage n Vermont Urban 4 17.4 (213) 3.0 (200) 5 26.2 (207) 5.5 (201) 6 39.8 (206) 3.1 (193) Washington Rural 4 15.6 (45) 0.0 (47) 5 27.0 (141) 7.7 (156) 6 49.0 (968) 13.0 (964) 7 52.0 (521) 16.0 (514) 8 58.9 (316) 20.1 (325) Washington Rural 10 73.5 (215) 30.9 (233) Waterloo, Canada Suburban/Rural 11 26.0 (281) 5.5 (444)

A'a Table 14 Kean Frequency of Smokeless Tobacco Use During Last 7 Days by Ethnicity by Male Respondents Prevalence Sa le Ethnicit n X California Asian 192 3.7 Suburban/ Rural Black 118 6.1 Grades 6-8 Hispanic ~ 188 11.2 White 1 1 1,046 11.4 Minnesota Suburban/ Asian 36 13.9 Urban Mur-_ay Black 201 4.0 Hispanic 24 45.8 Native Amer i can 38 18.4 e I t ~ 1,6 02 .6 19 New York 1 As ian ~ 119 2.5 New York ( City Black ~ 205 0.5 Grade 6 ~ Hispanic ( 510 1.0 ~ I 501 1.2 New York As ian 23 4.3 Suburban ( Grade 7 Black ~ 47 2.1 Hispanic ~ 39 2.6 Native ( P, m merican ( erican 26 3.8 White 1,796

Table 14 (continued) Prevalence Sa le Ethnicit n Oregon Asian 38 5.3 Suburban/Rural Grades 6-11 Black 33 15.2 Hispanic 61 16.4 Native 23.3 American 120 White 3,162 14.2 Oregon Asian 71 2.8 Suburban 1 Grades 6-9 Black 231 3.9 Hispanic 26 0.0 Native 48 12.5 American White 1,847 7.6 Southeas tern United States Black ~ 258 3.9 10 SMSA's White i 652 14.0 Washington Asian 148 6.1 Rural ~ Grades 4-8 Black ~ 119 1.7 Hispanic ~ 111 9.0 Native ~ American ( 179 30.7 White ~1,434 9.4 ~ ~ 1 ~ ~ ~ i ~ j

Figure 1 Manufacturing Trends: Quantities of Smokeless Tobacco Manufactured in the United States From 1961 to 1985 Expressed in Million Pounds Numbor Of Mitlion Pounds 1w-1 130 -~ tm. 110 -~ eo 70 60 1 ro ta 0 T-TTIi I- T-i i-1 i T 1 I 1 6 i d2 e3 6, 65 c6 67 68 69 ro 71 72 7] 74 75 76 77 'L 79 bU d t d1 iJ 84 85 Year : roial $m0=tMfS tOOKCO s Chtwlnq T0O7CC0 : Snulf = Mo.st SnWf = Ory $nulf 1-35

CHAPTER 2 CARCINOGENESIS ASSOCIATED WITH SMOKELESS TOBACCO USE

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1 EPIDEMIOLOGIC STUDIES AND CASE REPORTS OF ORAL CANCER IN RELATION TO SMOKELESS TOBACCO USE . . . . . . . . . . . 2-1. Data From North America and Europe . . . . . . . . . . . . . ... 2-1 Data From Asia . . . . . . . . . . . . . . . . . . . . . . . . . 2-7 S umma ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-8 EPIDEMIOLOGIC ST`UDIES OF OTHER CANCERS IN RELATION TO SMOKELESS TOBACCO USE . . . . . . . . . . . . . . . . . . . . . 2-20 Nasal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . 2-20 Esophageal Cancer . . . . . . . . . . . . . . . . . . . . . . . . 2-21 La ryng e a l Can c e r . . . . . . . . . . . . . . . . . . . . . . . . 2-22.,1 Stomach Cancer . . . . . . . . . . . . . . . . . . . . . . . . 2-23 Urinary Tract Cancer . . . . . . . . . . . . . . . . . . . . . . 2-23 Other Cancers . . . . . . . . . . . . . . . . . . . . . . . . . 2-25 S umma r y . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-26 References . . . . . . . . . . . . . . . ... . . . . . . . . . 2-26 CHEMICAL CONSTITUENTS, INCLUDING CARCINOGENS, OF SMOKELESS TOBACCO . . . . . . . . . . . . . . . . . . . . . . . 2-32 Chemical Composition of Smokeless Tobacco . . . . . . . . . . . 2-32 Carcinogens in Smokeless Tobacco . . . . . . . . . . . . . . . . 2-32 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-34 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-34 METABOLISM OF CONSTITUENTS OF SMOKELESS TOBACCO . . . . . . . . . 2-47 Metabolism of NAIK . . . . . . . . . . . . . . . . . . . . . . . 2-47 Metabolism of NNN . . . . . . . . . . . . . . . . . . . . . . . 2-48 Metabolism of NMOR . . . . . . . . . . . . . . . . . . . . . . . •2-49 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-49 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-49 EXPERIMENTAL STUDIES INVOLVING EXPOSING LABORATORY ANIMALS TO SMOKELESS TOBACCO OR ITS CONSTITUENTS . . . . . . . . . 2-58 Bioassays With Chewing Tobacco . . . . . . . . . . . . . . . . . 2-58 Bioassays With Snuff . . . . . . . . . . . . . . . . . . . . . 2-59 Bioassays With Constituents of Smokeless Tobacco . . . . . . . . 2-62 Mutagenicity Assays and Other Short-Term Tests . . . . . . . . . 2-63 Summa.ry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-64 References . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-64 t'~1 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-72 p © RESEARCH NEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . 2-72 N U'I C~D a ~ ~ i

INTRODUCTION This chapter presents the results of a systematic review of the world's medical literature describing experimental and human evidence pertinent to the evaluation of smokeless tobacco as a potential cause of cancer. Five catego- ries of research relevant to assessing the role of smokeless tobacco in cancer causation were defined: 1. Epidemiologic studies and case reports of oral cancer in relation to smokeless tobacco use. 2. Epidemiologic studies of other cancers in relation to tobacco use. smokeless 3. Chemical constituents of smokeless tobacco. 4. Metabolism of constituents of smokeless tobacco. 5. Experimental studies involving exposing laboratory animals to smokeless tobacco or its constituents. Consensus summaries of the literature in each of these categories were prepared and form the basis of this report. In addition, recommendations for future research to clarify suggestive findings or fill gaps in knowledge are made. EPIDEMIOLOGIC STUDIES AND CASE REPORTS OF ORAL CANCER IN RELATION TO SMOKELESS TOBACCO USE Because smokeless tobacco products used in different regions of the world vary considerably in composition and usage patterns, this section will consider North American and European data separately from Asian data.. Cita- tions to the literature from India and other Asian countries where quids containing tobacco and other ingredients are commonly used orally focus on articles that attempt to distinguish tobacco from other ingredients in the quids as possible determinants of cancer risk. , Data From North America and Europe Although about a dozen informative epidemiologic studies of smokeless tobacco use and oral cancer in North America or Europe have been reported, only a few were specifically designed to examine this relation. There are two major reasons for the relative paucity of studies. Apart from the recent increased prevalence in use of smokeless tobacco, the habit has not been widely practiced in America during this century, except in localized areas such as parts of the rural South (1,2). Furthermore, cancer of the mouth is uncommon in the Western Hemisphere, exacerbating the difficulty of conducting epidemiologic investigations, particularly cohort studies, into the relation between smokeless tobacco and oral cancer. The age-adjusted incidence rate for cancers of the buccal cavity and pharynx in the United States is approxi- mately 11 cases per 100,000 population per year, with these tumors accounting

for about 3 percent of all cancer deaths (3). Nevertheless, sufficient in- formation is available to evaluate whether the use of smokeless tobacco in- creases the risk of oral cancer. Case Studies In their review of 566 oral cancer patients treated in two hospitals -in Nashville, Rosenfeld and Callaway (4,5) noted that the proportion-of women (61 percent) with buccal and gingival carcinoma was higher than the proportion of men (36 percent). Approximately 90 percent of women with buccal and gingival carcinoma used snuff for 30 to 60 years; in contrast, 22 percent of women with cancers in other oral cavity subsites used snuff. Many of these women began practicing "snuff dipping," namely, the placement of tobacco snuff in the gingivobuccal sulcus, between the ages of 10 to 20 years. These reports are typical of numerous and sometimes large series of cases from the South., which reported that high percentages of patients with gingivobuccal cancers were snuff dippers or tobacco chewers (6-13). The articles describing these case series generally did not use comparison (control) groups, but the authors consistently commented on an apparently high prevalence of the use of snuff by the cancer patients. Clinicians also noted that the usual male predominance for epidermoid carcinomas of the oral cavity diminished or disappeared for the subgroup of gingivobuccal carcinomas occurring in geographic areas where there was relatively common use of snuff and chewing tobacco. Ahblom reported in the 1930's on a possible association between smoke- less tobacco and cancer in Sweden (14). Among male patients with cancers of various sites seen at the Radiumhemmet (Stockholm), the use of snuff or chew- ing tobacco was reported in 70 percent with buccal, gingival, and "mandibular" cancers as compared to 26 to 37 percent with cancers in other oral subsites, the larynx, pharynz, and esophagus. Axell•et al. reviewed medical records of male patients with squamous cell carcinoma in the oral cavity diagnosed between 1962 and 1971 and recorded in the Register of the Swedish Board of Health and Welfare (15). The authors were only able to determine a history for the pattern of use of snuff in 25 percent of eligible patients bun commented that two-thirds of patients who were verified snuff users had oral cancers in regions where the snuff was generally placed. Reports of a single or a few cases, usually among male tobacco chewers, in the northern United States and Canada also described buccal carcinomas that were often located precisely in the area where the tobacco was retained in the mouth (16-19). In the early 1940's, Friedell and Rosenthal associated the use of snuff or chewing tobacco with an exophytic, verrucous type of squamous carcinoma of the oral cavity (16). Ackerman described in detail the morphologic and clinical features of verrucous carcinoma of the oral cavity (20). Wh.ere the lesions originated in the buccal mucosa, a history of chronic use of chewing tobacco was elicited in 60 percent of the patients. The morphologic descrip- tion was that of a well-differentiated, locally invasive, papillary squamous carcinoma, often in association with leukoplakia. In more than half of these patients, there was poor oral hygiene and carious and missing teeth. In summary, clinical and pathological reports published during the past four decades in the United States and elsewhere have commented on the use of 2501258064

smokeless tobacco by oral cancer patients and have described the entity known as snuff-dipper's carcinoma (4,7,11), providing the basis for the hypothesis that the prolonged use of snuff or chewing tobacco is associated with an increased risk of low-grade, verrucal or squamous cell carcinoma of the buccal mucosa and gingivobuccal sulcus. Case Control Studies Most of the epidemiologic evidence comes from several case-control studies of oral cancer. The low prevalence of smokeless tobacco use in most North American populations contributes to a low statistical efficiency in most of these studies. Good information has been obtained, however, from studies that were either very large, conducted in an area of high prevalence of smokeless tobacco use, or analyzed according to site within the oral cavity (since the tissue affected by snuff use appears to be highly localized). One study, by Winn et al., with these characteristics consequently provides the most informative body of data on the carcinogenicity of smokeless tobacco in North America (21). The major concern for validity in the epidemiologic studies of smokeless tobacco and oral cancer is uncontrolled confounding. A small number of sub- jects in crucial categories prevented efficient adjustment for confounding by stratification in many of these studies. Many of the studies were conducted before the advent of sophisticated epidemiologic analyses and make no attempt to control confounding. The two primary confounding factors of concern are alcohol consumption and smoking (22). Alcohol consumption is a strong risk factor for oral cancer. It is not clear on a priori grounds, however, to what extent alcohol consumption would be correlated with smokeless tobacco use. The relation between smoking, also a strong risk factor for oral cancer (2), and smokeless tobacco use may be complex. Users of smokeless tobacco may be more likely to have been smokers at some time. On the other hand, heavy users of smokeless tobacco typically cannot be heavy users-of cigarettes, so that smoking is presumably negatively correlated with smokeless tobacco use. Failure to control confounding by smoking would therefore lead to under- estimates of the effect of smokeless tobacco. Chronologically, the first case-control study of smokeless tobacco was conducted by Moore et al. in Minnesota (23,24). Patients at the Univer- sity of Minnesota Tumor Clinic with a diagnosis of cancer of the mouth were interviewed about tobacco use as part of a general interview procedure for clinic patients. Surgical outpatients who received the same interviews served as controls. From the data that were reported by these authors, one can calculate a crude relative risk estimate for mouth cancer among smokeless tobacco users of 4.0 with a 95-percent confidence interval of 1.6-10 (table 1). An oddity was an apparent lack of effect for other forms of tobacco use. A partial explanation might be negative confounding between smokeless and smoked tobacco; indeed, 26 of the 40 cases of mouth cancer chewed tobacco. Still, the extent of disparity in crude effect estimates for smokeless tobacco (relative risk estimate 4.0) and smoked tobacco (all relative risk estimates < 1.0) is surprising. Wynder et al. reported on a case-control study of squamous cell cancers of the upper alimentary and respiratory tract that was conducted at Sweden's 2501250065 2-3

Radiumhemmet in 1952-55, including 33 tongue cancer patients, 14 lip cancer patients, 19 gingival cancer patients, and 8 patients with cancer of the buccal mucosa, among others (25). Controls were patients with cancers of the skin, head, and neck other than squamous cell carcinoma, stomach cancer, lymphoma, salivary-gland tumors, leukemia, sarcoma, cancers of the colon and rectum, and cancers of the female genital tract. A variety of risk factors was examined, including the use of chewing tobacco. The authors state that the data suggested that an increased risk is associated with the duration of chewing tobacco for cancers of the gingiva and oral cavity but not for cancers of the tongue, lip, hypopharynz, esophagus, or larynx, but the data as presented do not permit an estimation of risk. In addition, data were not adjusted for other potential confounders, including cigarette smoking. Wynder and colleagues also reported in 1957 data from a similar hospital-based case-control study of mouth cancer conducted in New York (26). Tobacco chewing was found to be more common among men with oral cavity cancers than among controls; but it was noted that almost all of these patients also drank alcoholic beverages and smoked, and no further analyses were attempted. Peacock et al. studied 56 cases of mouth cancer, including malignancies of the buccal mucosa, alveolar ridge, and floor of the mouth, and compared ,their tobacco histories with those of Wo control groups: 146 hospitalized controls with diagnoses other than cancer and 217 outpatients (27). Age- specific results using the hospitalized controls are summarized in table 2. The overall relative risk was estimated to be 2.0 (95-percent confidence interval 1.0-4.2); the relative risk se.emed to increase with age with an estimate of 3.7 for the 60 to 69 age group. The data were not reported in sufficient detail to control for confounding by smoking, which presumably led to underestimates of the relative risk. There was also insufficient detail reported to evaluate the relation between the risk of mouth cancer and the amount or duration of smokeless tobacco use. In Atlanta, patients with oral, pharynx, and larynx cancer were compared to three control groups having other mouth diseases, other cancers, or no cancer (28). Among urban women, 40 percent of the cases used snuff compare'd to 3 percent or less of the controls (table 3). Among rural women, 75 percent dipped snuff compared to 20 percent or less among controls. Cigarette smoking was common in urban women and not specifically controlled for. Few rural female cases smoked cigarettes (7 percent) so confounding by smoking was minimal. The association between snuff dipping and oral, pharynx, and larynx cancer in women was generally evident in most age groups. Among the cases, the proportion of snuff dippers was highest among oral cancer patients: 53/72 were dippers compared to 2/18 pharynx and larynx cancer patients. Among men, insufficient information was provided to obtain precise epidemiologic estimates of the effect of chewing tobacco, although data from one of the bar charts presented indicate that urban cases were more likely to be users of smokeless tobacco than controls, that rural men with oral, pharynx, and larynx cancer or mouth disease were more likely to chew than controls, and that oral cancer patients were more likely to chew than the pharynx and larynx cancer cases. Among men, confounding by smoking could not be ruled out. Vincent and Marchetta reported the results of a case-coatrol study of head and neck cancer according to anatomic site. Table 4 summarizes the findings for males (29). The oral cavity seems to be the anatomic site where 250125806f

the bulk of the effect is noted; only mild increases in risk were estimated for the larynx and pharynx, whereas users of smokeless tobacco were estimated to have a sevenfold greater risk for cancer of the oral cavity. These esti- mates are imprecise because of the small number of subjects and are uncon- trolled for age and smoking. Martinez reported on a case-control study in Puerto Rico of risk factors for cancers of the mouth, pharynx, and esophagus (30). This population- based study included 400 cases of epidermoid carcinomas of those sites and 1,200 controls matched on age (+ 5 years) and sex to the cases. One control per case was drawn from the same hospital or clinic and two from the same community. There were 153 cases of mouth cancer (115 male and 38 female) and 68 cases of pharyngeal cancer (55 male and 13 female). The authors concluded that "Patients with cancer of the mouth did not often use chewing tobacco disproportionately . . ." However, calculation of the relative risks of mouth cancer that are associated with chewing tobacco based on comparing the use of chewing tobacco only with no tobacco use suggests a strong effect for oral and pharyngeal cancer in males (data from table 13 in the paper). The estimated relative risks were 11.9 (95-percent confidence interval 2.5- 56.4) for oral cancer and 8.7 (95-percent confidence interval 1.4-54.5) for pharyngeal cancer among chewers. These numbers do not include the experience of the many study subjects whose use of tobacco was "mixed" (that is, those who used any combination of cigarette, cigar, and pipe smoking and chewing tobacco), and these calculations were based on unmatched data. Further evidence for the site specificity arose from a case-control analysis of multiple cancers using data from the Third National Cancer Survey (31). There were few female users of smokeless tobacco and scanty data by site within the head and neck region even for males; the findings do seem to indicate that the effect is greater for the site that is labeled gum-mouth as opposed to other head and neck sites (table 5). Browne et al. conducted interviews with 75 oral cancer patients, or (usually) their next of kin, and 150 living sex-, neighborhood-, and,occupa- tion-matched controls in the West Midlands area of the United Kingdom where oral cancer mortality rates were high and tobacco chewing was common among miners (32). Controls on average were born about 10 years earlier than the cases. The proportion of tobacco chewers was approximately the same among the 16 cases and 43 controls who were miners, although data on this variable were missing for one-fourth of the cases, and the authors apparently assumed that all cases with missing information were nonchewers. If the proportion of tobacco chewers among the cases with missing information was similar to those miners with known information, then the data would have shown a posi- tive association between chewing tobacco and oral cancer. A.].1 of the miners with oral cancer who chewed tobacco also smoked pipes, further complicating interpretation of this study. Additional evidence that a carcinogenic effect of smokeless tobacco may be greatest at the anatomic site of exposure came from Westbrook et al. who compared the medical records of 55 female patients with cancers of the alveolar ridge or buccal mucosa who were treated at the University of Arkansas with those of 55 randomly selected female hospital controls (33). Fifty of the cases, but only one control, were snuff dippers, with the tumors among 2501250067 2-5

the cases typically appearing at the site where the snuff was usually placed. No reliable estimates of risk can be derived from this study because of the strong possibility that there was not comparable elicitation of exposure in- formation for cases and controls. Two large case-control studies were not reported in a way that enables a meaningful quantitative assessment of the effect of smokeless tobacco in chewers and dippers compared to tobacco abstainers (34,35). The first study found that 10 percent, and the second 9 percent, of male oral cancer cases had ever chewed tobacco, while the corresponding figure for controls was 9 percent. These studies, like many of the others cited here, were not under- taken specifically to evaluate the carcinogenicity of smokeless tobacco. Although the data seem to indicate a weak relation, if any, between smokeless tobacco and cancer of the oral cavity, the findings are uncontrolled for age, race, geography, and smoking. The recent case-control study of Winn et al. is by far the most informa- tive study on the carcinogenicity of smokeless tobacco (21). The case series comprised 255 women with oral and pharyngeal cancer who were living in 67 counties in a high-risk (for oral cancer) region of North Carolina. Two female controls were obtained for all but a few cases and were individually matched for age, race, source of ascertainment (hospital or death certificate), and county of residence. There was a fourfold increased risk of oral-pharyn- geal cancer among nonsmoking white women who dipped snuff. The association could not be explained by smoking or alcoholic beverage consumption (21), denture wearing or poor dentition (36), diet (37), or mouthwash use (38). The data provided evidence for a strong relation between the duration of snuff use and risk for cancer, as we7.]" as a striking localization of the carcinogen- icity to the gum and buccal mucosa (table 6). For long-term chronic users of snuff, there was nearly a fiftyfold increase in risk for cancers of the gum and buccal mucosa. Indeed, almost alll of the patients with cheek and gum cancers had dipped snuff. Although some of the exposure information came from interviews with next of kin, when the analysis was restricted to interviews with study subjects, the association between snuff and oral cancer was even stronger (39). Matched conditional logistic analysis yielded similar results (39). Based on calcula- tions of attributable risk, the authors'estimated that 87 percent of these cancers were due to the patients' snuff-dipping habits. The authors also provided data that demonstrated the negative confounding by tobacco smoking in the population, raising the possibility of a serious validity problem with the other studies that did not control for smoking. If the negative correla- tion between the use of smokeless and smoked tobacco holds in other popula- tions, estimates of the carcinogenic effect of smokeless tobacco in studies without the control of smoking may be underestimates. The quantitative information that was provided by the Winn et al. study led its authors to conclude that the long-standing use of smokeless tobacco by Southern women was the principal cause of the elevated mortality from oral cancer among women in the Southern United States. Cohort Studies Few cohort studies of smokeless tobacco have been undertaken because of the rarity of both the exposure (smokeless tobacco use) and the outcome (oral 250125$068 2-6

cancer) of most interest. Bjelke and Schuman (40) reported on cancer mortality in cohorts of 12,945 Norwegian men and 16,930 American men and found increases in the risk of death for cancers of the buccal cavity, pharynx, and esophagus (relative risk estimates ranged from 2.6 to 3.1 (41); no further detail was given). They noted a negative association between smoking and chewing tobacco, confirming the pattern that was observed from the case-control research. In a 16-year followup of U.S. veterans, Winn et al. reported no deaths from oral or pharyngeal cancer among 951 smokeless tobacco users who did not use other forms of tobacco (about 0.5 deaths were expected) but a significant increase in both oral and pharyngeal cancers among smokeless tobacco users who were light smokers (42). These data, as well as those from Bjelke and Schuman (40), were reported only as abstracts in scientific journals or proceedings, with little or no detail as to the methods used, hindering interpretation of the results. Smith and colleagues followed a group of about 1,500 patients with changes in the oral mucosa to evaluate the effects of smokeless tobacco use (43,44). No oral cavity cancers were found in about 16,000 person-years'of followup. Based on the results of other studies, two or three should have been detected over the study period. Smith gave little documentation of the methods that were employed for followup; however, 12 percent of the original group (201 subjects) were lost without any data on outcome, and there was apparently no effort to trace them. It seems likely that persons who died and persons who developed cancer, including some with tumors of the oral cavity, may have been lost to followup. In fact, no deaths among cohort members were reported, whereas perhaps as many as 100 or more would have been expected among such a cohort of middle-aged adults, making Smith's data uninterpretable. Data From Asia The highest rates of oral cancer among the more than 100 that are listed from population-based registries around the world that report standardized cancer incidence statistics are found in India (45). In many areas.of Asia, hospital statistics suggest that oral cancer is extremely common and often accounts for 25 or more percent of all cancers (46-49), proportions that are far greater than in most areas of the United States where oral cancers typically comprise only 3 percent of all malignancies (3). It*has long been thought that the chewing of quids that contain tobacco and other substances is the cause of the increased risk of oral cancer in these areas (50). The smokeless tobacco products that are commonly used include tobacco with betel leaf, areca nut, and lime 'mixtures (often referred to as "pan"); Khaiai (powdered tobacco and slaked lime paste); mishri (powdered, partially burnt black tobacco); nass (tobacco, ash, and cotton or sesame oil; lime is used in Iran and certain Soviet Republics); and various preparations that vary locally throughout the Southeast Asia region. The inclusion of lime, areca nut, and other ingredients in many of the smokeless tobacco-containing quids hinders the evaluation of the contribution of tobacco per se to the increased risk of oral tumors. From five investiga- tions, however, relative risks of oral cancer among chewers of betel quids with versus without tobacco can be calculated. Data from these case-control 2501250069

studies, which were conducted in Calcutta, Madras, Karachi, Bombay, and several parts of India and Sri Lanka (47,51-55), reveal considerably higher risks of oral cancer for the use of tobacco-containing compared to nontobacco- containing quids (table 7). The findings thus suggest that the addition of tobacco contributes substantially to the elevated cancer risk among chewers, although other differences between those who use versus those who do not.use tobacco-containing quids could influence the differences. Smoking, however, is not such a difference, since most of the investigations referred to in table 7 demonstrated high relative risks of oral cancer (with excesses among tob4cco chewers often exceeding tenfold compared to nonquid users) among chewers who did not smoke, ruling out confounding by cigarette smoking. The studies also generally found that the large majority of oral cancer patients had been tobacco chewers and suggest that the habit of quid chewing accounts for most of the oral cancers in the diverse populations studied (55,56). Summary 0 Numerous case reports, especially in the South, have described oral cancers among smokeless tobacco users. The tumors often arose at anatomic locations where the tobacco was routinely placed. The number of epidemiologic investigations evaluating the relation between smokeless tobacco and oral cancer is not large, and several studies have methodologic limitations. The pattern of increased oral cancer risk among smokeless tobacco users, however, is generally consistent across studies, with evidence of an increasing risk with increasing duration of exposure, and with excess risks tending to be greatest for those anatomic sites where tobacco exposures are greatest. The best designed study was drawn from a female population in the Southern United States where exposure rates are high and potentially confounding variables could be taken into account. This study showed that chronic snuff users were at•substantially increased risk of oral cancers and that nearly all tumors of the cheek and gum were due to snuff use. Evidence from parts of Asia, where the prevalence of smokeless tobacco use is high and oral cancer is the most common tumor, indicates a strong association between the chewing of quids and oral cancer. Users of quids that contain tobacco have much higher oral cancer rates than users of quids that do not, and the association is not confounded by cigarette smoking, raising the possibility that tobacco per se contributes to the elevated oral cancer risk in this part of the world. In summary, users of smokeless tobacco face a strongly increased risk of oral cancer, particularly for the tissues that come in contact with the tobacco. References 1. Office of Smoking and Health. Smoking and health: A report of the Surgeon General. U.S. Department of Health, Education, and Welfare, Washington, D.C., U.S. Govt. Printing Office, 1979. 2. Blot, W.J., and Fraumeni, J.F. Geographic patterns of oral cancer in the United States: Etiologic implications. J. Chron. Dis. 30: 745-757, 1977. 3. Young, J.L., Percy, C.L., and Asire, A.J. Surveillance, epidemiology, and end results: Incidence and mortality data, 1973-77. NCI Monogr. 57, 1981. z501258a7a •

4. Rosenfeld, L., and Callaway, J. Snuff dipper's cancer. Am. J. Surg. 106: 840-844, 1963. 5. Rosenfeld, L., and Callaway, J. Squamous cell carcinoma of the oral cavity. South. Med. J. 56: 1394-1399, 1963. 6. Landy, J.L., and White, H.J. Buccogingival carcinoma of snuff dippers. Ann. Surg. 27: 442-447, 1961. 7. Wilkins, S.A., and Vogler, W.R. Cancer of the gingiva. Surg. Gynecol. Obstet. 105: 145-152, 1957. 8. Brown, R.L., Sun, J.M., Scarborough, J.E., Wilkins, S.A., and Smith, R.R. Snuff dippers intraoral cancer: Clinical characteristics and response to therapy. Cancer 18: 2-13, 1965. 9. Coleman, C.C. Surgical treatment of extensive cancers of the mouth and pharynz. Ann. Surg. 161: 634-644, 1965. 10. Fonts, E.A., Greenlaw, R.H., Rush, B.F., and Rovin, S. Verrucous squamous cell carcinoma of the oral cavity. Cancer 23: 152-160, 1969. 11. Hartselle, M.L. Oral carcinoma as related to the use of tobacco. Ala. J. Med. Sci. 14: 188-194, 1977. 12. McGuirt, W.F. Snuff dipper's carcinoma. Arch. Otolaryngol. 109: 757-760, 1983. 13. McGuirt, W.F. Head and neck cancer in women--a changing profile. Laryngoscope 93: 106-107, 1983. 14. Ahblom, H.E. Predisposing factors for epitheliomas of the oral cavity, larynx, pharynx, and esophagus. Acta Radiol. 18: 163-185, 1937 (in Swedish). 15. Axell, T., et al. Snuff dipping and oral cancer--a retrospective study. Tandlakartidningen 75: 2224-2226, 1978. 16. Friedell, H.L., and Rosenthal, L.M. The etiologic role of chewing tobacco in cancer of the mouth. JAMA 116: 2130-2135, 1941. 17. Moertel, C.G., and Foss, E.L. Multicentric carcinomas of the oral cavity. Surg. Gynecol. Obstet. 106: 652-654, 1958. 18. Sorger, K., and Myrden, J.A. Verrucous carcinoma of the buccal mucosa in tobacco-chewers. Can. Med. Assoc. J. 83: 1413-1417, 1960. 19. Stecker, R.H., Devine, K.D., and Harrison, E.G., Jr. Verrucose "snuff dippers" carcinoma of the oral cavity. JAMA 189: 838-840, 1964. 20. Ackerman, L.V. Verrucous carcinoma of the oral cavity. Surgery 23: 670-678, 1948. - 2501258071

21. Winn, D.M., Blot, W.J., Shy, C.M., et al. Snuff dipping and oral cancer among women in the Southern United States. N. Engl. J. Med. 304: 745-749, 1981. • 22. Rothman, K., and Keller, E. The effect of joint exposure to alcohol and tobacco oa risk of cancer of the mouth and pharyaz. J. Chron. Dis. 25: 711-716, 1972. 23. Moore, G.E., Bissinger, L.L., and Proehl, E.C. Tobacco and intra-oral cancer. Surg. Forum 3: 685-688, 1952. 24. Moore, G.E., Bissinger, L.L., and Proehl, E.C. Intra-oral cancer and the use of chewing tobacco. J. Am. Geriatr. Soc. 1: 497-506, 1953. 25. Wynder, E.L., Hultberg, S., Jacobsen, F., and Bruss, I.J. Environmental factors in cancer of the upper alimentary tract. Cancer 10: 470-487, 1957. 26. Wynder, E.L., Bruss, I.J., and Feldman, R.M. A study of the etiological factors in cancer of the mouth. Cancer 10: 1300-1323, 1957. 27. Peacock, E.E., Greenberg, B.G., and Brawley, B.W. The effect of snuff and tobacco on the production of oral carcinoma. Am. Surg. 151: 542-550, 1960. 28. Vogler, W.R., Lloyd, J.W., and Milmore, B.K. A retrospective study of etiological factors in cancer of the mouth, pharynz, and larynx. Science 15: 246-258, 1962. 29. Vincent, R.G., and Marchetta F. The relationship of the use of tobacco and alcohol to cancer of the oral cavity, pharynx, or larynx. Am. J. Surg. 106: 501-505, 1963. 30. Martinez, I. Factors associated with cancer of the esophagus,'mouth, and pharynz in Puerto Rico. J. Natl. Cancer Inst. 42: 1069-1094, 1969. 31. Williams, R.R., and Horm, J.W. Association of cancer sites with tobacco and alcohol consumption and socioeconomic status of patients: Inter- view study from the Third National Cancer Survey. J. Natl. Cancer Inst. 58: 525-547, 1977. 32. Browne, R.M., Camsey, M.C., Waterhouse, J.A.H., and Manning, G.L. Etiological factors in oral squamous cell carcinoma. Community Dent. Oral Epidemiol. 5: 301-306, 1977. 33. Westbrook, K.C., Sven, J.Y., Hawkins, J.M., and McKinney, D.C. Snuff dipper's carcinoma: Fact or fiction? In: H.E. Nieburg (ed). Preven- tion and Detection of Cancer. New York, Marcel Dekker, 1980, pp. 1367- 1371. 34. Wynder, E.L., and Stellman, S.D. Comparative epidemology of tobacco- related cancers. Cancer Res. 37: 4608-4622, 1977. 2501258a7Z

35. Wynder, E.L., Kabat, G., Rosenberg, G., and Levenstein, M. Oral cancer and mouthwash use. JNCI 70: 255-260, 1983. 36. Winn D.M., Blot, W.J., and Fraumeni, J.F. Snuff dipping and oral cancer. N. Engl. J. Med. 305: 230-231, 1981. 37. Winn, D.M., Ziegler, R.G., Pickle, L.W., Gridley, G., Blot, W.J., and Hoover, R.N. Diet in the etiology of oral and pharyngeal cancers among women from the Southern United States. Cancer Res. 44: 1216-1222, 1984. 38. Blot, W.J., Winn, D.M., and Fraumeni, J.F. Oral cancer and mouthwash. JNCI 70: 251-253, 1983. 39. Winn, D.M. Smokeless tobacco and oral-pharynx cancer: The role of cofactors. Banbury Report (in press). 40. Bjelke, E., and Schuman, L.M. Chewing tobacco and use of snuff: Relationships to cancer of the pancreas and other sites in two prospectives studies. Proceedings of the 13th International Congress on Cancer, 1982, p. 207. 41. International Agency for Research on Cancer. Tobacco habits other than smoking: Betel-quid and areca-nut chewing; and some related nitrosamines. IARC Monogr. 37: 103-104, 1985. 42. Winn, D.M., Walrath, J., Blot, W., and Rogot, E. Chewing tobacco and snuff in relation to cause of death in a large prospective cohort (Abstract). Am. J. Epidemiol. 116: 567, 1982. 43. Smith, J.F., Mincer, H.A., Hopkins, K.P., and Bell, J. Snuff-dippers lesion: A cytological and pathological study in a large population. Arch. Otolaryngol. 92: 450-456, 1970. 44. Smith, J.F. Snuff dippers lesion, a ten-year follow-up. Arch.. Otolaryngol. 101: 276-277, 1975. 45. Waterhouse, J., Muir, C., Shanmugaranam, K., and Powell, J. Cancer incidence in five continents, Vol. IV. International Agency for Research in Cancer, Lyon, France, 1982. 46. Pindborg, J.J. Epidemiologic studies of oral cancer. Int. Dent. J. 27: 172-178, 1977. 47. Wahi, P.N. The epidemiology of oral and oropharyngeal cancer. Bull. WHO 38: 495-521, 1968. 48. Hirayama, T. An epidemiologic study of oral and pharyngeal cancer in Central and Southeast Asia. Bull. WHO 34: 41-69, 1966. 49. Paymaster, J.C. Cancer and its 1964. distribution in India. Cancer 17: 1026, 50. Orr, I.M. Oral cancer in betel nut chewers in Travancore. Lancet 2: 575-580, 1933. 2501258073 2-11

51. Chandra, A. Different habits and this relation with cheek cancer. Bull. Cancer Hosp. Natl. Cancer Res. Center 1: 33, 1962. 52. Shanta, V., and Krishnamurthi, S. A study of aetiological factors oral squamous cell carcinoma. Br. J. Cancer 13: 381, 1959. in in Karachi, 54. Jussawalla, D.J., and Deshpande, V.A. Evaluation of cancer risk in tobacco chewers and smokers: An epidemiologic assessment. Cancer 28: 244-252, 1971. 53. Jafary, N.A., and Zaidi, S.H. Carcinoma of the oral cavity Pakistan: An appraisal. Trop. Doct. 6: 63, 1976. 55. Gupta, P.C., Pindborg, J.J., and Mehta, F.S. Comparison of carcino- genicity of betel quid with and without tobacco. An epidemiological review. Ecol. Dis. 1: 213-219, 1982. 56. Jayant, K., Balakrishnan, V., Sanghvi, L.D., and Jussawalla, D.J. Quantification of the role of smoking and chewing tobacco in oral, pharyngeal, and esophageal cancers. Br. J. Cancer 35: 232-235, 1977.

Table 1 Smokeless Tobacco and Mouth Cancer, Case-Control Data From Moore et al. (23,24) Smokeless Tobacco Mouth Cancer Cases Controls Users 26 12 Nonusers 14 26 Totals 40 38 Crude RR = 4.0 95% confidence interval: 1.6-10

Table 2 Smokeless Tobacco and Mouth Cancer, Case-Control Data From Peacock et al. (27) Age 40 - 49 50 - 59 60 - 69 Smokeless Tobacco Case Controls Case Controls Case Controls User 0 16 7 13 18 20 Nonuser 5 14 6 16 9 37 Total 5 60 13 29 27 57 RR - 0 RR - 1.4 RR - 3.7 RRMH = 2.0 95% confidence interval : 1.0-4.2

Table 3 Estimated Relative Risks Associated With Snuff Use for Cancers of the Oral Cavity, Pharynx, and Larynx, Case-Control Data From Vogler et al. (28), Females Only Oral/Pharynx/ Larynx Other Mouth Disease Other Cancer No Cancer Urban Us er 15 1 5 4 Nonuser 23 56 165 373 Crude Relative Risk Estimate 60.8 1.7 2.8 1.0* Rural User 41 4 26 17 Nonuser 14 33 103 133 Crude Relative Risk Estimate 22.9 0.9 2.0 1.0* *Reference category.

Table 4 Smokeless Tobacco and Head and Neck Cancer by Anatomic Site, Case-Control Data From Vincent and Marchetta (29), Males Only x Smokeless Tobacco Use Control Larynx Pharynx Oral Cavity A11 Head and Neck User 5 2 3 9 14 Nonuser 95 21 30 24 75 To tal 100 23 33 33 89 Relative Risk Es timate 95% Confidence Interval 1.8 1.9 7.1 3.5 0.3-9.8 0.4-8.3 2.4-21 1.3-9.8 J.

Table 5 Estimated Relative Risk for Cancer of the Head and Neck From Smokeless Tobacco Use by Anatomic Site, Third National Cancer Survey (31), Males Only Relative Risk Estimate Anatomic Site Low Exposure High Exposure Gum-mouth 5.6 3.9 Pharyna Lip-tongue 0.6 0.3 1.1 Larynx 2.0 1.7

Table 6 Estimated Relative Risk of Oro-Pharyngeal Cancer According to Duration of Snuff Use and Anatomic Site, Winn et al. (21) Anatomic Site Duration of Snuff Use (yr) Relative Risk Estimate 95% Confidence Interval Gum and Buccal Mucosa 0 1.0 --- 1- 24 13.8 1.9 - 98 25 - 49 12.6 2.7 - 53 , >_ 50 48.0 9.1 - 250 Other Mouth 0 1.0 --- and Pharqaz 1- 24 1.7 0.4 - 7.2 25 - 49 3.8 1.5 - 9.6 > 50 1.3 0.5 - 3.2

Table 7 Relative Risk of Oral Cancer From Betel Quid With and Without Tobacco (With 95 Percent Confidence Limit) Study Location (References) Betel Quid With Tobacco Betel Quid Without Tobacco No Chewing Habit Calcutta, Cases 138 46 135 India (50,54) Controls 61 70 256 Relative risk estimates 4.3 (.3.0-6.1) 1.2 (0.8-1.9) Madras, Cases 219 33 25 India (51,54) Controls 35 144 99 Relative risk 25 (15-41) 0.91 (0.5-1.6) estimates Karachi, Cases 339 40 88 Pakistan (52,54) Controls 474 216 1,690 Relative risk 14 (11-17) 3.6 (2.4-5.2) estimates Bombay, Cases 238 44 129 India (53) Controls 513 152 1,340 Relative risk 4.8 (3.9-6.0) 3.0 (2.1-4.3) estimates India and Cases 120 3 6 Sri Lanka (47) Controls 63 8 47 Relative risk 15 (7.0-32) 2.9 (0.6-14) estiinates Remarks Smokers not included in these data. Only buccal mucosa cancers considered. Smokers not included in these data. Only buccal mucosa and tongue cancer cases included. Numbers reconstructed from percentages and totals. Smokers not included in these data. Separate analyses indicate that elevated risks of oral cancer associated with tobacco chew- ing are found among nonsmokers well as smokers. Smokers not included in these data. Only buccal mucosa cancer considered. I.oO$Szl0'Sz

,i EPIDEMIOLOGIC STUDIES OF OTHER CANCERS IN RELATION TO SMOKELESS TOBACCO USE The epidemiologic studies reported in the preceding section that show an association between the use of smokeless tobacco and oral cancers, particularly malignancies of the cheek and gum, indicate that the topical exposure of tissues to tobacco can cause cancers at the site of the exposure. In the United States, the tissues in direct prolonged contact with the tobacco are generally those of the oral cavity. Smokeless tobacco may occasionally come in contact with other tissues. One case has been reported of squamous cell carcinoma that developed in the ear of an individual in Minnesota who habitually placed snuff in his ear for 42 years at the site where the neoplasm developed (1). Although but a single report, this highly unusual observation raises the possibility of a carcinogenic potential of smokeless tobacco at other anatomic sites when exposure is direct and prolonged. Nasal Cancer In some areas of the world snuff is inhaled, so that tissues of the nasal cavity come in contact with the tobacco powder. The earliest report that links any form of tobacco to cancer was published over two centuries ago when what were probably nasal cancers were described in several patients in England who were heavy inhalers of snuff (2). There have been no systematic evaluations of snuff inhalation and nasal cancer in the United States, United Kingdom, or other European countries, most likely because both the sniffing habit and nasal cancer are uncommon. Sniffing snuff has been reported, however, to be a frequent habit among Bantu men, whose rates of nasal cancer have been reported to be high (3). In case-control studies of nasal sinus cancer reported in 1955, 80 percent of patients with tumors of the maxillary antrum were prolonged and heavy snuff users, in contrast to about one-third of Bantu men with other cancers (4,5). The snuff used by the Bantu is thought to contain aloe plant ash, trace elements such as nickel and chromium, and other ingredients in addition to tobacco (6). Snuff use (presumably by inhalation) was reported not to account for the high rates of nasal adeno- carcinoma among furniture makers in studies in England and Denmark, but evaluations of snuff itself as a risk factor were not undertaken (7,8). One case-control study of cancers of the nasal cavity and paranasal sinuses in the United States addressed the issue of smokeless tobacco (9). A total of 193 cases were identified in four hospitals in Virginia and North Carolina over a 10-year period. No association between sinonasal cancers and chewing tobacco was found (relative risk 0.7, 95-percent confidence interval 0.4-1.5). However, a relative risk of 1.5 was observed for users of snuff (95-percent confidence interval 0.8-2.8). Risk was increased in snuff users for both adenocarcinomas (relative risk 3.1) and squamous cell carcinomas (relative risk 1.9) but not for other histologic types (relative risk 0.6) and was found for both sexes. The implications of the findings are not clear since the snuff used by the cases and controls was oral snuff not coming in contact with nasal tissues. Animal experiments, however, suggest that tumors distant to the site of exposure may result from exposure to constituents of snuff (see the section on animal studies). An apparent excess of posterior nasal space tumors was reported among certain tribes in Kenya, and 6 of 12 cases interviewed were found to be 2501250a82

chronic "liquid snuff" users (10). Multiple subsites of the respiratory tract were considered, however, increasing the likelihood of a chance associa- tion. No increased risk of nasopharyngeal cancer associated with snuff use was noted in a case-control study in Singapore (11). Esophageal Cancer Other tissues that come in contact with constituents of smokeless tobacco in more dilute concentrations include the linings of the esophagus, larynx (supraglotic portion), and stomach. The results of studies of cancers of these three sites in relation to smokeless tobacco are inconclusive (38). The studies are generally of limited power to detect small increases in risk, and many did not control for relevant, potentially confounding variables. However, some studies of these three cancers do show an increase in risk in relation to the use of smokeless tobacco. As shown in table 1, elevated relative risks of esophageal cancer up to twofold or higher were found in two hospital-based case-control studies in the United States involving 150 and 183 cancer patients (12,13) and one in Puerto Rico (described in the previous section) with 179.cases (14). One of the studies by Wynder and colleagues, however, found no evidence of an increase in risk with duration of exposure, and all chewers were also smokers (12). The effect of smoking was not adjusted for in the other study (13). Another case-control study involving 120 black male cases of esophageal cancer was conducted in Washington, D.C. (15). Few of the cases or controls had used either chewing tobacco or snuff, suggesting that it did not contribute to the high rates of esophageal cancer observed in the area. Finally, data from a prospective (cohort) study of U.S. veterans were analyzed to determine whether mortality rates of specific diseases were increased in users of smokeless tobacco (16). In the absence of smoking, the standardized mortality ratio for esophageal cancer was found to be 228, but this value was based on only one death. In a cohort study of 12,945 Norwegian and 16,930 American men followed over 10 years, the risk of esophageal cancer was reported to be significantly increased among men who used chewing tobacco or snuff, after controlling for age, residence, and smoking habits (17,18). Unfortunately, the results of both cohort studies have been published only as abstracts, so additional details are not available. Some evidence that the chewing of quids may increase the risk of esopha- geal cancer arises from studies in southeast Asia. In a series of 237 cases of esophageal cancers in Sri Lanka, interview information from 111 revealed that 90 (81 percent) habitually used betel containing tobacco leaf (19). This percentage was considerably higher than the frequency of betel chewing in the general population (30 percent). Betel chewing was more common among women. Esophageal cancer also was more common among women, an unusual obser- vat' on since this cancer occurs more frequently among men in almost all areas of the world that report standardized cancer statistics (20). Since few women were reported to smoke or use alcohol, the possibility of an etiologic role of chewing is increased. However, the potential effects of tobacco as opposed to other ingredients in the quids cannot be distinguished. In a case-control investigation in Bombay involving interviews with 305 esophageal cancer patients and nearly 2,000 population controls of age, sex, and religions similar to all head and neck cancer cases, a 2.5-fold increased risk of esophageal malignancy was observed (p < .01) among nonsmokers who chewed pan, a mixture usually consisting of tobacco, betel, lime, and other ingredients 2501250083 2-21

a`~!1 '. Y . .~ i p':~'~'~Y`~C~ imw (21). The excess was higher, however, among those chewing quids without tobacco (relative risk 3.5) than with tobacco (relative risk 2.1). A more recent analysis (22) in Bombay based on 649 patients with esophageal cancer and 649 controls yielded similar qualitative findings, but the excess among users of pan without tobacco (relative risk 12.1) was accentuated compared to users of tobacco-containing chews (relative risk 2.8). On the other hand in an earlier case-control investigation in southern India of several upper di- gestive tract tumors, including 93 esophageal cancers, increases-in esophageal cancer risk were much greater among men who used betel with tobacco (calculated relative risk 11) than without tobacco (calculated relative risk 2) (23). The chewing of nass was not associated with esophageal cancer risk in a case-control study conducted in an area of Iran with among the World's highest rates for this cancer (24). Of 638 identified cases of esophageal cancer, interviews were completed with 344 and with two neighborhood controls matched to each case. The relative risk associated with ever using nass was 0.9, with an upper limit of the 95-percent confidence interval of 1.5, suggesting that any major effect of nass on the origins of this cancer could be excluded. Laryngeal Cancer In a case-control analysis of the interview data from the Third National Cancer Survey (TNCS), Will.iams and Horm compared the prior use of smokeless tobacco products (in the aggregate) in persons with a variety of iadividual types of cancer (including laryngeal cancer) with the history of such use in persons with the remaining cancers thought not to be related to tobacco use (25). Prior experience with smokeless tobacco was divided into two levels of exposure. The estimates of the relative risks were controlled for age, race, and smoking. Relative risks of laryngeal cancer in men of 2.0 and 1.7 were found among individuals with low and high levels, respectively, of exposure to chewing tobacco or snuff. These estimates were not significantly different from'one. They are based on 106 cases, 11 with relatively low exposure and five with higher exposure, and 2,102 controls of which 98 had low exposure and 71 had high exposure. Only 13 female laryngeal cases were available•for analy- sis in this study, which was insufficient to provide any meaningful results. A case-control study by Wynder and Stellman included 387 male cases of laryngeal cancer and 2,560 hospital controls (13). The percentages that had previously used chewing tobacco and snuff were 11.9 and 3.9, respectively, for the cases, and 9.0 and 2.7, respectively, for the controls. Based on these findings, crude relative risks of 1.4 for chewing tobacco and 1.5 for snuff were obtained. Neither estimate differs significantly from one. No control for smoking or alcohol was done, although the authors state that cigarette smoking in users and nonusers of chewing tobacco was similar. Interviews with 560 laryngeal cancer patients and 2,000 controls from the general population of Bombay revealed significantly increased risks, com- pared to nonchewers, among chewers of betel without tobacco (relative risk 2.5) than with tobacco (relative risk 2.6) (21). Laryngeal cancer was noted to comprise an unusually high proportion of all cancer diagnoses in a hospital series in eastern India where pan chewing is common, but no assessment of the role of tobacco was made (26). 2501258084

Stomach Cancer Zacho et al. noted that, in Denmark, both gastric cancer and use of chewing tobacco and snuff are directly related to age, more common in men than women, more prevalent in rural than urban areas, and inversely related to socioeconomic status (27). On the basis of these observations, they hypothesized that use of smokeless tobacco increases the risk of stomach cancer. Obviously, other differences among individuals within Denmark could also explain these findings. Weinberg et al. conducted a case-control study of stomach cancer in a coal mining region of Pennsylvania (28). Cases who had died of stomach cancer from 1978 through 1980 were compared with three control groups: persons who died of other cancers of the digestive system, persons who died of arterial sclerotic heart disease, and persons who lived in the same neigh- borhood as the case. A11 controls were matched to individual cases on age, sex, race, and location of residence. Data on the use of various forms of.,, tobacco were obtained by interviewing aext-of-kin or (for neighborhood con- trols) the subjects themselves. About 16 percent of all men in the study had used chewing tobacco. This percentage did not differ significantly among the cases and the three control groups. No women in this study had chewed tobacco. This study provides some evidence to suggest that chewing tobacco does not increase the risk of gastric cancer, although a small increase in risk could have been missed due to lack of statistical power. The case-control analysis of the interview data from the TNCS found a relative risk of stomach cancer of 1.7 in men in the highest level of use of chewing tobacco and snuff, no increase in men in the lower use category, and no increase in women (25). These results are based on 120 male cases, 12 of which were users, and 82 female cases, two of which were users. The power of this analysis to detect a true increase in risk is obviously low. The relative risk of 1.7 was not significantly greater than one. In an abstract describing a cohort mortality study of U.S. veterans, the standardized mortalit;r ratio for stomach cancer among nonsmoking users of smokeless tobaccq was 151, but no study details were provided (16). Urinarv Tract Cancer Constituents of smokeless tobacco can enter the blood stream, and some are excreted in the urine. The kidney and bladder are thus potentially exposed to these agents but presumably in lower concentrations than are tissues of the upper aerodigestive tract. In a hospital-based case-control study in Seattle, Washington, patients who chewed tobacco were reported to be at nearly a fivefold increased risk of renal cancer compared to nontobacco users (29). Only 6 percent of the 88 male cases were chewers. No association between the use of smokeless tobacco products and either renal cell or renal pelvis cancer was reported in a case-control study of these tumors in England (30). Among 106 renal cell cancer case-control pairs in this study, 10 cases versus 11 controls had at sometime used smokeless tobacco. Among 33 renal pelvis cancer-control pairs, 2 cases and 3 controls reported ever using smoke- less tobacco products. In a large population-based study in Minnesota involv- ing 495 cases and 697 controls, a nonsignificantly increased relative risk of renal cell cancer of 1.7 (95-percent confidence interval 0.5-6.0) was found 2501250085 2-23

~i: among snuff users after adjusting for smoking (31). There was a deficit in risk, however, associated with ever using chewing tobacco (relative risk 0.4, 95-percent confidence interval 0.1-2.6). A review of eight epidemiologic investigations revealed no consistent evidence that the risk of bladder cancer is altered in users of smokeless tobacco products (table 2) (13,25,32-39). The National Bladder Cancer Study is the largest of the investigations of bladder cancer considered in this review (37). Cases for this study were selected through 10 population-based cancer registries in the United States. Controls were a random sample of the same population from which the cases came. Information was obtained fro'm interviews of 2,982 cases and 5,782 controls. Analyses of smokeless tobacco use were restricted to the 340 cases and 1,227 controls who claimed never to have smoked cigarettes. Of these, 11 percent of the cases and 10 percent of the controls had ever used chewing tobacco, and 3 percent of the cases and 4 percent of the controls had ever used snuff. The relative risks of bladder cancer in users of chewing tobacco and snuff were estimated to be 1.0 (0.7-1.5) and 0.8 (0.4-1.6), respectively. A Wynder et al. conducted a hospital-based study of 300 male bladder can- cer cases (32). Eleven percent of the 300 cases and 8 percent of the 300 hospital controls had ever used chewing tobacco; 2 percent of the cases and 3 percent of the controls had used snuff. The percentage of users was not sig- nificantly different in cases and controls, and no attempt was made to analyze the data further. flunham'et al. interviewed 493 bladder cancer patients and 527 hospital- ized controls in New Orleans (33). Among nonsmokers, there was an increased relative risk associated with chewing tobacco use among males but a deficit in risk associated with snuff use among females, but the numbers of cases involved were.small (four males and three females). Cole et al. interviewed 470 cases from the Boston area and 500 popula- tion-based controls (34).. Forty-six of the cases had used chewing tobacco and three had used snuff. Based on the prior experience with smokeless tobacco in the controls (controlling for age and sex), 42.3 and 7.9 cases would have been expected to have used chewing tobacco and snuff, respectively. Some increase in the risk of bladder cancer was found in the TNCS survey, but none of the risks from this study are significantly different from one (table 1) (25). In addition, no evidence of a dose response is seen. In a second hospital-based case-control study (13) of similar design to the first (32), Wynder and Stellman found that 8 percent and 1.9 percent of 586 cases had used chewing tobacco and snuff, respectively, compared to 9 percent and 2.7 percent of 2,560 controls who had used these two products. When analyses were restricted to nonsmokers in a continuation of this study, a significant excess risk of bladder cancer was associated with snuff use among women, but only 3 of 76 cases were users (35). A population-based case-control study was conducted in three Canadian provinces by Howe et al. (36). Controls were matched to individual cases on neighborhood, age, and sex. The ratio of male pairs discordant for the use of chewing tobacco was 29/34, giving a relative risk of 0.9 (95-percent 2501258086 2-24

confidence interval, 0.5-1.6). This estimate was not altered by controlling for smoking. No female cases or controls gave a prior history of use of smokeless tobacco. In Denmark, 165 male and 47 female patients with cancer of the urinary bladder from a hospital serving a specific geographical area were interviewed, as were geographically-matched controls (38,39). The estimated relative risk associated with tobacco chewing was 2.0 (1.2-3.4) based on 39 exposed cases. In a logistic model containing variables for tobacco chewing, smoking, and other major correlates of bladder cancer, the relative risk associated with chewing was 1.7 and statistically significantly higher than 1.0. The authors estimated that tobacco chewing might account for 9 percent of the bladder cancer diagnoses in the area. Although two studies did report elevated relative risks associated with smokeless tobacco use, on balance these studies provide little evidence to suggest that smokeless tobacco alters the risk of bladder cancer. It is possible that a small increase in risk has not been detected by the studies not reporting increases due to lack of statistical power. Other Cancers All other organs of the body are likely exposed to even lower concentra- tions of products of smokeless tobacco via the blood. In a large prospective study in Norway, 16,713 individuals were inter- viewed to obtain information on the use of.tobacco and alcohol and were followed up for development of pancreatic cancer (40). Sixty-three persons in the cohort developed this neoplasm during a 10-year followup. After controlling for cigarette smoking and alcohol consumption, a relative risk of 2.9 was observed in regular users of chewing tobacco or snuff (compared to nonusers). The 95 percent confidence limits of this value include one. Risk was greater in regular users than former or occasional current users, and a trend of increasing risk with amount used was of borderline statist4cal significance (P=.06). The case-control analysis of the interview data from the TNCS (24) with respect to pancreas cancer is based on only 91 male cases (3 exposed to smokeless tobacco) and 85 female cases (none exposed); and although no increase in relative risk of pancreatic cancer in relation to smokeless tobacco was observed, the power of this study to detect such an increase is low. Other cancer sites were found to be related to the use of smokeless to- bacco in the case-control analysis of the interview data from the TNCS (24). Relative risks for colon cancer at low and high levels of exposure were found to be 0.9 and 1.5 for men and 0.4 and 2.0 for women, respectively. Relative risks of cervical cancer in users of these two levels of exposure were 3.1 and 2.3. No studies have been conducted to confirm or refute these findings. In view of the large numbers of possible associations investigated, these results should be considered of value only in generating hypotheses for fur- ther investigation. 2501258a87

X Summary The epidemiologic studies showing an association between the use of snuff and oral cancers indicate that topical exposure of tissues to smokeless tobacco can cause cancers at the site of the exposure. Case reports of neo- plasms developing in the ear and nose of individuals who used snuff at these sites raise the possibility that direct exposure may increase the risk in locations besides the oral cavity. Other tissues that come in contact with constituents of smokeless tobacco in more dilute concentrations include the linings of the esophagus, larynx (supraglotic portion), and stomach. Results of studies of cancers of these three sites in relation to smokeless tobacco are inconclusive; many are of limited power to detect small increases in risk and did not control for relevant, potentially confounding variables. However, some studies of these three cancers do show an increase in risk in relation to the use of smokeless tobacco. Constituents of smokeless tobacco can enter the bloodstream, and some are excreted in the urine. The kidney and bladder are thus potentially exposed to these products and their metabolites but pre- sumably in lower concentrations than are tissues of the upper aerodigestive tract. Evidence suggests that the risk of bladder cancer is not altered to any large extent in users of smokeless tobacco products, but results from studies of kidney cancer are inconsistent. Information regarding the risks of other cancers in relation to smokeless tobacco use is sparse. References 1. Root, H.D., Aust, J.B., and Sullivan, A. Snuff and cancer of the ear. N. Engl. J. Med. 262: 819-820, 1960. 2. Redmond, D.E. Tobacco and-cancer: The first clinical report, 1761. N. Engl. J. Med. 282: 18-23, 1970. 3. Higginson, J., and Oettle, A.G. Cancer incidence in the Bantu and Cape colored races of South Africa: Report of a cancer survey in the Transvaal. J. Natl. Cancer Inst. 24: 589-671, 1960. - 4. Shapiro, M.P., Keen, P., Cohen, L., and de Moor, N.G. Malignant disease in the Transvaal, III. Cancer of the respiratory tract. S. &fr. Med. J. 29: 95-101, 1955. 5. Keen, P., de Moor, N.G., Shapiro, M.P., and Cohen L. The aetiology of respiratory tract cancer in the South African Bantu. Br. J. Cancer 9: 528-538, 1955. 6. Baumslag, N. Carcinoma of the maxillary antrum and its relationship to trace metal content of snuff. Arch. Environ. Health 23: 1-5, 1971. 7. Acheson, E.D., Hadfield, E.H., and Macbeth, R.G. Carcinoma of the nasal cavity and accessory sinuses in woodworkers. Lancet 1: 311-312, 1967. 8. Anderson, H.C., Anderson, I., and Solgaard, J. Nasal cancers, symptoms, and upper airway function in woodworkers. Br. J. Int. Med. 34: 201-207, 1977. 2501258088

9. Brinton, L.A., Blot, W.J., Becker, J.A., et al. A case-control study of cancers of the nasal cavity and paranasal sinuses. Am. J. Epidemiol. 119: 896-905, 1984. 10. Hou-Jensen, R. On the occurrence of post nasal space tumors in Kenya. Br. J. Cancer 18: 58-68, 1964. 11. Shanmugaratnam, K., and Higginson, J. Etiology of nasopharyngeal car- cinoma: Origin and structure. In: C. Muir, K. Shanmugaratnam (eds.). Cancer of the Nasopharynx. UICC Monogr. 1: 153-162, 1967. 12. Wynder, E.L., and Bross, I.J. A study of etiological factors in cancer of the esophagus. Cancer 14: 389-413, 1961. 13. Wynder, E.L., and Stellman, S.D. Comparative epidemiology of tobacco- related cancers. Cancer Res. 37: 4608-4622, 1977. 14. Martinez, I. Factors associated with cancer of the esophagus, mouth, and pharynx in Puerto Rico. J. Natl. Cancer Inst. 42: 1069-1094, 1969. 15. Pottern, L.M., Morris, L.E., Blot, W.J., et al. Esophageal cancer among black men in Washington, D.C., 1. Alcohol, tobacco, and other risk factors. JNCI 67: 777-783, 1981. 16. Winn, D., Walrath, J., Blot, W., and Rogot, E. Chewing tobacco and snuff in relation to cause of death in a large prospective cohort (Abstract). Am. J. Epidemiol. 116: 567, 1982. 17. Bjelke, E., and Schuman, L.M. Chewing tobacco and use of snuff: Relationships to cancer of the pancreas and other sites in two prospectives studies. Proceedings of the 13th International Congress on Cancer, 1982, p. 207. 18. International Agency for Research on Cancer. Tobacco habits other than smoking: Betel-quid and areca-nut chewing; and some related nitrosamines. IARC Monogr. 37: 103-104, 1985. 19. Stephen, S.J., and Uragoda, C.G. Some observations on oesophageal carcinoma in Ceylon, including its relationship to betel chewing. Br. J. Cancer 24: 11-15, 1970. 20. Waterhouse, J., Muir, C., Shanmugaratnam, and Powell, J. Cancer incidence in five continents, Vol. IV. International Agency for Research on Cancer, Lyon, France, 1982. 21. Jussawalla, D.J., and Deshpande, V.A. Evaluation of cancer risk in tobacco chewers and smokers: An epidemiologic assessment. Cancer 28: 244-252, 1971. 22. Jussawalla, D.J. Oesophageal cancer in India. J. Cancer Res. Clin. Oncol. 99: 29-33, 1981. 2-27

23. Shanta, V., and Krishnamurthi, S. Further study in aetiology of carcinomas of the upper alimentary tract. Br. J. Cancer 17: 8-23, 1963. 24. Cook-Mozaffari, P.J., Azordkegan, F., Day, N.E., Ressicaud, A., Sabai C., and Aramesh, B. Oesophageal cancer studies in the Caspian littoral of Iran: Results of a case-control study. Br. J. Cancer 39: 293-309, 1979. 25. Williams, R.R., and Horm, J.W. Association of cancer sites with tobacco and alcohol consumption and socioeconomic status of patients. Interview study from the Third National Cancer Survey. J. Natl. Cancer Inst. 58: 525-547, 1977. 26. Sarma, S.N. A study into the incidence and etiology of cancer of the larynx and adjacent parts in Assam. Indian J. Med. Res. 46: 525-533, 1958. ~ 27. Zacho, A., Nielsen, J., and Larsen, V. On the consumption of unburned tobacco in patients with cancer of the stomach. Acta Chir. Scaad. 134: 272-274, 1968. 28. Weinberg, G.B., Kuller, L.H., and Stehr, P.A. A case control study of stomach cancer in a coal mining region of Pennsylvania. Cancer 56: 703-713, 1985. i 29. Bennington, J.L., Campbell, P.B., and Ferguson B.R. Epidemiologic studies of carcinoma of the kidney, II. Association of renal adenocarcinoma with smoking. Cancer 22: 821-823, 1968. 30. Armstrong, B., Garrod A., and Doll R. A retrospective study of renal cancer with special reference to coffee and animal protein consumption. Br. J. Cancer 33: 127-136, 1976. 31. McLaughlin J.K., Mandel J.S., Blot, W.J., Schuman, L.M., *ieh1, E.S., and Fraumeni, J.P. Population-based case-control study of renal• cell carcinoma. JNCI 72: 275-284, 1984. 32. Wynder, E.L., Onderdonk, J., and Mantel, N. An epidemiological invest- igation of cancer of the bladder. Cancer 11: 1388-1406, 1963. 33. Dunham, L.J., Rabson, A.S., Stewart, H.L., Frank, A.S., and Young, J.L. Rates, interview and pathology study of cancer of the urinary bladder in New Orleans, Louisiana. J. Natl. Cancer Inst. 41:683-709, 1968. 34. Cole, P., Monson, R.R., Haning, H., and Friedell, G.H. Smoking and cancer of lower urinary tract. N. Engl. J. Med. 284: 129-134, 1971. 35. Kabat, G.C., Dieck, G.S., and Wynder, E.L. Bladder cancer in nonsmokers. Cancer 57: 362-367, 1986. 36. Howe, G.R., Burch, J.D., Miller A.B., et al. Tobacco use, occupation, coffee, various nutrients, and bladder cancer. JNCI 64: 701-713, 1980. 2501258090

37. Hartge; P., Hoover, R.,'and Kantor, A. Bladder cancer risk and pipes, cigars, and smokeless tobacco. Cancer 55: 901-906, 1985. 38. Mommsen, S., Aagaard, J., and Sell, A. An epidemiologic study of bladder cancer in a predominantly rural district. Scand. J. Urol. Nephrol. 17: 307-312, 1983. 39. Mommsen, S., and Aagaard, J. Tobacco as a risk factor for bladder cancer. Carcinogenesis 4: 335-338, 1983. 40. Heuch, I., Kvale, G., Jacobsen, B.K., and Bjelke, E. Use of alcohol, tobacco and coffee, and risk of pancreatic cancer. Br. J. Cancer 48: 637-643, 1983.

Table 1 Relative Risks of Esophageal Cancer in Persons Exposed to Chewing Tobacco and Snuff: Summary of Four Case-Control Studies First Type of Level of Cases Controls Relative Author Ezposure Exposure Sex No. X Exposed No. X Exposed Risk* Wynder (12) Chewing Any M 150 20 150 10 2.3 < 10 yrs. 14 4 3.9 2:10 yrs. 6 6 1.2 Williams (24) Chewing Level 1 M 38 5.2 1,788 5.4 0.9 or snuff Level 2 0 0 --- Wynder (13) Chewing Any M 183 10.9 2,560 9.0 1.2 Snuff Any M 4.4 2.7 1.7 Martinez (14) Chewing** Any M 120 2.5 360 3.6 1.2 F 59 11.9 177 7.3 2.7 *Calculated from published report if not provided by author. **Restricted to nonsmokers.

Table 2 Estimates of Relative Risks of Bladder Cancer in Persons Who Have Ever Used Chewing Tobacco and Snuff Relative Risks First Years Author Cases (ref.) Diagnosed Sex Chewing Tobacco Both Snuff Wynder (32) 1957-63 Male 1.4* 0.7* Dunham et al. 1958-64 Male 5.3*# 0.9*# - (33) Female 1.1*# - 0.3*# Cole et al. 1966-68 Both 1.1* 1.0* (34) Williams and 1969-71 Male-level 1 1.61 Horm (25) -level 2 1.15 Female-level 1 0 -level 2 1.78 Wynder and 1974-75 Males Stellman (13) 0.9 0.7 Howe et al. 1974-76 Males 0.9 (36) Hartge, et al. 1977-78 Males 1.02 0.77# (37) *Estimated from published report. #Based on analysis of nonsmokers only. N LrI ~ ~ N Ln C~0 a ~ w

CHEMICAL CONSTITUENTS, INCLUDING CARCINOGENS, OF SMOKELESS TOBACCO K Chemical ComDosition of Smokeless Tobacco To date, at least 2,500 known compounds have been identified in processed tobacco (1). Besides polysaccharides and protein, tobacco contains Nicotiana alkaloids (0.5-5.0 percent), alkanes (0.1-0.4 percent), terpenes (0.1-3.0 per- cent), polyphenols (0.5-4.5 percent), phytosterols (0.1-2.5 percent), carboxylic acids (0.1-0.7 percent), aromatic hydrocarbons, aldehydes, ketones, aminqs, amides, nitriles, N- and 0-heterocyclic compounds, chlorinated organic com- pounds, alkali nitrates (0.2-5.0 percent), and at least 30 metal compounds (2,3). - The most important habituating agent in tobacco is nicotine, the major representative of the alkaloids that constitute 0.5-5 percent of the leaf depending on the strain, variety, and agricultural practices that 4re employed during the tobacco cultivation. In total, the alkaloids are composed of 85 to 95 percent nicotine (4) and of other major alkaloids such as the secondary amines nornicotine, anatabine, and anabasine with lesser amounts of cotinine, myosmine, nicotyrine, 2,3'-dipyridyl, and N'-oxynicotine (5). Carcinogens in Smokeless Tobacco At present, three classes of carcinogens are known to occur in smokeless tobacco products: N-nitrosamines, polynuclear aromatic hydrocarbons (PAH), and polonium-210 (210Po). Although chemical-analytical data are lacking, some smokeless tobacco mixtures contain or are suspected to contain traces of cadmium and nickel compounds (6), formaldehyde, and coumarin, all of which are known animal carcinogens (7,8). N-Nitrosamines Tobacco leaves contain an abundance of amines in the form of proteins and alkaloids. Tobacco also contains up to 5 percent nitrates and traces of nitrite. Thus there is the potential for the formation of N-nitrosamines from the nitrate, nitrite, and amines during the processing of smokeless tobacco products. In tobacco, we distinguish between volatile nitrosamines, nonvola- tile nitrosamines, and tobacco-specific nitrosamines (figure 1). With the exception of some N-nitrosamino acids, the nitrosamines in tobacco are animal carcinogens that are formed after harvesting of the tobacco during curing, fermentation, and/or aging. The N-nitrosamino acid, N-nitrosoproline, occurs- in processed food and can also be formed in humans by endogenous nitrosation of proline. This nitrosamino acid is not carcinogenic on the basis of presently available data (9-12). Table 1 summarizes the available data for the volatile nitrosamines in smokeless tobacco. Only one of the volatile nitrosamines, NDMA, has been found in U.S. looseleaf tobacco, but four nitrosamines have been found in American snuff. N-Nitrosomorpholine is formed during tobacco processing or aging from morpholine, a cyclic amine that is not known to occur in uncontaminated tobacco (13,14) but originates from packing materials and/or flavor additives. Table 2 lists the presently known nonvolatile nitro- samines in smokeless tobacco. N-Nitrosodiethanolamine (NDELA) in U.S. tobacco originates primarily from residues on tobacco leaves of the sucker-growth in- hibitor maleic hydrazidediethanolamine (MH-30). Use of this formulation of 2501250094

~_~~ 1xLa_~r,•.. -. . ;. the agricultural spray was banned in the United States in 1981, and the con- centration of NDELA in smokeless tobaccos has markedly decreased since then (14,15). Figure 2 presents the formation of the tobacco-specific N-nitrosamines (TSNA) from the alkaloids. There is progressive nitrosation of the alkaloids during curing and processing and even during the shelf life of the commercial products (16). Table 3 summarizes the presently available quantitative data for four out of five TSNA's that are present in smokeless tobacco. The nitrosamines are detectable in snuff and tobacco products from various parts of the world. Analyses of Swedish snuff brands manufactured between 1980 and 1985 have revealed a significant decrease of the levels of TSNA; such a trend has not been observed for U.S. snuff brands (14,16,17). It has been suggested that the lowering of TSNA levels in Swedish snuff brands is due to better control of the bacterial content of the tobacco products. Reduced bacterial activity will probably reduce nitrite levels and, consequently, inhibit nitrosamine formation (17). NNK and NNN are powerful carcinogens in mice, rats, and hamsters, NAB is moderately carcinogenic, and NAT is inactive in rats in doses up to 9 mmol/kg (table 3, 2-83) (3). The daily exposure of an "average" snuff dipper to carcinogenic N-nitro- samines exceeds by at least 2 orders of magnitude the estimated exposure of U.S. residents to aitrosamines in products other than tobacco products (table 4) (18,19). Furthermore, the concentrations of carcinogenic nitrosamines in snuff exceed very significantly the permissible limits for individual nitro- samines in consumer products (table 5). During snuff dipping or chewing of tobacco, the TSNA's are extracted by the saliva. Consequently, the saliva of snuff dippers are reported to contain 5.0-420 ppb of NNN, up to 96 ppb of NNK, and 6.6-555 ppb of NAT (16). The saliva analyses of Indian tobacco chewers showed the presence of 1.2-220 ppb of :TNN, 3.2-51.7 ppb of NAT, and up to 2.3 ppb of NNK (20,21). Recently, three additional TSNA's have been isolated from U.S. commercial snuff: 4- (methylnitrosamino)-1-(3-pyridyl)butanol-1 (NNAL), 4-(methylnitrosamino)-l- (3-pyridyl)butene-1 (NNO) and 4-(methylnitrosamino)-4-(3-pyridyl)butanol-1 (Red NNA) (figure 3) (22). Additional amounts of TSNA's are most likely also formed by aitrosation processes that occur in the oral cavity during chewing (19-21,23). Polynuclear Aromatic Hydrocarbons A number of naphthalenes have been identified in processed tobacco and especially in Latakia, which is flavor enriched by treatment with wood smoke (24,25). FTh.ile smoking tobaccos were found to contain 300-5,000 ppb of phenanthrene, 110-4,200 ppb of anthracene, 76-1,800 ppb of pyrene, 15-14,000 ppb of fluoranthene, and 8.5 ppb of benzo(a)pyrene (BaP) (26,27), analyses of British snuff in 1957 showed levels of 260 ppb of pyrene, 335 ppb of fluoranthene, and 72 ppb of BaP (28). In the five most popular snuff brands in the United States that were analyzed in 1985, BaP ranged from 0.1 to 63 ppb (29). Polonium-210 This alpha-emitting element has long been incriminated as a human carcinogen (30). The levels of 210Po in dozens of U.S. and foreign cigarette tobaccos 2501250095 2-33

were between 0.1 and ~.0 pCi/g (31). In recent samples of the five leading N. snuff brands, 21 Po ranged from 0.16 to 1.22 pCi/g (29). It appears that Po in tobacco leaves stems partially from certain types of fertilizers and airborne particles that are taken up by the trichomes (glandular hair) of the tobacco leaf (31-33). Summary In processed tobacco, more than 2,550 chemical compounds have beea identi- fied. Among these are traces of known carcinogens such as PAH, 210Po, and N-nitrosamines. The most prevalent organic carcinogens are the tobacco-specific N-nitrosamines that are formed from the Nicotiana alkaloids during the proces- sing of tobacco leaves. Their concentrations ia snuff exceed the levels of aitrosamines in other consumer products by over 100-fold. During snuff dipping or chewing of tobacco, the aitrosation process continues within the mouth stimulated by ora]l bacteria. References 1. Dube, M.F., and Green, C.R. Methods of collection of smoke for analytical purposes. Recent Advan. Tobacco Sci. 8: 42-102, 1982. 2. Wynder, E.L., and Hoffmann, D. Tobacco and tobacco smoke. Studies in Experimental Carcinogeaesis. New York, Academic Press, 1967, p. 730. 3. International Agency for Research on Cancer. Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Tobacco habits other than smoking: Betel-quid and areca-nut chewing; and some related nitro- samines. IARC Moaogr. 37: 291, 1985. 4. Sisson, V.A., and Serverson, R.F. Alkaloid composition of the Nicotiana species, 24. Tobacco Chemists Res. Coaf., p. 10, 1984. 5. Piade, J.C., and Hoffmann, D. Quantitative determination of alkaloids in tobacco by liquid chromatography. J. Liquid Chromatogr. 3: 1505-1515, 1980. 6. Baumslag, N., Keen, P., and Petering, H.G. Carcinoma of the maxillary antrum and its relationship to trace and metal content in snuff. Arch. Environ. Health 23: 1-5, 1971. 7. Vainio, H., Hemminke, K. and Wilburn, 4. Data on the carcinogenicity of chemicals in the IARC Monographs Programme. Carcinogenesis 6: 1663-1665, 1985. - Grigg, G.W. Genetic effects of coumarins. Mut. Res. 47: 161-181, 1977/78. Ohshima H., and Bartsch H. Quantitative estimation of endogenous nitrosation in humans by monitoring N-nitroso proline excreted in t FV he GJ9 urine. Cancer Res. 41: 3658-3662, 1981. Q r Hoffmann D., and Brunnemann R.D. Endogenous formation of N-nitroso proline in cigarette smokers. Cancer Res. 43: 5570-5574, 1983. r~j E.n Da Q ~c CN

~•4bY..~1 . ~, .. _. .. .. . . ~ 'v r.v.... I •lP,tMaV..4 ~ 11. International Agency for Research on Cancer. Monograph on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 17. Some N-nitroso compounds. Lyon, France, 1978, p. 365. 12. Preussmann R., and Stewart B.W. N-nitroso carcinogens. In: C.E. Searle (ed.). Chemical Carcinogens, Second Edition. Am. Chem. Soc. Monogr. 182: 643-828, 1984. 13. Brunnemann, K.D., Scott, J.C., and Hoffmann, D. N-Nitrosomorpholine and other volatile N-nitrosamines in snuff tobacco. Carcinogenesis 3: 693-696:. 1982. 14. Brunnemann, K.D., Genoble, L., and Hoffmann, D. N-nitrosamines in chewing tobacco: An international comparison. J. Agr. Food Chem. 33: 1178-1181, 1985. 15. Brunnemann, K.D., and Hoffmann, D. Assessment of the carcinogenic N-nitro- sodiethanolamine in tobacco products and tobacco smoke. Carcinogenesis 2: 1123-1127, 1981. 16. Hoffmann, D., and Adams, J.D. Carcinogenic tobacco-specific N-nitro- samines in snuff and in the saliva of snuff dippers. Cancer Res. 41: 4305-4308, 1981. 17. Osterdahl B.G., and Slorach S. N-nitrosamines in snuff and chewing tobacco on the Swedish market in 1983. Food Additiv. Contamin. 1: 299-305, 1984. 18. National Research Council. In: The health effects of nitrate, nitrite and N-nitroso compounds (Ch. 7, Pt. 1). Washington, D.C., National Academic Press, 1981, p. 51. 19. Hoffmann, D., and Hecht, S.S. Nicotine-derived N-nitrosamines and tobacco-related cancer. Current status and future directions. Cancer Res. 45: 935-944, 1985. ' 20. Nair, J., Ohshima, H., Friesen, M., Croisy, A., Bhide, S.V., and Bartsch, H. Tobacco-specific and betel nut-specific N-nitroso compounds. Occur- rence in saliva and urine of betel quid chewers and formation in vitro by nitrosation of betel quid. Carcinogenesis 6: 295-303, 1985 21. Wenke, G., Rivenson, A., Brunnemann, K.D., and Hoffmann, D. Formation of N-nitrosamines during betel quid chewing. IARC Sci. Publ. 57: 859- 866, 1984. 22. Brunnemann, K.D., Chou, D., Adams, J.D., and Hoffmann, D. On the isolation and identification of new tobacco-specific N-nitrosamines (Abstract). 39th Tobacco Chemists Ref. Conf., Montreal, October 2-5, 1985. 23. Sipahimalani, A.T., Chada, M.S., Bhide, S.V., Pratap, A.I., and Nair, Y. Detection of N-nitrosamines in the saliva of habitual chewers of tobacco. Food Chem. Toxicol. 22: 261-264, 1984. 2501250097

24. Schmeltz, I., Tosk, J., and Hoffmann, D. Formation and determination of naphthalenes in cigarette smoke. Anal. Chem. 48: 645-650, 1976. 25. Nicolaus, G., and Elmenhorst, H. Nachweis and quantitative Bestimmung von Alkylnaphthalinen in Latakia-Tabak. Beitr. Tabakforsch. 11: 133-140, - 1982. 26. Campbell, J.M., and Lindsey, A.J. Polycyclic hydrocarbons extracted from tobacco: The effect upon total quantities found in smoke. -Br. J. Cancer 10: 649-652, 1956. 27. Onishi, I., Nagasawa, M., Tomita, H., and Fukuzumi, T. Studies on the essential oil of tobacco leaves, Part XVI. Neutral fraction (3). Polycyclic aromatic hydrocarbons of Burley tobacco leaf. Buil. Agr. Chem. Soc. Japan 22: 17-20, 1958. 28. Campbell, J.M., and Lindsey, A.J. Polycyclic aromatic hydrocarbons in snuff. Chem. Ind. London, 951, 1957. 29. Hoffmann, D., Harley, N.H., Fisenne, I., Adams, J.D., and Brunnemann, K.D. Carcinogenic agents in snuff. JNCI (in press). 30. Lundin, F.E., Jr., Wagoner, J.K., and Archer, V.E. Radon daughter expo- sure and respiratory cancer. Quantitative and temporal aspects. U.S. Dept. of Health, Education, and Welfare; Joint NIOSH/NIEHS Monogr. 1, 1971. 31. Harley, N.H., Cohen, B.S., and Tso, T.C. Polonium-210: A questionable risk factor in smoking-related carcinogenesis. Banbury Report. 3: 93-104, 1980. - 32. Martell, E.A. Radioactivity of tobacco trichromes and itisoluble cigarette smoke particles. Nature 249: 215-217, 1974. 33. Tso, T.C., Harley, N.H., and Alexander, L.T. Source of lead-to-tin and polonium-to-tin. Science 153: 880-882, 1966. 34. Brunnemann, K.D., Scott, J.C., and;Hoffmann, D. N-Nitrosoproline, an indicator for N-nitrosation of amines in processed tobacco. J. Agr. Food Chem. 31: 905-909, 1983. 35. Palladino, G., Adams, J.D., Brunnemann, K.D., Haley, N.J., and Hoffmann, D. Snuff-dipping in college students: A clinical profile. Military Med. (in press). 36. Oesterdahl, B.G., and Slorach, S.A. Volatile N-nitrosamines in snuff and chewing tobacco on the Swedish market. Food Chem. Toxicol. 21: 759-762, 1983. - 37. Brunnemann, K.D., Yu, L., and Hoffmann, D. Assessment of carcinogenic volatile N-nitrosamines in tobacco and in mainstream and sidestream smoke from cigarettes. Cancer Res. 37: 3218-3222, 1977. 2501258a98

38. Nair, J., Ohshima, H., Malaveille, C., Friesen, M., Bhide, S.V., and Bartsch, H. N-Nitroso compounds (NOC) in saliva and urine of betel quid chewers: Studies on occurrence and formation. Carcinogenesis 6: 295-303, 1985. 39. Ohshima, H. Identification and occurrence of new N-nitrosamino acids in human urine and environmental samples. Presented at the Conference on Organic and Biological Chemistry of Carcinogenic and'Carcinostatic Agents Containing Nitrogen-Nitrogen Bonds, Harper's Ferry, West Virginia, May 17-21, 1985. 40. Ohshima, H., Nair, J., Bourgade, M.C., Friesen, M., Garreen, L., and Bartsch, H. Identification and occurrence of two new N-nitrosamino acids in tobacco products: 3-(N-nitroso-N-methylamino)propionic acid and 4-(N-nitroso-N-methylamino)butyric acid. Cancer Lett. 26: 153-162, 1985. 41. Hoffmann, D., Hecht, S.S., Ornaf, R.M., Wynder, E.L., and Tso, T.C. Nitrosonornicotine: Presence in tobacco, formation and carcinogenicity. IARC Sci. Publ. 14: 307-320, 1976. 42. Munson, J.W., and Abdine, H. Determination of N-nitrosonornicotine in tobacco by gas chromatography/mass spectroscopy. Anal. Letters 10: 777- 786, 1977. 43. Adams, J.D., Brunnemann, K.D., and Hoffmann, D. Rapid method for the analysis of tobacco-specific N-aitrosamines by gas-liquid chromatography with a thermal energy analyzer. J. Chromatogr. 256: 347-351, 1983. 44. U.S. Department of Agriculture, Food Safety and Quality Service. Nitrates, nitrites and ascorbates (or isoascorbates) in bacon. Fed. Reg. 43: 20992-20995, May 16, 1978. 45. U.S. Food and Drug Administration. Dimethyl nitrosamine in malt beverages; availability of guide. Fed. Reg. 45: 39341-39342, 1980. , 46. U.S. Food and Drug Administration. Action levels of total volatile N- nitrosamines in rubber baby bottle:nipples; availability of revised compliance policy guide. Fed. Reg. 49: 50789-50790, 1984.

Abbreviations BaP Benzo(a)pyrene NAB N'-Nitrosoanabasine NAT N' -Nitrosoanatabine ND Not detected NDEA Nitrosodiethylamine NDELA Nitrosodiethanolamine NDMA Nitrosodimethylamine NMBA Nitrosomethylbutyric acid NMOR Nitrosomorpholine NMPA Nitrosomethylpropionic acid NNAL 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol• NNK 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone NNN N'-Nitrosonornicotine NNO 4-(Methylnitrosami.no)-1-(3-pyridyl)butene-1 NPIC Nitrosopipecolic acid NPIP Nitrosopiperidine NPIPAC Nitrosopiperidine-acetic acid NPRO Nitrosoproline NPYR Nitrosopyrrolidine NPYRAC Nitrosopyrrolidiae-acetic acid PAH Polynuclear aromatic hydrocarbons 210Po Polonium-210 ' Red NNA 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol TSNA Tobacco-specific nitrosamines

Table 1' Volatile Nitrosamines in Smokeless Tobacco (ppb)* Product NDMA N PYR NPIP NMOR Reference U.S. Looseleaf** ND - 380 (4) ND - 1.2 (4) ND (4) ND - 2.5 (4) 13,14,17,34 Snuff ND - 215 (26) ND - 291 (16) ND - 107 (16) ND - 690 (26) 13,14,17,20, 29,34-37 Sweden Chewing Tobacco ND - 0.6 (4) 0.9 - 3.7 (4) ND - (2) ND - 0.8 (2) 17,36 Snuff ND - 60 (53) ND - 210 (27) ND - 0.5 (37) ND - 1.2 (53) 14,17,36 Canada Snuff 23 - 72.8 (2) 321 - 337 (2) 14 Denmark ~ Chewing Tobacco ND - 8.6 (6) 7.0 - 25.5 (6) ND - (4) ND - 32.8 (6) 17,36 w ~ Norway Chewing Tobacco 37 - 220 (2) 84.0 - 280 (2) 2.8- 15 (2) 28 - 37 (2) 17 India Chewing Tobacco ND - 0.56(4) 1.55 - 4.48 (4) ND (4) 14 U.S.S.R. Nass*** ND - (4) 1.74 - 8.82 (4) ND (4) 14 *Number in parentheses, number of samples analyzed. **One sample also'contained 8.6 ppb NDEA. ***Also contained ND - 69.6 NDEA (14). t0l8SZl0SZ

Table 2 Nonvolatile N1trosa.lnea 1n Swokelea• Tobacco (ppb)* Toba.•co Pruduct NI1EI.A PN1PA NWBA NPRO NPIfRAC NPIC U.S. Loureleaf 224 - 680 (3) 450 - 463 (2) Snuff 160 - 6,800 (1'1) 1,250 - 7,420 (S) 120 - 2,240 (5) 500 - 50,900 (13) ND - 2,000 (5) ND - 6,100 (5) SweJen Snuff 230 - 391) (8) 510 - 4,404) (12) ND - 260 (12) 890 - 29,500 (12) 100- 300 (5) ND - 5,560 (12) Canada Plug Tobacco I1/) (1) 100 (1) Snuff 1,180 - 2,720 (3) 8,800 - 16,600 (2) Cerrany Plug Tobacco 50 (2) 500 - 700 (2) IrIKhew ChewlnR Tobacco 1,6tN1 (1) 1/)0 (1) 3.300 (1) 200 (1) 100 (1) U.S.S.R. Naaa 40 (4) ND - 180 (4) lndla Chewln8 Tobacco 30 - 110 (4) 190 - 410 (4) eNunl.er In parentherce, muwber of ra.pleu aualyzeJ. Z04QSMS2 . NPIPAC Reference 13,14,34 ND - 1,500 13-15 34,38,39 100 - 200 (5) 14,15,38,40 14 14 14 200 (i) 40

Table 3 Tobacco-Specific N-Nitrosamines in Smokeless Tobacco (ppb)* Product NNN NNK NAT NAB Reference U.S. Looseleaf 620- 8,200 (9) ND 380 (4) 130- 2,300 (5) ND- 140 (5) 14,17,41,42 0 3 0 4 3) 43 Plug Tobacco 3,4 0- , 0 ( Snuff 1,600-135,000 (21) 100-13,600 (21) 1,560-338,000 (21) 10-6,700 (12) 6,14,16,17,38,42,43 Sweden Snuff 3,050-154,000 (34) 510- 2,950 (34) 1,600- 21,400 (34) 110- 150 (19) 14,16,17,38 Plug Tobacco 350- 2,090 (3) ND- 240 (3) 690- 1,580 (3) ND- 100 (3) 14,17 Canada Snuff 50,420- 79,100 (2) 3,200- 5,800 (2) 152,000-170,000 (2) 4,000-4,800 (2) 14 Norway ~ Snuf f 13,000- 29,000 (2) 2,700- 3,900 (2) 9,100- 16,000 (2) 1,000-2,400 (2) 17 Denmark Snuff 4,460- 8,000 (3) 1,350- 7,030 (3) 2,680- 6,170 (3) 16 Chewing Tobacco 210- 1,400 (4) ND- 210 (4) 300- 2,800 (4) ND- 60 (4) 17 Germany Plug Tobacco 1,420- 2,130 (2) 30- 40 (2) 330- 500 (2) 30- 50 (2) 14 Snuff 6,080- 6,700 (2) 1,500- 1,540 (2) 3,920- 4,370 (2) 16 U.S.S.R. Nass 120- 520 (4) 20- 130 (4) 32- 300 (4) 8- 30 (4) 14 India Chewing Tobacco 470- 2,400 (5) 130- 230 (4) 300- 450 (4) 30- 70 (4) 14,41 Belgium Chewing Tobacco 7,38() (1) 970 (1) 130 (1) 38 *Number in parontljeses, i-umbcr of sainples anal yzed. EQ 1$Sz1OSz

Table 4 Estimated Exposure of U.S. Residents to Nitrosamines* Source of Exposure Nitrosamines Primary Exposure Daily Intak Route ug/person e Beer NDMA Ingestion 0.34 Cosmetics NDELA Dermal Absorption 0.41 Cured meat; cooked bacon NPYR Ingestion 0.17 Scotch whiskey Cigarette smoking NDMA VNA** NDELA Ingestion 0.03 Inhalation 0.3 Inhalation 0.5 NNN NNK NAT+NAB Inhalation 6.1 Inhalation 2.9 Inhalation 7.2 16.2 -------------------------------------------------------------------------------- Snuff Dippingt VNA Ingestion 3.1 NDELA Ingestion 6.6 NN~t Ingestion 75.0 154.5 vNK Ingestion 16.1 NAT+NAB Ingestion 73.4 *From "The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds" Natl. Res. Council, 1981 (18), amended by data for snuff dipping (13). In addition it has been established that upon inhalation of the air in cars with new leather upholstery daily exposure amounts to 0.50 ug of NDMA and 0.20 ug of NDEA (18). **VNA, NDi4A + NE;iA + NDEA + >1PYR (37). tBrunnemann, et al. (13); average values LErom the leading 5 U.S. fine-cut tobaccos used for snuff dipping in 1981; assumed daily consumption 10 g/day of snuf f; VNA = NDMA + WPYR + NMOR.

Table 5 Permissible Limits for Individual N-Nitrosamines in Consumer Products Product Permissible Limit ppb (ug/kg) Agency Bacon (Meat) 5 USDA* Beer 5 FDA** Rubber Nipples of Baby Bottles 10 FDAt Range of Individual Nitrosamines Present in Snuff Tobaccos ppb (pg/kg) NNN 5,800 - 64,000 NNK 100 - 3,100 Range in the leading N AT 3,300 - 215,000 5 U.S. brands (1984/85) NAB 200 - 6,700 NDELA 160 - 6,800 Range in 13 U.S. brands (1980-1985) ' *No "confirmable levels of nitrosamines"' (44). **Regulation set for N-nitrosodimethylamine (45). tReguLation set for any individual volatile v-nitrosamine (46).

t. '~., ~i? ~,:~r'.?e`~'~ ~.:t':.<es Figure 1. N-ilIZ'RCSAlI7iES IP S?lORELESS SO8aCC0 1. Volatil. Nitrosamin.s ~N-NO R R--CH3 NDKA n Rm-C28s NDEA 2R NPIP 2. Nonvolatile Nitrosamin.s BO-CHZ-CHZ SO-CBZ-CH H3C-N-R 1 NO -NO NDELA Rs-CHZ-CHZ-COOB NMPA R--CH2-CH2-CHZ-C0OB NrlBA 3. Tobacco-Soecific Nitrosamiaes 2501250106 NAT NAB NMOR NO Rs-COOB NPRO R-C00H NPIC Rs-CHZCOOB NPYRAC R-CHZCOOH NPIPAC

UtT1l03JT70M 01/ MMLL ~•+e0 I ~ •2M Figure 2, IO]tK>?ZON 07 TOEaCCD-SPECZFIC 2RTROSA?fIHES M/Cat'txt lIOAlp=llt LNAUS1Mt AXaTjVtlt

Figure 3. Tobacco Spacific N-Nitrosamines In Snuff U.S. Brands-1985 © Nitrssaainss Coacattraiioa in SAni( tYs/9) (Ory Wsight) (e Raa A B NNN O . NAa ~ O p NAT Q • r M .a AL ~~. 'I N NAL Q . rN =0 CQ M p Gy R Aj a .. `/NA `Cx=O» . Reiatiw Catciwoyteicity +++ + ± +++ + 7 3.3 1.1 64 6.7 44 1.8 0.3 trace 5 1.3 1. +++Tumors with I mmol/kg; + tumors with 9 mmol/kg; tumors induced see table 4, page 2-19). + insignificant number of tumors with 9 mmol/kg; ? 2. Isolated amounts only. 3. <0.01 ug/g l 215 3.1 0.14 . tracti 1.8 (for type of not tested , 2-46

METABOLISM OF CONSTITUENTS OF SMOKELESS TOBACCO The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-l- butanone (NNK) and N'-nitrosonornicotine (NNN) are quantitatively the major known carcinogens that are present in snuff and other types of smokeless tob- acco. Molecular changes that are induced in the genetic material of tobacco chewers are most likely to arise from the metabolism of these two nitrosamines. Although present in similar quantities, N'-nitrosoanabasine (NAB). and N'-nitro- soanatabine (NAT) are less carcinogenic than NNR and NNN and are less likely to play an important role in the induction of oral cancer in man. Some snuff products contain considerable amounts of N-nitrosomorpholine (NMOR) and N-nitro- sodiethanolamine (NDELA); the former is a potent carcinogen. The levels of benzo[a]pyrene (BaP)'and 210po in snuff tobacco are low compared to those of the aitrosamines (see previous section). This section will focus on the routes of metabolic activation of the compounds that are most likely to be involved in the induction of tumors that are related to snuff use-NNK, NNN, and NMOR. , Metabolism of NNK The overall metabolic scheme for NNK, as determined by in vivo and in vitro studies in F-344 rats, Syrian golden hamsters', and A/J mice, is illustrated in figure 1 (1-4). A key feature of this metabolic scheme is the conversion of NNK to the aipha-hydrozy intermediate 4, which is unstable and undergoes spontaneous conversion to the keto aldehyde 8 and, most likely, methyl diazo- hydroxide (9). The latter is a methylating agent that is well known for its ability to methylate DNA forming 7-methylguanine, 06-methylguanine, 4-methyl- thymidine, and a spectrum of other products (5). Among these, 06-methylguanine, which is generated from precursors such as N-methylnitrosourea (~4U) or N- nitrosodimethylamine, has been unequivocally shown to be able to induce mis- coding during DNA replication, and the resulting point mutation is sufficient to activate proto-oncogenes (6,7). Many studies have demonstrated a correlation between 06-methylguanine persistence in replicating tissues and the initiation of the carcinogenic process, although it is clear in other cases tha~ additional factors are also involved (8,9). Recent studies have demonstrated that NNK can methylate target tissue DNA of rats; 7-methylguanine and 05-methylguanine have been detected in the DNA of rat lung, nasal mucosa, and liver but not in the nontarget tissues, kidney, and esophagus (10-14). These studies have also shown that, in the case of NNK, 06-methylguanine formation alone is not sufficient for tumor induction since persistent levels of 06-methylguanine in the lung were less than those observed upon treatment with equivalent quantities of N-nitrosodimethylamine, but the latter did not induce lung tumors (13). It is clear from these, and related studies with NNN, that DNA adducts are also formed via pyridyloxobutylation or related processes. Regardless of the mechanism, it is significant that YNK causes DNA methylation; this creates a mechanistic link between nicotine, the habituating factor in tobacco, and 06-methylguanine formation in DNA, as illustrated in figure 2. Immunoassay methods are currently being developed to detect 06-methylguanine in the NJ exfoliated oral cells of snuff dippers. Its presence can be inferred from LA the animal studies that are discussed above and by the demonstration that a human tissues, including buccal mucosa, can metabolize NNK by alpha-hydroxy- ~ lation (15). In this respect, it is significant that injection of Syrian ar golden hamsters with the methylating agent MNU, combined with irritation of ~ the buccal mucosa, resulted in the induction of oral cavity tumors (16). a ~

i4".`F A~1IR~ :~ " ~:a, %w"w~ 1'Y The pathway of NNK metabolism leading to the alpha-hydroxy intermediate 3 is also considered to be important in NNK carciaogenesis. This pathway gives rise to the electrophilic diazohydroxide 7. The properties of this intermediate have been investigated by using a model compound, 4-(carbethoxy- nitrosamino)-1-(3-pyridyl)-1-butanone (CNPB). Generation of 7 from CNPB is strictly analogous to the well-known ability of :VMU to generate methyl diazo- hydroxide. Mutagenicity assays in S. typhimurium of CNPB have shown that it is more mutagenic than NMU (17). Chemical model studies have demonstrated that it modifies the N2-position of deoxyguanosine (18). This adduct and other adducts that may be formed from the diazohydroxide 7 and related intermediates are likely to play an important role in tumor induction by NNK. Autoradiographic studies have demonstrated that radioactivity from [carbonyl-14C]NNK is firmly bound to target tissues of rats and hamsters (4,19) and to tissues of the Marmoset monkey (20). A third key feature of NNK metabolism is its rapid conversion in vivo and in cultured tissues from experimental animals and humans to its a'educed form, NNA1, which has similar tumorigenic activity to that of NPiK (1,3,4,15,21). NNA1 is slowly metabolized as indicated in figure 1 and also by reconversion to NNK. Like NNK, it methylates DNA in vitro and in vivo. While the full details of the NNIX-NNA1 equilibrium have not yet been elucidated, it is clear that NNA1 can act as a circulating source of NNK metabolites. It may play an important role in tissue-specific carcinogenesis by NNK. Metabolism of NNN Metabolic pathways of NNN are illustrated in figure 3. These pathways have been elucidated by in vivo and in vitro studies in rats, hamsters, and mice (2,3,22-29). The stable metabolite NNN-1-N-oxide (1) has tumorigenic activity somewhat less than that of NNN but is still an effective carcinogen in F-344 rats (30). Metabolism of NNN to the 2'- and 5'-hydroxy intermediates 2 and 5 constitutes a major pathway in vivo and in vitro in experimental animals, human liver microsomes (31), and cultured human tissues, includ;ng bucca.I mucosa (15). Of particular interest is the ability of two NNN'target tissues, lingual mucosa and esophageal mucosa, to carry out preferential 2'-hydroxylation of NNN (27,32). The intermediate that is formed by 2'- hydro3rylation of NNN is diazohydroxide 8, which is identical to that formed by methyl hydroxylation of NNK (7, figure 1). As described above, this inter- mediate is highly mutagenic and'this or related intermediates appear to play an important role in carcinogenesis by both NNN and NNK. The intermediate 9 is significantly less mutagenic than 8 in S. typhimurium (33), and various Lines of evidence indicate that it is less important in NNN tumorigenesis than is 8 (33,34). Autoradiographic studies have demonstrated that radioactivity from [2'-14C]NNN is bound to tissues of mice, rats, and Marmoset monkey (20,35-37). Immunoassays are currently being developed for the putative DNA adducts that that are produced by 2'-hydroxylation of NNN and methyl hydroxylation of :WK; it will be important to assess the levels of these adducts in the exfoliated oral cells of snuff dippers. Their levels may relate to the susceptibility of individuals to the effects of smokeless tobacco. The metabolic pathways that lead to these intermediates can be affected by alcohol consumption and dietary components (32,38-43). 2501258110

Metabolism of NMOR The metabolic pathways of NMOR are illustrated in figure 4. These have been elucidated by in vitro and in vivo studies in rats (44-47). Structure activity studies had shown that 3-hydroYylation of NMOR, leading to inter- mediate 4, was likely to be important in NMOR carcinogenesis (48). This path- way could result in the formation of glyoxal-deoxyguanosine adducts (49); 2-hydroxylation of NMOR also occurs, giving the mutagenic product 2. The latter• also forms glyoxal-deoxyguanosine adducts (50). These adducts, which are likely to have miscoding properties, also should be present in the DNA of snuff dippers since human tissues are capable of metabolizing NMOR (51). Summa Persuasive evidence exists that the carcinogenic nitrosamines that are present in high quantities in snuff and other forms of smokeless tobacco are metabolized by target tissues of experimental animals and by human tissues to intermediates that can modify the genetic material of the cell. References 1. Hecht, S.S., Young, R., and Chen, C.B. Metabolism in the F-344 rat of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Cancer Res. 40: 4144-4150, 1980. 2. Hoffmann, D., Castonguay, A., Rivenson, A., and Hecht, S.S. Comparative carcinogenicity and metabolism of 4-(methyl-nitrosamino)-l-(3-pyridyl)-l- butanone and N'-nitrosonornicotine in Syrian golden hamsters. Cancer Res. 41: 2386-2393, 1981. 3. Castonguay, A., Lin, D., Stoner, G.D., Radok, P., Furuya, K., Hecht, S.S., Schut, H.A.J., and Klaunig, J.E. Comparative carcinogenicity in A/J mice and metabolism by cultured mouse peripheral lung of N'-aitro- sonornicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and their analogues. Cancer Res. 43: 1223-1229, 1983. ' 4. Castonguay, A., Tjalve, H., and Hecht, S.S. Tissue distribution of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone, and its metabolism in F-344 rats. Cancer Res. 43: 630-638, 1983. - 5. Singer, B., and Grunberger, D. Molecular biology of mutagens and carcinogens. New York, Plenum Publishing Corp., 1983, pp. 45-96. 6. Loechler, E.L., Green, C.L., and Essigmann, J.M. In vivo mutageas by 06-methylguanine built into a unique site in a viral genome. Proc. Natl. Acad. Sci. USA 81: 6271-6275, 1984. 7. Sukumar, S., Nofario, V., Martin-Zanca, D., and Barbacid, M. Induction of mammary carcinomas in rats by nitrosomethylurea involves malignant activation of H-ras-l locus by single point mutations. Nature 306: 658- 662, 1983. - 2501258111 2-49

8. Pegg, A.E. Methylation of the 06-position of guanine in DNA is the most likely initiating event in,carcinogenesis by methylating agents. Cancer Invest. 2: 223-231, 1984. 9. Singer, B. Alkylation of the 06 of guanine is only one of many chemical events that may initiate carcinogenesis. Cancer Invest. 2: 233-238, 1984. 10. Castonguay, A., Tharp, R., and Hecht, S.S. Kinetics of DNA methylation by the tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone in the F344 rat. In: I.K. O'Neill, R.C. Von Borstel, C.T. Miller, J. Long, and H. Bartsch, (eds.). N-Nitroso Compounds: Occurrence, Biological Effects and Relevance to Human Cancer. IARC Sci. Publ. 57: 805-810, 1984. 11. Chung, F-L., Wang, M., and Hecht, S.S. Effects of dietary indoles and ' isothiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone alpha-hydroxylation and DNA methylation in rat liver. Carciaogenesis 6: 539-543, 1985. 12. Foiles, P., Trushin, N., and Castonguay, A. Measurement of 06-methylde- oxyguanosine in DNA methylated by the tobacco-specific carcinogen 4- (methylaitrosamino)-1-(3-pyridyl)-1-butanone using a biotin-avidin enzyme-linked immunosorbent assay. Carcinogenesis 6: 989-993, 1985. 13. Hecht, S.S., Trushin, N., Castonguay, A., and Rivenson, A. Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine. Cancer Res. 46: 498-502, 1986. 14. Belinsky, S.A., White, C.M., Boucheron, J.A., Richardson, F.C., Swenberg, J.A., and Anderson, M.W. Accumulation of DNA adducts in hepatic and respiratory tissue following multiple administrations of the tobacco- specific carcinogen 4-(N--methyl-i3-aitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Proc. Am. Assoc. Cancer Res. 26: 100, 1985. 15. Castonguay, A., Stoner, G.D., Schut, H.A.J., and Hecht, S.S. Metabolism of tobacco-specific N-nitrosamines by cultured human tissues. Proc. Natl. Acad. Sci. USA 80: 6694-6697, 1983. 16. Konstantinidus, A., Smulow, J.B., and Sonnenschein, C. Tumorigenesis at a predetermined oral site after one intraperitoneal injection of Y- nitroso-N-methylurea. Science 216: 1235-1237, 1982. 17. Hecht, S.S., Lin, D., and Castonguay, A. Effects of alpha-deuterium sub- stitution on the mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). Carcinogenesis 4: 305-310, 1983. 18. Hecht, S.S., Lin, D., Chuang, J., and Castonguay, A. Reactions with deoxyguanosine of 4-(carbethoxynitrosamino)-1-(3-pyridyl)-1-butanone, a model compound for alpha-hydroxylation of tobacco specific nitrosamines. J. Am. Chem. Soc. (in press). 2501258112

19. Tjalve, H., and Castonguay, A. The in vivo tissue distribution and in vitro target tissue metabolism of the tobacco-specific carcinogen 4 methylnitrosamino)-1-(3-pyridyl)-1-butanone in Syrian golden hamsters. Carcinogenesis 4: 1259-1265, 1983. 20. Castonguay, A., Tjalve, H., Trushin, N., d'Argy R., and Sperber, Y. Metabolism and tissue distribution of tobacco-specific N-nitrosamines in the marmoset monkey (Callithria jacchus). Carcinogenesis 6: 1543-1550, 1985. - 21. Adams, J.D., LaVoie, E.J., and Hoffmann, D. On the pharmacokinetics of tobacco-specific N-nitrosamines in Fischer rats. Carcinogenesis 6: 509- 511, 1985. 22. Chen, C.$., Hecht, S.S., and Hoffmann, D. Metabolic alpha-hydroxylation of the tobacco-specific carcinogen N'-nitrosonornicotine. Cancer Res. 38: 3639-3645, 1978. 23. Chen, C.B., Fung, P.T., and Hecht, S.S. Assay for microsomal alpha-hydro:cy- lation of N'-nitrosonornicotine and determination of the deuterium isotope effect for alpha-hydroxylation. Cancer Res. 39: 5057-5062, 1979. 24. Hecht, S.S., Chen, C.B., and Hoffmann, D. Metabolic beta-hydroxylation and N-oxidation of N-'nitrosonornicotine. J. Med. Chem. 23: 1175-1178, 1980. 25. McCoy, G.D., Chen, C.B., and Hecht, S.S. Influence of mixed function oxidase inducers on the in vitro metabolism of N'-nitrosonornicotine by rat and hamster liver microsomes. Drug. Metab. Disp. 9: 168-169, 1981. 26. Hecht, S.S., Lin, D., and Chen, C.B. Comprehensive analysis of urinary metabolites of N'-nitrosonornicotine. Carcinogenesis 2: 833-838, 1981. 27. Hecht, S.S., Reiss, B., Lin, D., and Williams, G.M. Metabolism of :1'- nitrosonornicotine by cultured rat esophagus. Carcinogenesis 3:, 453-456, 1982. - 28. Hecht, S.S., and Young, R. Regiospecificity in the metabolism of the homologous cyclic nitrosamines, N'-nitrosonornicotine and N'-nitrosoana- basine. Carcinogenesis 3: 1195-1199, 1982. 29. Brittebo, E.B., Castonguay, A., Furuya, K., and Hecht, S.S. Metabolism of tobacco-specific nitrosamines by cultured rat nasal-mucosa. Cancer Res. 43: 4343-4348, 1983. 30. Hecht, S.S., Young, R., and Maeura, Y. Comparative carcinogenicity in F344 rats and Syrian golden hamsters of N'-nitrosonornicotine and N'- nitrosonornicotine-l-N-oxide. Cancer Lett. 20: 333-340, 1983. 31. Hecht, S.S., Chen, C.B., McCoy, G.D., Hoffmann, D., and Domellof, L. Aipha-hydroxylation of N-nitrosopyrrolidine and N'-nitrosonornicotine by human liver microsomes. Cancer Lett. 8: 35-41, 1979. 2501250113

32. Castonguay, A., Rivenson, A., Trushin, N., Reinhardt, J., Stathopoulos, S., Weiss, C.J., Reiss, B., and Hecht, S.S. Effects of chronic ethanol consumption on the metabolism and carcinogenicity of N'-nitrosonornicotine in F344 rats. Cancer Res. 44: 2285-2290, 1984. 33. Hecht, S.S., and Lin, D. Comparative mutagenicity of 4-(carbethoxynitro- samino)-4-(3-pyridyl)-butanol and 4-(carbethoxynitrosamino)-1-(3-pyridyl)- 1-butanone, model compounds for alpha-hydroxylation of N'-nitrosonornicotine. Carcinogenesis (ia press). 34. Hecht, S.S., Castonguay, A., Rivenson, A., Mu, B., and Hoffmann, D. Tobacco-specific nitrosamines: Carcinogenicity, metabolism, and possible role in human cancer. J. Environ. Health Sci. 1: 1-54, 1983. 35.' Brittebo, E.B., and Tjalve H. Autoradiograhpic observations in the dis- tribution and metabolism of N-[14C] nitrosonornicotine in mice. J. Cancer Res. Clin. Oncol. 98: 233-242, 1980. 36. Waddell, W.J. and Marlowe, C. Localization of (14C] nitrosonornicotine in tissues of the mouse. Cancer Res. 40: 3518-3520, 1980. 37. Brittebo E.B., and Tjalve, H. Formation of tissue-bound N-nitrosonornico- tine metabolites by target tissues of Sprague-Dawley and Fischer rats. Carcinogenesis 2: 959-963, 1981. 38. McCoy, G.D., Chen, C.B., and Hecht, S.S. Influence of modifiers of MFO activity on the in vitro metabolism of cyclic nitrosamines. In: M.J Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette, and P.J. O'Brien (eds.). Microsomes, Drug Oxidations, and Chemical Carcino- genesis, Vol. II. New York, Academic Press, 1980, pp. 1189-1192. 39. McCoy, G.D., Katayama, S., Young, R., Wyatt, M., and Hecht, S.S. Influence of chronic ethanol consumption on the metabolism and carciao- genicity of tobacco-related nitrosamines. In: H. Bartsch, I.K.'0'Yeill, M. Castegnaro, M. Okada, and L. Davis (eds.). N-Nitroso Compounds: Occurrence and Biological Effects. :IARC Sci. Publ. 41: 309-318, 1982. 40. Waddell, W.J., and Marlowe, C. Inhibition by alcohols of the carcino- genicity of radioactive nitrosonornicotine in sites of tumor formation. Science 221: 51-52, 1983. 41. Chung, F-L., Juchatz, A., Vitarius, J., and Hecht, S.S. Effects of dietary compounds on target tissue alpha-hydroxylation of N-nitrosopyrrolidine and N'-nitrosonornicotine. Cancer Res. 44: 2924-2928, 1984. 42. Chung, F-L., Wanv, M., and Hecht, S.S. Effects of dietary indoles and isothiocyanates on N-nitrosodimethylamine and 4-(methylnitrosamino)-1-(3- pyridyl)-l-butanone aipha-hydroxylation and DNA methylation in rat liver. Carcinogenesis 6: 539-543, 1985. 43. Swann, P.F. Effect of ethanol on nitrosamine rnetabolism and distribution. Implications for the role of nitrosamines in human cancer and for the influence of alcohol consumption on cancer incidence. IARC Sci. Publ. 57: 501-512, 1984. Z5a1Z50114

44. Manson, D., Cox, P.J., and Jarman, M. Metabolism of N-aitrosomorpholine by the rat in vivo and by rat liver microsomes and its oxidation by the Fenton system. Chem. Biol. Interact. 20: 341-354, 1978. 45. Hecht, S.S., and Young, R. Metabolic alpha-hydroxylation of N-nitrosomor- pholine and 3,3,5,5,-tetraduetero-N-nitrosomorpholine in the F-344 rat. Cancer Res. 41: 5039-5043, 1981. 46. Hecht, S.S. N-Nitroso-2-hydroxymorpholine, a mutagenic metabolite of N-nitrosodiethanolamine. Carcinogenesis 5: 1745-1747, 1984. 47. Lofberg, B., and Tjalve, H. Tissue specificity of N-nitrosmorpholine metabolism in Sprague-Dawley rats. Food Chem. Toxicol. 23: 647-657, 1985. 48. Linjinsky, W., Taylor, H.W., and Keefer, L.K. Reduction of rat liver carcino- genicity of 4-nitrosomorpholine by alpha-deuterium substitution. J. Nati. Cancer Inst. 57: 1311-1313, 1976. 49. Chung, F-L., Palladino, G., and Hecht, S.S. Reactions of N-nitro- somorpholine metabolites with deoxyguanosine and DNA. Proc. Am. Assoc. Cancer Res. 26: 89, 1985. 50. Chung, F-L., and Hecht, S.S. Formation of the cyclic 1,N2-glyoxal- deoxyguanosine adduct upon reaction of N-aitroso-2-hydroxymorpholine with deoxyguanosine. Carcinogenesis, 6: 1671-1673, 1985. 51. Bartsch, H., Camus, A., and Malaveille, C. Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat liver tissue fractions. Mutat. Res. 37: 149-162, 1976.

N0 0 1 1 ~ NI CH3 N 0 ~ 1 ~ N=NOH l + JN T 1 OH 12 I 13 4 15 HCHO i COZ 13 Figure 1. Metabolic Pathways of NNK NO 1 _~ N.CH3 5 10 I OH i C H30H OH OH 6 L N=NOH 1 OH q N OH OH 0 ., 14 15 + HCHO

Figure 2. Scheme Linking Nicotine to Formation of the Promutagenic DNA Adduct, 06-Methylguanine ivuAUw rKmtsbmu ~ IMETAaOLIC ~ ~0 N-N=O FCH3NNOH-1 -0, 7-11ETHYLGUANINE CH3 NICOTINE ACTIVATION OR C~ CH 6 3 O E -UTHYLGUANINE CIGARETTE " METHYLDIA20- HYDROXIDE IN DNA SMOKING NNK itt8SZIUSz

I Figure 3. Metabolic Pathways of NNN 6 i I ON $ttgsz.tasz 0 ~ (n-r -oH -oN 14 i- -a$- 12 i

Figure 4. Ptetabolic Pathways of NMOR Cp~OH N 1 2 N•0 lt H0~Nf0 ~ N•0 2,4- ONP 4- 6 6tt8SZlOSZ i HO 7 I0 0 ON ~ •--- ~ 1 5 C 0 ~ HO` O,,OH 0 pH l N 4•0 9

EXPERIMENTAL STUDIES INVOLVING EXPOSING LABORATORY ANI.KALS TO SMOKELESS TOBACCO OR ITS CONSTITUENTS This section reviews bioassays evaluating the carcinogenicity in animals of smokeless tobacco and its constituents, particularly the tobacco-specific nitrosamiaes (TSNA) described in the section on the chemical constituents of smokeless tobacco. The bioassays involved multiple routes of administration of chewing tobacco, snuff, or extracts of these products and of several TSNA. Studies of chewing tobacco, snuff, and TSNA are summarized in tables 1 to 3 respectively, with comments on the individual investigations provided below. Bioassays With Chewing Tobacco Oral Administration An alcohol extract of Indian chewing tobacco diluted 1:50 (group 1) or 1:25 (group 2) was gavage-fed to male Swiss mice over 15 to 20 months. In another group of mice, a mixture of the tobacco extract with standard labora- tory diet was administered o.ver 21 to 25 months (group 3). This treatment produced tumors in 8 of 15 mice at risk in group 1 including 5 mice with lung tumors and 2 with liver tumors; 4 of 10 mice at risk in group 2 developed lung and liver tumors. The feeding experiment (group 3) resulted in 8 of 10 mice with tumors, specificall y 4 with tumors of the lung and 4 with liver tumors. Despite the high toxicity of.the tobacco extracts and certain short- comings of the methodology, these assays indicate that the extract of chewing tobacco is carcinogenic in mice (1). Application to the Oral Mucosa and Cheek Pouch Three different extracts of an Indian chewing tobacco were applied daily for up to 18 months to the buccal mucosa of strain A and Swiss mice. No excess of tumors was observed (2). The oral mucosa of a group of weanling Wistar rats was painted twice weekly with a 2-percent alkaloid-free extract of an Indian chewing tobacco. No tumors were observed at the application site even though applications were continued throughout the lifespan of the rats (3). A group of 12 male Syrian golden hamsters received topical applications on the buccal mucosa of a dimethyl sulfoxide (DMSO) extract of an Indian chewing tobacco three times weekly for 21 weeks. None of the treated hamsters developed tumors in the oral mucosa; however, 8 of 12 treated animals had leukoplakia. These changes were not seen in the oral mucosa of the animals treated with DMSO alone (4). In another bioassay, 12 male hamsters received applications to the cheek pouch of a IXVSO extract of Indian chewing tobacco three times weekly over their entire lifespan. Tumors were not observed in the treated group or the control group (5). When 1 mg of a paste made of a chewing tobacco extract was applied topically to the mucosa of the cheek pouches twice daily over a 6-month period, and animals were maintained with- out further treatment for another 6 months, the incidence of hyperplasia in the buccal pouches was 17.6 percent, that of dysplasia was 29.4 percent, and that of squamous cell papilloma or carcinoma was 17.6 percent in 17 ham- sters. There were no tumors in the 20 control animals (6) 2501250120 .

Fifty hamsters received implantations of a 2 cm3 plug of chewing tobacco in their cheek pouches. The opening of the cheek pouch was ligated and the animals were observed for 18 months. After 13 months, 21 of 50 animals had survived. No tumors were recorded upon termination of the assays (7). Although the studies cited above had some inherent weaknesses due to short application time or low dose, it appears, nevertheless, that both the oral mucosa of rats and the cheek pouches of Syrian golden hamsters are relatively resistant to the carcinogenic activity of the extracts of chewing tobacco. Subcutaneous Application Seventeen C57 black mice were subcutaneously injected with 1 ml of a 2-percent solution of either partly or completely alkaloid-free extracts of an Indian chewing tobacco once a month for 1 to 24 months. One squamous carcinoma at an unspecified site developed in one mouse receiving the partly alkaloid-free extract (8). Skin Application A large number of studies have been published regarding the tumorigenicity on mouse skin of various extracts of chewing tobacco. Most of these bioassays failed to produce skin tumors. The negative results appear to be due primarily to the low dose applied or the short duration of the applications (9,10). The negative results indicate also that the concentrations of TSNA and PAH in these extracts do not suffice to induce tumors upon topical application (11). However, the application of methanol or DMSO extracts of cigarette tobacco in- duced a low but significant number of benign tumors in the skin of CAF'1 and Swiss mice when these extracts were applied three times weekly for up to 24 months to the shaved backs of the mice (12,13). A number of studies have reported tumor-promoting activity of the extracts of chewing tobacco when these were applied to mouse epidermis previously treated with a tumor ;nitiator (8,12,14-16). The bioassay data with chewing tobacco are summarized in table 1. Bioassays With Snuff Oral Administration For 2 years, 50 male BIO 15.16 and 50 male BI0 87.20 hamsters were each maintained on a standard diet containing 20 percent moist, fresh snuff. Controls consisted of 50 male BI0 15.16 hamsters and 50 male 310 87.20 hamsters on a diet containing 20 percent cellulose (of caloric value similar to the snuff-containing diet). The spectrum of tumors observed was nearly identical in both groups. Hamsters of both strains gavaged 60 times with 5 mg of the carcinogen 3-methylcholanthrene (MC) had a significantly increased incidence of both benign and malignant tumors of the forestomach and large intestine. Hamsters of the BI0 87.20 strain also had an increased incidence of stomach cancers while the BI0 15.16 strain developed tumors of the skin. To assay the cocarcinogenic activity of snuff, 50 hamsters of each strain received the diet containing 20 percent snuff plus 50 times 0.5 mg of MC. Compared to the control group (diet containing 20 percent cellulose), the tumor yield was not increased in the two experimental groups indicating a lack of carcinogenic 2501250121 2-59

activity as well as of cocarcinogenic activity of the snuff in this setting (17). Application to the Lip, Oral Mucosa, or Cheek Pouch The upper lips of 20 male BALB mice were painted 3 times a day for 5 days weekly over a 2-month period with a concentrated water extract of snuff (group 1). In another group of 20 male mice, the upper lips were inoculated with herpes simplex virus type 1(HSV-1) and were subsequently painted with a concentrated snuff extract for 2 months (group 2). As control served a group of 20 male mice receiving inoculation of the upper lips with HSV-l and painting with water (group 3). Two months' exposure to snuff extract (group 1) or HSV-1 inoculation (group 3) alone did not induce dysplasia in the epithelium of the labial mucosa, while HSV-l inoculation combined with painting of snuff extract produced epithelial dysplasia and other histomorphologic changes (18). In respect to this and other studies in which animals are infected with herpes virus in addition to treatment with snuff extracts, it should be noted that 20 to 40 percent of the U.S. population have periodic occurrences of labial herpes (19). Male F344 rats were treated for up to 30 months by swabbing the oral cavity•with either a concentrated water extract of snuff (group 1; 13.2 ug NNN and 2.8 ug NNK per milliliter snuff extract solution), snuff extract enriched with the tobacco-specific nitrosamiaes NNN and NNK (group 2; 148 ug NNN and 30 µg NNK per milliliter snuff extract solution), NNN and NNK alone in concentrations corresponding to those applied in group 2 (group 3; 135 ug NNN and 27.6 ug NNK per milliliter test solution), or with water alone (group 4). Groups 1, 2, and 3 consisted of 30 male rats each and group 4 (control) of 21 rats. The incidence of tumors in groups 1 and 2 was not significantly increased over that in the control group. In the group of 30 rats treated with NNN and NNR alone, 8 animals had oral tumors (6 papillomas in the cheek, 4 papillomas in the hard palate, and 1 papilloma of the tongue), and 4 animals had lung carcinoma. This study indicates that snuff contains carcinpgenic N-nitrosamines; however, when they are being tested in an admixture with other components in the water extract of snuff, their carcinogenic activity may be suppressed (20). A group of 21 male and 21 female Sprague-Dawley rats was treated with snuff placed in a surgically created canal in the lower lip. Approximately 0.2 g of a standard Swedish snuff (pH 8.3) was given twice daily 5 days per week for 9 to 22 months. The mean retention time of the snuff in the canal was 6 hours, and the estimated daily dose was 1 g of snuff/kg b.w. Using the same methodology, another group of 5 male and 5 female rats was treated with alkaline snuff in the surgically created canal (pH 9.3). One of the 42 rats treated with regular snuff developed a squamous carcinoma in the oral cavity after 8.5 months. The exposure to the regular snuff resulted in mild to moderate hyperplasia of the epithelium, hyperorthokeratosis, and acanthosis. Among rats exposed to snuff for 18 to 22 months, 16 of 42 showed vacuolated cells penetrating deeper into the epithelium with hyperplastic and atropic lesions. Rats exposed to alkaline snuff differed little from those in the group treated with regular snuff. Outside the area of treatment, squamous 2501250122

cell hyperplasia of the forestomach was found in rats exposed to snuff for 18 to 22 months (21). In another bioassay using the same methodology as described by Hirsch and Johansson (21), the surgically created canal in the lower lip of F344 rats was filled five times each week over 28 months with either U.S. snuff (average 0.2 g per application; n=30), snuff enriched with its own water extract (a=30), or the extracted residue of snuff (a=21). Ten rats with the surgically created lip canal, and otherwise untreated, served as controls. The incidence of nonspontaneous tumors in each group was the following: rats treated with snuff had one squamous carcinoma of the oral cavity, one squamous cell papilloma of the hard palate, and one meningioma; treatment with enriched snuff led to one squamous cell papilloma of the floor of the mouth and one nasal olfactory tumor; treatment with extracted snuff induced one squamous cell papil- loma of the hard palate. There were no tumors in the control group (20). Four groups of female Sprague-Dawley rats with surgically created canals in the lower lip, received the following treatments beginning at 3 months of age: group 1 was infected with herpes simplex virus type 1(HSV-1) by scarification and topical application followed 10 days later by administration of snuff into the canal morning and night on 5 days per week; group 2 was infected with virus and received no other treatment; group 3 was sham-infected with sterile saline followed by snuff treatment; and group 4, not given virus or snuff, served as controls. The HSV-1 infection was repeated once after a 1-month interval, and snuff treatment was continued for 18 months after which time all animals were killed. Three animals in each of groups 1 and 2 died from encephalitis shortly after the second infection with HSV-1. Squamous- cell carcinomas of the oral cavity developed in two of seven rats, and a retroperitoneal sarcoma was seen in one of seven rats exposed to HSV-l plus snuff. In the group exposed to snuff alone, 1 of 10 animals developed a squamous carcinoma of the anus and 1 of 10 a retroperitoneal sarcoma (22). In several studies, various forms of snuff were installed in the cheek pouches of Syrian golden hamsters for up to 20 months. The application of snuff did not lead to the induction of tumors in the cheek pouches nor at any other site of the oral cavity in any of these studies even though malignant tumors were induced in the oral cavity with high doses of 7, 12-dimethylbenz(a) anthracene and 3-methylcholanthrene (7,23-26). In an assay for the joint action of HSV-virus and snuff, the buccal pouches of 125 Syrian hamsters were inoculated with HSV-1, HSV-2, or culture medium. The control and HSV inoculations were done once a month for 6 con- secutive months. Then 25 hamsters with HSV-inoculated pouches received instal- lations of commercial snuff twice daily into both the right and left pouches. One month after the last HSV-inoculation and 6 months after continuous snuff application, the assay was terminated. The buccal pouches were removed for histopathologic examination. Neither the application of snuff to the cheek pouches nor HSV infection alone induced neoplastic changes in hamster buccal pouches. However, HSV infection in combination with snuff resulted in epithe- lial dysplasia and in squamous carcinoma of the buccal pouches in 11 out of 25 hamsters (27). This investigation provides the strongest evidence to date that snuff may increase cancer risk in animals; however, full evaluation is precluded since the findings have been published only in abstract form. 2501250123 2-61

Subcutaneous Administration A Swedish snuff was extracted with 60 percent alcohol and resulted in 18 percent dry extract, which was injected subcutaneously into rats with 70 percent ethanol and tri-a-caprylin (1:1) as vehicle. The rats received a total dose of 4.2 g of extract with 84 weekly doses of 50 mg of extract. No tumors were observed at the area of injection (28). This result is quite different from an earlier one by the same investigators in which an alcohol extract from cigarette tobacco (20 percent yield) was injected into 75 rats with 70 percent alcohol and glycerol as solvent (1:3). Per week, 45 mg extracts were injected until the total dose amounted to 3.2 g/rat. After 25 months, 18 of 75 rats had developed malignant tumors, primarily sarcomas at the injection site (29). The bioassay data with snuff are summarized in table 2. Bioassays With Constituents of Smokeless Tobacco At least three types of carcinogens occur in smokeless tobacco: poly- nuclear aromatic hydrocarbons (PAH), polonium-210 (210Po), and N-nitrosamines. One of the PAH.identified in smokeless tobacco, benzo(a)pyrene (up to 72 pp~), has long been recognized as an animal carcinogen (18,24,30). Levels of Z1 Po in processed tobacco amount to 0.1-1.0 pCi per gram and to 0.18-1.22 pCi/g in commercial U.S. snuff products. Ionizing radiation can cause multiple types of canc r in animals and humans raising the possibility that the alpha- radiation of ~lOPo may contribute to the carcinogenic potential of smokeless tobacco and especially snuff (31,32). Three groups of N-nitrosamines have been identified in smokeless tobacco. All of the 4 volatiYe nitrosamines thus far identified are carcinogenic in animals (33). These are nitrosodimethylamine (0 to 215 ppb), nitrosopyrrolidine (0 to 291 ppb), nitrosopiperidine (0 to 107 ppb), and nitrosomorpholine (0 to 690 ppb). Seven nonvolatile nitrosamines have also been identified in smokeless tobacco. (7f these, only nitrosodiethanolamine (30 to 6,800 ppb) is a known carcinogen in mice, rats, and hamsters (33). Swabbing of the oral cavity of 20 male and 20 female hamsters with solutions of these agents three times weekly for 45 weeks (20 mg per application) induced tumors of the nasal cavity in 17 animals, tumors of the trachea in 6, and a tumor of the larynx in 1 of the hamsters (34). The most abundant carcinogens in smokeless tobacco yet identified are the tobacco-specific aitrosamines (TSNA). These are formed during the proces- sing of tobacco from its alkaloids. So far, seven TSNA have been identified in smokeless tobacco. Of these, N'-nitrosonornicotine (NNN; 470-135,000 ppb) and 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK; 30-13,600 ppb) are powerful carcinogens in mice, rats, and hamsters (table 1; 1,9). Table 3 summarizes results from bioassays administering TSNA to test animals. A variety of tumors were produced, particularlty in the esophagus, nasal cavity, and lung. In a recently completed investigation, daily swabbing for up to 30 months of the oral cavity of F344 rats with a saline solution containing 135 ppm NNN and 28 ppm NNK led to the development of benign oral tumors in 8 and lung carcinoma in 4 of 30 rats. Neither oral tumors nor tumors of the lung were observed in the negative control group (20). This study suggests that NNN and NNk may be tumorigenic at the site of exposure as well as systemically. Full evaluations of these results are precluded, however, since the original manuscript is now under journal review and not published. 2501258124 2-62

It is noteworthy that some of the bioassays indicated that relatively low doses of the TSNA could induce tumors. In hamsters, a total dose of only 0.2 mmol/ks of NNK induced a significant incidence of tumors (35), whereas in F344 rats, 60 subcutaneous injections of a total dose of 20 mg (0.33 mmollkg) of NNK induced tumors of the liver in 10, tumors of the lung in 13, and tumors of the nasal cavity in 6 of 30 rats. Subcutaneous applications to 27 rats of the same molar dose (0.33 mmol/kg) of nitrosodimethylamine, resulted in 6 animals with tumors of the liver and 1 rat with a tumor of the nasal cavity (36). For NNN, high tumor incidences were produced in F344 rats by a total dose of 1.0 mmol/kg (37). Based on daily use for 30 years of 10 g of snuff containing 3.1 ppm of NNK, the estimated NNK exposure of a snuff dipper would be approximately 0.02 mmol/kg. Exposure to NNN from the same brand would be 0.4 mmol/kg (figure 3, chapter II). Hence, the bioassays indicate that exposures in the dose range actually experienced by long-term snuff dippers induce tumors in animals. This is a distinctive and potentially important finding, since for most chemical carcinogens their carcinogenicity was detected following exposure at doses much higher than usually received by humans. Of the other five TSNA, besides NNN and NNK, N'-nitrosoanabasine (NAB; 10-6,700 ppb) and 4(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL; 140-300 ppb) were moderately active carcinogens, and N-nitrosoanatabine (NAT; 300-338,000 ppb) was inactive when tested at the low dose level of 9 mmol/kg (9,38). Recently, two additional TSNA have been identified in snuff: 4-(methyl- nitrosamino)-4-(3-pyridyl)-1-butanone (1,300-1,800 ppb) and 4-(methylnitros- amino)-(3-pyridyl)butene-1 ( 10 ppb; 6). These two nitrosamines have not yet been tested for carcinogenicity. Mutagenicity Assays and Other Short-Term Tests Chewing Tobacco Nicotiana rustica is a tobacco variety that is widely cultivated and used throughout India. Its ethanol extracts induced mutations in Salmonella typhimurium TA98 and in V79 cells of Chinese hamsters. The addition of 7liver homogenate from Aroclor pretreated rats enhanced the mutagenic effect. No mutations were induced in TA100, TA1535, or TA1538 in the presence of the S9 homogenate. This ethanol extract of tobacco also induced micronuclei in bone marrow cells of Swiss mice (1,39,40). An ethyl acetate extract of Indian chewing tobacco induced sister chromatid exchange (SCE) in human lymphocytes and in a human lymphoblastoid cell line. In the latter system, S9 rat liver homogenate enhanced the effect. When the tobacco extract was tested in the absence of the S9 homogenate it did not induce ouabain-resistance in Chinese hamster V79 cells. The same extract, another ethyl acetate extract and an ethanol extract of tobacco induced cell transformation in Syrian hamster embryo cells (41,42). The incidence of micronucleated oral mucosa cells in 27 Indians using khani chewing tobacco was 2.2 percent (0.8-4.9 percent). The incidence of micronuclei in exfoliated cells of nonchewers of similar ethnic backgrounds and dietary habits was 0.47 percent (0.0-0.9 percent) (43). 2-63

Snuff The residue of organic solvent extracts from a U.S. commercial snuff was dissolved in DMSO and tested for the induction of SCE's in human peripheral lymphocytes. The organic snuff extract induced significant SCE's with a 0.05 percent concentration in lymphocytes of one of three donors, with a 0.15 percent concentration in lymphocytes in two of three donors, and with a 0.5 percent concentration in lymphocytes of aLl three donors (44). - Tobacco-Specific N-Nitrosamines (TSNA) Of the seven TSNA so far identified in smokeless tobacco, only NNN and NNK were also tested for genotozicity in short-term tests. In the presence of a liver microsomal preparation from Aroclor-induced rats, NNN and NNK caused dose-dependent mutations in Salmonella typhimurium TA100 and TA1535. Increased mutation frequencies were observed in the case of NNN at 2.5 u mol and at 5.65 umol/plate and in the case of NNK at 1.4 u mol/plate (45-47,~). NNN and NNK at 10-3 and 10-2 molar concentration each induced unscheduled DNA synthesis in freshly isolated hepatocytes from adult rats (48). Summary Chewing tobacco and extracts from various chewing tobaccos have been tested by oral administration in mice, topical application to the oral mucosa of mice, rats, and hamsters, and by subcutaneous administration and skin application to mice. The investigations failed to demonstrate significantly increased tumor production. Short application times and low-dose exposures, however, limit the evaluation of the carcinogenicity of chewing tobacco or its extracts. Bioassays of snuff have likewise generally shown no excess cancer, although some experiments suggest that it may cause oral tumors in rats and hamsters that are infected with herpes simplex virus. Among the chemical coiaponents of snuff, the tobacco-specific nitrosamines YNN and NNK are powerful carcinogens. The doses of NNN and NNK that produce tumors in experimental animals are close to the doses estimated from lifetime exposure among human snuff dippers. References 1. Shah, A.S., Sarode, A.V., and Bhide, S.V. Experimental studies on mutagenic and carcinogenic effects of tobacco chewing. J. Cancer Res. Clin. Oncol. 109: 203-207, 1985. 2. Mody, J.K., and Ranadive, J.K. Biological study of tobacco in relation to oral cancer. Ind. J. Med. Sci. 13: 1023-1037, 1959. 3. Gothoskar, S.V., Sant, S.M., and Ranadive, K.J. Effect of tobacco and lime on oral mucosa of rats fed on vitamin B deficient diet. Int. J. Cancer 12: 424-429, 1975. 4. Suri, K., Goldmaa, H.M., and Wells, H. Carcinogenic effect of a dimethylsulphoxide extract of betel nut on the mucosa of the hamster buccal pouch. Nature 230: 383-384, 1971. 2501250126

5. Ranadive, K.J., and Gothoskar, S.V. Betel quid chewing and oral cancer: Experimental studies. In: Prevention and Detection of Cancer, Part I, Vol. 2. H.E. Nieburgs (ed.). Marcel Dekker, New York, 1976, pp. 1745- 1766. 6. Rao, A.R. Modifying influences of betel quid ingredients on B(a)P- induced carcinogenesis in the buccal pounch of hamster. Int. J. Cancer 33: 581-586, 1984. 7. Peacock, E.E., Jr., and Brawley, B.W. An evaluation of snuff and tobacco in the production of mouth cancer. Plast. Reconstr. Surg. 23: 628-635, 1959. 8. Ranadive, K.J., Gothoskar, S.V., and Khanolka, V.R. Experimental studies on the etiology of cancer types specific to India. A. Oral cancer, B. Kangri cancer. Acta Unio. Internatl. Contra Cancrum 19: 634-639, 1963. 9. International Agency for Research on Cancer. Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Tobacco habits other than smoking; Betel-quid and areca-nut chewing; and some related nitro- samines. IARC Monogr. 37: 291, 1985. 10. Wynder, E.L., and Hoffmann, D. Tobacco and tobacco smoke. Studies in experimental carcinogenesis. New York, Academic Press, 1967, pp. 198-202. 11. Hoffmann, D., Hecht, S.S., Ornaf, R.M., and Wynder, E.L. Nitrosonornicotine: Presence in tobacco, formation and carcinogenicity. IARC Sci. Publ. 14: 307-320, 1976. 12. Wynder, E.L., and Hoffmann, D. A study of tobacco carcinogenesis, X. Tumor promoting activity. Cancer 24: 289-301, 1969. 13. Wynder, E.L., and Wright, G.A. A study of tobacco carcinogenesis, I. The primary fractions. Cancer 10: 255-271, 1957. 14. Bock, F.G., Moore, G.E., and Crouch, S.K. Tumor promoting activity of extracts of unburned tobacco. Science 145: 831-833, 1964. .15. Bock, F.G., Shamberger, R.J., and Meyer, H.K. Tumor promoting agents in unburned cigarette tobacco. Nature 208: 584-585, 1965. 16. Van Duuren, B.L., Sivak, A., Segal, A., Orris, L., and Langseth, L. The tumor-promoting agents of tobacco leaf and tobacco smoke condensate. J. Natl. Cancer Inst. 37: 519-526, 1966. 17. Homburger, F., Hsueh, S.S., Russfield, A.B., Laird, C.W., and Van Dongen, C.G. Absence of carcinogenic effects of chronic feeding of snuff in inbred Syrian hamsters. Toxicol. Pharmacol. 35: 515-521, 1976. 18. Park, N.H., Herbosa, E.G., Ninkian, K., and Shklar, G. Combined effect of herpes simplex virus and tobacco on the histopathologic changes in lips of mice. Oral Surg. 59: 154-158, 1985. 2501258127

19. Barker, R., Burke, J., and Zieve, P. (eds.). Principles of Ambulatory Medicine. Williams and Wilkins, Baltimore, 1983, p. 1074. 20. Hecht, S.S., Rivenson, A., Braley, J., DiBello, J., Adams, J.D., and Hoffmann, D. Induction of oral cavity tumors in F344 rats by tobacco- specific nitrosamines and snuff. Submitted (1986). 21. Hirsch, J.M., and Johansson, S.L. Effect of long-term application of snuff on the oral mucosa: An experimental study in the rat. J. Oral Pathol. 12: 187-198, 1983. 22. Hirsch, J.M., Johansson, S.L., and Vahlne, A. Effect of snuff and herpes simplex virus-1 on rat oral mucosa: Possible associations with the development of squamous cell carcinoma. J. Oral Pathol. 13: 52-62, 1984. r 23. Dunham, L.J., and Herrold, K.M. Failure to produce tumors in the cheek pouch by exposure to ingredients of betel quid; histopathological changes in the pouch and other organs by exposure to known carcinogens. J. Natl. Cancer Inst. 29: 1047-1067, 1962. 24. Dunham, L.J., Muir, C.S., and Hamner, J.E., III. Epithelial atypia in hamster cheek pouches treated repeatedly with calcium hydroxide. Br. J. Cancer 20: 588-593, 1966. 25. Dunham, L.J., Snell, K.C., and Stewart, H.L. Argyrophilic carcinoids in two Syrian hamsters (Mesocricetus auratus). J. Natl. Cancer Inst. 54: 507-513, 1975. 26. Homburger, F. Mechanical irritation, polycyclic hydrocarbons, and snuff. Effects on facial skin, cheek pouch, and oral mucosa in Syrian hamsters. Arch. Pathol. 91: 411-417, 1971. 27. Park, N.H., Herbosa, E.G., and Sapp, J.P. Oral cancer induced in hamsters with herpes simplex infection combined with simulated snuff-dipping (Abstract 10). International Herpes Virus Workshop, Ann Arbor, August 11- 16, 1985, p. 297. 28. Schmahl, D. Prufung von Kautabakextract auf cancerogene Wirkung bei Ratten. Arzneimittel-Forsch., 15: 704-705, 1965. 29. Druckrey, H., Schmahl, D., Beuthner, H., Muth, F. Vergleichende Prufung von Tabakrauch-Kondensaten, Benzopyren und Tabakextract auf carcinogene Wirkung bei Ratten. Naturwissenschaften 47: 605-606, 1960. 30. Campbell, J.M., and Lindsey, A.J. Polycyclic aromatic hydrocarbons in snuff. Chem. Ind. London 951, 1957. 31. Harley, N.H., Cohen, B.S., and Tso, T.C. Polonium-210. A questionable risk factor in smoking-related carcinogenesis. Banbury Report 3: 93-104, 1980. M" 32. Hoffmann, D., Harley, N.H., Fisenne, I., Adams, J.D., and Brunnemann, K.D. Carcinogenic agents in snuff. J. Natl. Cancer Inst. (in press). 2501258128

33. International Agency for Cancer Research. Monograph on the evaluation of the carcinogenic risk of chemicals to humans, Vol. 17. Some N-nitroso compounds. Lyon, France, 1978, p. 365. 34. Hoffmann, D., Rivenson, A., Adams, J.D., Juchatz, A., Vinchkoski, N., and Hecht, S.S. Effects of route of administration and dose on the carcinogenicity of N-nitrosodiethanolamine in the Syrian golden hamster. Cancer Res. 43: 2521-2524, 1983. 35. Hecht, S.S., Adams, J.D., Numoto, S., and Hoffmann, D. Induction of respira- tory tract tumors in Syrian golden hamsters by a single dose of 4-(methyl- nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the effect of smoke inhala- tion. Carcinogenesis 4: 1287-1290, 1983. 36. Hecht, S.S., Trushin, N., Castonguay, A., and Rivenson, A. Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine. Cancer Res. 46: 498-502 (Feb) 1986. 37. Hoffmann, D., Rivenson, A., Amins, S., and Hecht, S.S. Dose-response study of the carcinogenicity of tobacco-specific N-nitrosamines in F344 rats. J. Cancer Res. Clin. Oncol. 108: 81-86, 1984. 5: 501-503, 1984. 38. Hoffmann, D., and Hecht, S.S. Perspectives in cancer research. Nicotine- derived N-nitrosamines and tobacco-related cancer: Current status and future directions. Cancer Res. 45: 935-944, 1985. 39. Bhide, S.V., Shah, A.S., Nair, J., and Nagarajrow, D. Epidemiological and experimental studies on tobacco related oral cancer in India. IARC Sci. Publ. 57: 851-857, 1984. 40. Shirname, L.P., Monon, M.M., and Bhide, S.V. Mutagenicity of betel quid and its ingredients using mammalian test systems. Carcinogenesis' 41. Umezawa, K., Fujie, S., Sawamur, M., Matsushima, T., Koath, Y, Tanaka, K., and Takayama, S. Morphological transformation, sister chromatid exchange and mutagenicity assay of betel constituents. Toxicol. Lett. 8: 17-22, 1981. 42. Umezawa, K., Takayama, S., Fujie, S., Matsushima, T., and Sugimura, T. In vitro transformation of hamster embryo cells by betel tobacco extracts. Toxicol. Lett. 2: 243-246, 1978. 43. Stich, H.F., Curtis, J.R., and Parida, B.B. Application of the micro- nucleus test to exfoliated cells of high cancer risk groups: Tobacco chewers. Int. J. Cancer 30: 553-559, 1982. 44. Tucker, J.D., and Ong, T. Induction of sister chromatic exchanges by coal dust and tobacco snuff extracts in human peripheral lymphocytes. Environ. Mutagen. 7: 313-324, 1985. 45. Andrews, A.W., Thibault, L.H., and Lijinsky, W. The relationship between mutagenicity and carcinogenicity of some nitrosamines. Mutat. Res. 51: 319-326, 1978. 2501250129~ 2-67

46. Bartsch, H., Malaveille, C., Camus, A.M., Martel-Planche, G., Brun, G., Hautefeuille, A., Sabadie, N., Borlin, A., Kuroki, T., Drevon, C., Piccoli, C., and Montesano, R. Validation and comparative studies on 180 chemicals with S. typhimurium and V79 Chinese hamster cells in the presence of metabolizing systems. Mutat. Res. 76: 1-50, 1980. 47. Hecht, S.S., Lin, D., and Castonguay, A. Effects of alpha-deuterium substitu- tion on the mutagenicity of 4-(methylnitrosamino)-l-(3-pyriayl)-1-butanone. Carcinogenesis 4: 305-310, 1983. 48. Williams, G.M., and Laspia, M.F. The detection of various nitrosamines in the hepatocyte primary culture/DNA repair test. Cancer Lett. 6: 199- 206, 1979.

Table 1 Bioassays for Carcinogenic Activity of Chewing Tobacco or Chewing Tobacco Extracts* Oral- mice, M intubation Oral-feeding mice, M Skin- mice, M+F topical Oral- mice, M+F swabbing Oral- rats (NS)*** swabbing Oral-pouch hamsters implantation (NS) Oral-pouch hamsters (NS) Oral-pouch swabbing Duration of Fraction of Animals With Tumors Test Material Exposure Refer- and Dose (Months) Exposed Controls ence extract diluted 1:25 4/1/2 diluted 1:50 15-20 0.2% extract in diet 21-25 DMSO extract (dose ?) 21-22 extracts applied up to 18 daily, dose not given 4/10** lung adenocarcinoma 8/15** and liver carcinoma 8/10** lung adenocarcinoma 0/10 0/7 no excess tumors compared to controls 2% alkaloid-free Lifespan 0/10 extract dose not given- 0/12 + lime 2-cm3 plug up to 30 0/50 DMSO-extract three 18-24 0/12 times weekly hamsters, M DMSO-extract three times weekly, dose not given 5 0/12 Oral-pouch hamsters, F 2% tobacco extract 6 3/17 swabbing in water; twice daily application Subcutaneous mice (NS) 2Z tobacco extract 10-23 1/17 squamous-cell carcinoma injection partially or com- IEI$rCZIDSZ pletely free of alkaloids; 25 solu- tlon once a month *Abbrevlatl.on: Dt4S0, rl[met:hyl sulluxlde. **An[rnal:j at risk. *k*(N',) = nnt :t;itvd. (site not specified) 0/20 1 2 0/10 3 0/14 7 Oh 5 0/11 4 0/10 6 8

Table 2 Bioassays for Carcinogenic Activity of Snuff or Snuff Extracts* Route of Application Species, Sex Test Material and Dose Oral- feeding haasters, `t S;20S of the diet Lips- painting Hice, M SE, dose not given Oral- Rats, H SE+ti swabbing SE (approx. 30%) SE (approx. 30%) + (NNN+NNX) NNN+NMG L1 p canal- Racs, F S instillation S H S+H Lip canal, Rats, H S instillation S+Se ES Cheek pouch Hamsters S instillation (YS)r S M S+H Subcutaneous Rats, :4+F SE Injection Rats (NS) TE Duration of Fraction of Animals «ith Tumors Exposure ReEer- Acplications (Months) Exposed Controls e^ce_ Once daily 24 0/100** 0/100 1' 3 x daily 2 0V 20 0/20 !3 0.5 al daily 2 0.5 t1 daily up to 30 0/20 0/20 la 0/30 1/21 (lung =J adenoma) 0.5 al daily up to 30 S/30 (3 papillosa in 1/21 (Lung =0 oral cavity, 2 adenoaa) lung adenosa) 0.5 st1 daily up to 30 13/30 (8 papillosa in 1/21 (lung :3 oral cavity, 5 adenoaa) luns adenosa) 200 sg 9-22 twice daily 200 ag 18 twice daily 200 ng 18 twice daily 18 SO a+= up to 30 daily 50 ag daily 50 ag daily up to 30 up to 30 up to 30 6 6 6 50 mg, 84 26 weekly ap- plicacions weekly ap- plications 45 mg, 70 21 + 4 1/42** (oral carcinosu) 0/20 -L 1/10 (oral carcinoma) 0/7 2/7 (2 oral carcinoma) 0/10 0/10 0/10 3/32 (papillona and 1 carcinoma in cest 0/10 -0 canal, I oral papil lossa ) 1/32 (oral papilloaa) 2/21 (oral papilloma) 0/10 0/10 :0 '_0 0/50 0/5J 0/25 0/25 L1/25 (papilloma and carcinoma of the oral cavity) • 0/82 0/82 :~ 18/75 1/75 :? *Abbrevtations: ES, extracted snuff; li, infected with herpes siopLex virus; NYK, .-(methylnicrosamino)-1-(-3-pyridyl)-:- butanone; NNN, Y'-nitrosonornicocine; S, snuff; SE, snuff extract. **No tu®ors of the oral cavity, esophagus, nasopharynx and Larynx; all other tumors nearly identical to those in control aninals. f(:IS) - not stated.

. Table 3 Carcinogenicity of Tobacco-Specific Nitrosamines* Nitros- amine Species and Route of Strains Application Principal Target Organs Dose NNN K AT NAB NNA A/J mouse F344 rat prague-Dawley rat Syrian golden hamster A/J mouse F344 rat Syrian golden hamster F344 rat F344 rat Syrian golden hamster A/J mouse i.p. s.c. p.o. P.O. s.c. i.p. s.c. S.C. S.C. p.o. s.c. i.p. lung nasal cavity esophagus eso phagus nasal cavity nasal cavity trachea nasal cavity lung nasal cavity lung, liver trachea, lung, nasal cavity none esophagus none none 0.12 mmol/mouse 0.2-3.4 mmol/rat 1.0-3.6 mmol/rat 8.8 mmol/rat 0.9-2.1 mmol/aamste 0.12 mmol/mouse 0.1-2.8 mmol/rat 0.9 mmol/hamster 0.005 mmol/hamster 0.2-2.8 mmol/rat 3-12 mmol/rat 2 mmol/hamster 0.12 mmol/mouse *Hoffmann and Hecht, 1985 (11).

CONCLUSIONS 1. The scientific evidence is strong that the use of smokeless tobacco can cause cancer in humans. The association between smokeless tobacco use and cancer is strongest for cancers of the oral cavity. 2. Oral cancer has been shown to occur several times more frequently among snuff dippers than among nontobacco users, and the excess risk of cancers of the cheek and gum may reach nearly fiftyfold among long-term snuff users. 3. Some investigations suggest that the use of chewing tobacco also may increase the risk of oral cancer. 4. Evidence for an association between smokeless tobacco use and cancers outside of the oral cavity in humans is sparse. Some investigations suggest that smokeless tobacco users may face increased risks of tumors of the upper aerodigestive tract, but results are currently inconclusive. 5. Experimental investigations have revealed potent carcinogens in snuff and chewing tobacco. These include nitrosamines, polycyclic aromatic hydrocarbons, and radiation-emitting polonium. The tobacco-specific nitrosamines N-nitrosonornicotine and 4-(methylnitrosamino)-l-(3-pyridyl)- 1-butanone have been detected in smokeless tobacco at levels 100 times higher than the regulated levels of other nitrosamines found in bacon, beer, and other foods. Animals exposed to these tobacco-specific nitrosamines, at levels approximating those thought to be accumulated during a human lifetime by daily smokeless tobacco users, have developed an ezcess of a variety of tumors. The nitrosamines can be metabolized by target tissues to compounds that can modify cellular genetic material. 6. Bioassays exposing animals to smokeless tobacco, however, have generally shown little or no increased tumor production, although some bioassays suggest that snuff may cause oral tumors when tested in animals that are infected with herpes simplex virus. RESEARCH NEEDS It has been established beyond reasonable doubt that smokeless tobacco use can increase the risk of cancer. The experimental and epidemiologic evidence is strongest for the association between oral cancer and the chronic use of snuff. Additional studies are needed to determine whether the patterns of risk differ according to the form of smokeless tobacco, including research evaluating cancer risks that are associated with chewing tobacco and dry versus moist snuff, and to quantify further the levels of risk in relation to differing levels of smokeless tobacco exposure. The influence of smoking, alcohol, and other factors (including viral exposures) on the smokeless tobacco-associated risk of oral cancer also should be explored further with an emphasis on detecting possible interac- tions between these factors and smokeless tobacco. 2501250134

Inhaled snuff may increase the risk of nasal carcinoma. The feasibility of initiating studies in areas where snuff sniffing is common should be ascertained, and studies should be launched to confirm and quantitate this possible relationship. There have been few studies of smokeless tobacco and esophageal, laryngeal, and gastric cancers. These investigations have provided equivocal results, but in the aggregate, their findings raise the possibility of some increase in risk among smokeless tobacco users. Additional case-control studies of these neoplasms should be encouraged. These studies should be large enough to assess the risks that are associated with smokeless tobacco use while control- ling for the potential confounding effects of smoking, alcohol, and other risk factors. Isolated reports have associated smokeless tobacco with cancers of the cervix, pancreas, and other anatomic sites. Investigators with existing data from case-control studies of these neoplasms should be encouraged to perform analyses to determine whether associations with smokeless tobacco exist. Similarly, existing data from cohort studies with information on smokeless tobacco use should be analyzed. Reports from two relatively large cohort studies have been published only as abstracts. These should be expanded with detailed descriptions of both the methods used and the findings for various cancers and should be updated to include followup into the 1980's. Recommen- dations for additional studies of the role, if any, of smokeless tobacco in the etiology of cancers outside of the upper aerodigestive tract should await the results of these analyses. On the basis of current knowledge, it can be assumed that chewing tobacco and snuff contain several unknown nitroso compounds that may be contributors to the carcinogenic potential of these products. Indepth analytical studies are needed for the identification of these unknowns. Furthermore, mechanisms of their in vitro and endogenous formation should be studied together with those of t-ie n t=oso compounds that are already known to occur in smokeless tobaccos. For the validation of the uptake of the major carcinogens by tobacco chewers and snuff dippers, markers should be measured in the target tissues and in physiological fluids. Major emphasis should be placed on the identification and assays of DNA-adducts: with tobacco-specific compounds in tissues of the oral cavity. Finally, trends over time in age-specific oral cancer incidence and mortality rates should be monitored to determine whether the increasing use of smokeless tobacco by Americans is influencing national or regional cancer patterns. Changes in the prevalence of use and in the characteristics of smokeless tobacco products should also be documented. Such monitoring will provide a base upon which future investigations of associations between smokeless tobacco and cancer can be built. 2-73

CHAPTER 3 NONCANCEROUS AND PRECANCEROUS ORAL HEALTH EFFECTS ASSOCIATED WITH SMOKELESS TOBACCO USE

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1 THE EFFECTS OF SMOKELESS TOBACCO USE ON ORAL LEUKOPLAKIA/MUCOSAL PATHOLOGY AND THE TR.ANSFORMATION OF ORAL SOFT TISSUES . . . . . . . . . . . . . . . . . 3-2 Oral Leukoplakia/Mucosal Pathology . . . . . . . . . . . . . ... . . 3-2 Transformation of Oral Soft Tissues . . . . . . . . . . . . . . . . 3-5 THE EFFECTS OF SMOKELESS TOBACCO USE ON THE GINGIVA, PERIODONTAL TISSUE, AND SALIVARY GLANDS . . . . . . . . . . . . . . .3-14 Background and Definitions . . . . . . . . . . . . . . . . . . . . .3-14 Gingival and Periodontal Tissues . . . . . . . . . . . . . . . . . .3-15 Salivary Glands . . . . . . . . . . . . . . . . . . . . . . . . . . 3-17 THE EFFECTS OF SMOKELESS TOBACCO USE ON TEETH . . . . . . . . . . . . 3-18 Background and Definitions . . . . . . . . . . . . . . . . . . . . .3-18 Dental Caries . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-19 Other Hard Tissue Effects . . . . . . . . . . . . . . . . . . . . . 3-20 CONCLUS IONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2 0 RES EARCH NEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-21 REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-2 2 i

INTRODUCTION This chapter addresses the health effects of smokeless tobacco use on the oral tissues through a systematic review of the relevant scientific litera- ture of animal and human studies. The major areas addressed are the effects of smokeless tobacco use on the oral soft tissues, the periodontium, and the teeth. This chapter also reviews information regarding the potential of oral tissue altered by smokeless tobacco use to transform to dysplasia and malignancy. Within each area, except for the section on the transformation of oral soft tissues, those tissues or conditions that are suspected to be most affected by smokeless tobacco use, or that hold the greatest potential for health effects, are considered initially. Where contradictory evidence exists, these data are also presented. Studies that were judged to meet stringent selection criteria* are presented first, followed by data from less rigorous study designs and case reports. Within the section on the transformation of oral soft tissues, the presentation of the evidence is grouped according to clinical reports, cohort studies, and case-control studies. This was done so as to be consistent with the format used in the chapter on Carcinogenesis Associated With Smokeless Tobacco Use (chapter 2). In some cases, studies referenced in this chapter are the same as those used in chapter 2. The reader should review both chapters in order to obtain all pertinent information contained in these studies. Only studies from the United States and Scandinavia are included for the sections on oral leukoplakia/mucosal pathology, gingival and perio- dontal tissues, and salivary glands. This assures that studies dealing with similar types of smokeless tobacco are used for comparison purposes. However, the section on the'transformation of oral soft tissues includes a fuller range of studies that have reviewed the histopathologic changes associated with smokeless tobacco-induced lesions. Studies investigating the histopatho- logic transformation of nonsmokeless tobacco-induced lesions have not been included. A summary of selected studies that addresses study sample, methods, and observations is provided in table 1 as a ready alphabetical reference to the text. In addition, a summary of selected case reports is provided in table 2. Emphasis has been placed on the issues of prevalence of oral tissue changes, types of changes, site-specificity of changes, and the effects of dose-response. *See Introduction, Overview, and Conclusions for discussion of criteria for causality. 3-1

THE EFFECTS OF SMOKELESS TOBACCO USE ON ORAL LEUKOPLAK.IA/MUCOSAL PATHOLOGY AND THE TRANSFORMATION OF ORAL SOFT TISSUES Oral Leukoplakia/Mucosal Pathology Background and Definitions Various oral soft tissue effects of smokeless tobacco use-have been re- ported in the literature. These include oral leukoplakia/mucosal pathology. The actual terms used and the definitions employed to describe these con- • ditions vary widely from study to study (table 3). The World Health Organi- zation (WHO) defines oral leukoplakia as a white patch or plaque that cannot be characterized clinically or pathologically as any other disease (1). The mucosal pathology that is found in smokeless tobacco users also has been referred to as hyperkeratosis, an oral mucosal lesion that exhibits an abnormal whitish (keratinized) appearance clinically. The authors' terms are employed when a specific study's findings are described. However, in the discussion portion of the report, the general terms of oral leukoplakia/mucosal pathology are used. The association between smokeless tobacco use and oral leukoplakia/ mucosal pathology has been moderately studied. The WHO has stated that tobacco is an etiologic agent for the formation of oral leukoplakia (1). This was reaffirmed at an International Seminar on Oral Leukoplakia and Associated Lesions Related to Tobacco Habits (2). In a review of the effects of tobacco habits other than smoking, the use of smokeless tobacco/snuff was associated with the presence of leukoplakia (3). Studies in the United States Six studies have addressed the prevalence of oral leukoplakia/mucosal .pathology in smokeless tobacco/snuff users (4-9). In two of these studies, blindness of the examiners toward the tobacco habits of the subjects was maintained, and oral tissue findings in smokeless tobacco users anti nonusers were compared (7,9). Three of these studies investigated adults (4-6) and three investigated adolescents (7-9). In addition, several case reports have described oral leukoplakia/mucosal pathology findings in smokeless tobacco users (10-13). Highlights of these studies and reports are summarized below. Offenbacher and Weathers investigated the oral tissue effects of smoke- less tobacco use in adolescent males from the greater metropolitan area of Atlanta, Georgia (9). They used oral examinations and self-administered questionnaires on tobacco use. Of the 565 males who were examined, 75 (13.3 percent) used smokeless tobacco. The difference in the prevalence of mucosal pathology in smokeless tobacco users (22.7 percent) was statistically signif- icant compared to that of nonusers (4.7 percent); however, the authors did not provide specific diagnostic criteria in this assessment. The range of mucosal pathologic findings included such conditions as morsicatio (cheek biter's lesion), ulcer, keratosis/leukoplakia, vesiculobullous, petechiae, abscess, erythema, mucocele, and pericoronitis. Although 50 percent of the smokeless tobacco users with mucosal pathology had keratosis/leukoplakia 25a1258139 3-2

compared to 3.8 percent of the nonusers with mucosal pathology, the authors did not identify the locations of the mucosal pathologies. Peacock, Greenberg, and Brawley reported a significant relationship between chronic tobacco use and the presence of oral leukoplakia+ in a study of 1,388 textile mill workers in North Carolina (5). The 362 employees who reported using smokeless tobacco had a significantly higher prevalence of leukoplakia (34 percent) than did the 457 nonusers (7.4 percent). In addi- tion, the authors noted a direct leukoplakia and age effect. In a study conducted in Denver, Colorado, Greer and Poulson examined 1,119 teenagers in grades 9 to 12 in order to assess the relationship between oral tissue alterations and the use of smokeless tobacco (7). Smokeless tobacco was used by 117 (10.5 percent) of these teenagers. Of these, 42.7 percent had oral mucosal lesions* in the area of tobacco placement. Forty-six percent of the teenagers: with mucosal lesions also had concomitant periodontal tissue degeneration.** Poulson, Lindenmuth, and Greer examined a sample of 445 teenagers in 5 rural Colorado towns to assess the relationship between oral tissue altera- tions and smokeless tobacco use (8). Smokeless tobacco was used by 56 (12.6 percent) of the teenagers. Of these, 58.9 percent had oral mucosal lesions in the area of habitual tobacco placement. Concomitant periodontal degeneration was noted in 39.4 percent of those with oral mucosal lesions. Contrasting the results of rural versus urban adolescent smokeless tobacco users, Poulson, Lindenmuth, and Greer suggested that the duration of use may be critical in the development of "oral lesions" (8).*** Those adolescents with oral lesions used smokeless tobacco longer (an average of 3.3 years in the rural and urban groups) than those without lesions in both the rural and urban groups (2.3 years and 2.2 years, respectively). In addition, the authors noted similar effects of different levels of smokeless tobacco use in daily exposure. Users with oral lesions were exposed 205 minutes per day in the rural and 177 minutes per day in the urban group compared with users with no oral lesions (110 minutes (rural) and 53 minutes (urban), respectively). Also, more:than twice as many marked oral mucosal lesions were identified in the rural population as in the urban population. Smith et al. examined a population of 15,500 snuff users by cytological, histological, and visual means (6). Of these users, 1,751 (11.3 percent) demonstrated oral mucous membrane changes. Although no definitions were provided, these changes were described as "cloudy or gray glistening" areas +Leukoplakia was defined as a "pearly white plaque on the mucous membrane which could not be scraped off with a tongue blade." *The authors used a modification of the classification method that was de- veloped by Axell et al. that identifies the oral mucosal lesions according to color, wrinkling, and thickening (14). **The authors define this degeneration as "site-specific gingival recession with apical migration of the gingiva to or beyond the cementoenamel junction,' with or without clinical evidence of inflammation." ***The term "oral lesions" used here includes periodontal tissue degeneration and oral mucosal lesions.

having "wrinkled appearance(s)" and presenting "white or red granular appearance(s)." The authors reported that when snuff was withdrawn, the tissue returned to normal appearance. Moore, Bissinger, and Proehl investigated the relationship between tobacco use and oral cancer in male patients ages 50 years and older who attended the General Tumor Clinic in Minneapolis, Minnesota (4). The authors noted that a significant number of the patients who manifested oral leukoplakia (18 of 23--78.3 percent) used smokeless tobacco. A tobacco user in this study was defined as a person who used the tobacco product for 20 or more years. Apparently, some of these 23 patients were also pipe, cigar, or cigarette smokers, although the exact number was not specified. The authors indicated that the most severe patches of leukoplakia were seen in patients who chewed "strong" tobacco and over a longer duration (no quantification reported). In most instances in which patients had stopped using smokeless tobacco, leuko- plakia disappeared. Several case reports (table 2) have described oral leukoplakia/mucosal pathology at the site of smokeless tobacco/snuff placement (10-13). These cases represent males of various ages with differing years of smokeless tobacco/snuff use. Hoge and Kirkham reported that, in one patient, with- drawal of snuff resulted in a reversal of the hyperkeratotic lesions (12). Studies in Scandinavia Studies of smokeless tobacco from Scandinavia have investigated the prevalence of oral leukoplakia/mucosal pathology in users (15-19). xe All found 1,444 smokeless tobacco users (predominantly men) in the 20,333 Swedes who were examined for soft tissue lesions (17). Of these users, 116 (8 percent) had "snuff-dipper's lesion" (see table 3 for defini- tions). The prevalence of oral leukoplakia among the total study population was 3.6 percent. Hirsch, Heyden, and Thilander (18) graded oral mucosal lesions on an established four-point scale (14) and correlated these findings with the snuff habits in 50 Swedes ages 15 to 84 years who used snuff routinely. Younger patients were found to have lower degrees of pathologic changes, while a significant predominance of older patients was noted with higher degrees. The authors reported that patients with oral mucosal lesions of the highest degree had used snuff an average of 34.7 years compared with the 9.2- to 13.6-year average for patients with lower degrees of pathologic changes. They also noted that patients with high degrees of pathologic changes dipped twice as long per day (an average of 10.1 and 10.6 hours per day) as patients with lower degrees of pathologic changes (5.2 and 6.5 hours per day, respectively). Although these patients reported multiple tobacco habits, the authors stated that no differences in clinical grading were found between patients who used snuff only and those who used snuff and other tobacco products. In addition, several case reports have described oral leukoplakia/ mucosal pathology (table 2). In Sweden, Frithiof et al. examined 21 male 2501258141

snuff users ages 31 to 79 years (19). A11 had snuff-induced lesions that were localized to the area in the oral cavity where the tobacco was held. Similarly, leukoplakia lesions were found at the site of snuff placement in all 12 male users of snuff ages 39 to 83 years in a study in Denmark (15). In this latter study, 3 weeks after one of the patients discontinued,snuff use, the clinical appearance of the mucous membrane had returned to normal. In another report, four of seven Danish male users of snuff exhibited leukoplakia at the site of snuff placement (16). . Discussion The studies from the United States and Scandinavia demonstrate that oral leukoplakia/mucosal pathology is associated with smokeless tobacco/ snuff use. In two studies, a higher prevalence of oral leukoplakia/mucosal pathology was.found in users compared to nonusers of smokeless tobacco- 22.7 percent compared to 4.7 percent (9) and 34.0 percent compared to 7.4 percent (5). In all of these studies, between 8 and 59 percent of smokeless tobacco/snuff users were found to have oral leukoplakia/mucosal pathology. It appears that the oral leukoplakialmucosal pathology noted in smoke- less tobacco/snuff users is found commonly at the habitual site of tobacco/ snuff placement. Using a similar grading classification for snuff-induced lesions (7,14), all of the mucosal pathology that was noted in four studies was at the site of habitual tobacco placement (7,8,17,18). Similarly, the majority of the oral leukoplakia/mucosal pathology that was described in the case reports was found where the tobacco/snuff was usually placed. The duration of use (in years) and daily exposure (in hours or minutes) to smokeless tobacco appear to be critical in the development and severit7 of oral leukoplakia/mucosal pathology. Three studies using similar approaches to the definition of oral leukoplakia/mucosal pathology and to the measure- ment of exposure noted this effect (7,8,18). Only two studies were designed to study the concomitant findings of oral leukoplakia/mucosal pathology and other tissue changes. The authors reported that 39.4 (8) and 46.0 (7) percent, respectively, of smokeless tobacco users with oral leukoplakia/mucosal pathology also had periodontal tissue degeneration (gingival recession). These oral soft tissue changes also were found at the site of habitual tobacco placement. In several studies where individuals had stopped smokeless tobacco use, the oral leukoplakia/mucosal pathology disappeared (4,6,12,15). Transformation of Oral Soft Tissues Background and Definitions The previous section that discussed smokeless tobacco-induced leukoplakia noted that clinically observable changes in soft tissue morphology do occur as a result of smokeless tobacco use. Smokeless tobacco-associated lesions- that have been traditionally classified as leukoplakias (white lesions) offer varying clinical degrees of differentiation and may persist or progress with continued smokeless tobacco use. Additionally, some leukoplakias have been 2501250142 3-5

observed to resolve clinically upon the cessation of smokeless tobacco use. This section of the report addresses the transformation of oral soft tissues. It discusses the potential for smokeless tobacco-induced lesions to regress, persist, or continue to progress to lesions with higher malignant potential or to malignancy. There are varying clinical and histologic definitions in the scientific literature related to tobacco-induced changes (transformation) of oral soft tissues. The following definitions represent those most frequently encountered. It will be noted when significant variation of these definitions occurs in studies cited. • Oral leukoplakia--a white patch or plaque that cannot be characterized clinically or pathologically as any other disease (1). • Snuff dipper's leukoplakia-a leukoplakia associated with the use of smokeless tobacco. These are further characterized as to differing morphologic forms. • Erythroplakia-a lesion present as a bright red patch or plaque that cannot be characterized clinically or pathologically as any other condition, such as carcinoma, infection, etc. • Precancerous condition-a generalized state that is associated with an increased risk of cancer based on epidemiologic or histologic evidence. Precancerous lesion-a morphologically altered tissue in which cancer is more likely to occur than in its apparently normal counterpart. • Acanthosis -an increased thickness of the spinous cell layer of the epithelium. • Hyperkeratosis --an increased thickness of the keratinized layer of the epithelium. • Hyperparakeratosis-an increased thickness of a normally parakeratotic layer of the epithelium, i.e., surface cells with retained nuclei. • Hyperorthokeratosis---an increased thickness of a normally keratotic layer of the epithelium, i.e., surface cells without retained nuclei. • Chevron keratinization--a keratinization pattern typified by vertical streaks of parakeratinization that extend to the epithelial surface and create surface irregularities by extensions of the outer surface layer. • Dysplasia--abnormal tissue development characterized by varying numbers and degrees of morphologic cell changes that reflect grades of severity. • Dysplastic changes include the following: 3-6

-Pleomorphism in the size and shape of cells and their nuclei. -Abnormal numbers of cells undergoing mitotic activity (discrepancy in maturation). --Atypical mitotic cells. -Cytoplasmic atypicalities (altered nuclear to cytoplasmic ratio). -Hyperchromasia. -Irregular nuclear borders. -Basal cell hyperplasia. -Loss of polarity. • Carcinoma in situ--a significant number of dysplastic epithelial cell changes that extend from the basal layer to the surface layer without violation of the basement membrane. • Verrucous carcinoma-a clinically verruciform cancer of epithelial tissue that tends to be slowly and locally invasive with a metastasis and mortality potential that is lower than classic squamous cell carcinomas. The cells are well differentiated. • Squamous cell carcinoma-a cancer of the stratified squamous epithe- lium that has varying clinical appearances, is invasive, extending beyond the basement membrane, and has a great potential for metastasis. Evidence of the relationship between smokeless tobacco use and the transformation of o=a1 soft tissues is represented by the following: 1. Clinical reports describing tobacco habits of persons 'with graded oral lesions. 2. Followup (cohort) studies of tissue changes, including transforma- tion to malignancy, among patients with leukoplakia. 3. Case-control studies or case series of oral cancer describing con- comitant leukoplakia. A review of the evidence in each of these study areas follows: Clinical Reports of Oral Lesions in Association 'With Smokeless Tobacco Use Hirsch, Heyden, and Thilander (18) graded oral snuff-induced mucosal lesions in 50 patients on a four-point scale according to criteria developed by Axell (14): 25a1258144

• Degree 1: A superficial lesion with a color similar to the surround- ing mucosa, slight wrinkling, and no obvious thickening. • Degree 2: A superficial whitish or yellowish lesion with wrinkling and no obvious thickening. • Degree 3: A whitish-yellowish to brown lesion with wrinkling, intervening furrows of normal mucdsal color, and obvious thickening. • Degree 4: A marked white-yellowish to brown lesion with heavy wrinkling, intervening deep and reddened furrows, and heavy thickening. Snuff habits and drinking habits of the patients were obtained from questionnaires. Patients in the degree 4 category had been snuff dippers significantly longer than the rest of the patients. Also, patients in de- grees 3 and 4 dipped approximately twice as long per day as did patients in degrees I and 2. The daily exposure to snuff was significantly longer in degree 4 (10.6 hours) than in degrees 1 (5.2 hours) and 2 (6.5 hours). When total exposure was compared between the four clinical groups taking into account hours of use per day as well as years of use, significant differences were found. In this study, significant difference could be found with regard to clinical grading and histological appearances between patients with multiple habits (snuff, smoking, and drinking) and those who only used snuff. The four clinical degrees of lesions exhibited an age-dependent effect with younger patients usually found in clinical degrees 1, 2, and 3 and a significant pre- dominance of older patients noted in degree 4. Degree 4 lesions included an increased number of mitotic figures, edema, and slight to moderate inflamma- tion compared to the other three degrees. Eighteen'percent of the patients exhibited slight epithelial dysplasia, and lesions with slight epithelial dysplasia were found in all categories. Patients in the dysplastic group had been snuff dippers longer on average (23.9 years) as compared with those without dysplasia (19.5 years). No case of moderate or severe dysplasia was noted. (The authors referenced the WHO Collaborating Center for Oral Precan- cerous Lesions as the definition for dysplasia (1).) • Aaell, Mornstad, and Sundstrom obtained biopsies of the oral mucosal lesions of 114 male dippers aged 20 to 88 years from a sample of 1,200 Swedish snuff dippers (14). Clinically, lesions were graded (degrees 1 through 4) based on color and morphology. Lesions of higher clinical degrees were associated with greater daily exposure to snuff in terms of hours and grams of exposure. All but one of the biopsies showed increased epithelial thick- ness. The outer layers appeared vacuolated with occasional remnants of cell nuclei. Lesions in degrees 3 and 4 had more pronounced surface layers. Acanthosis was evident in all of the clinical groups. None of the biopsies showed changes that were interpreted as cellular atypia or epithelial dysplasia. The cessation of snuff dipping for a few days was reported to result in clinical regression of the lesions with loss of the vacuolated layer. 2501258145

Greer et al. reviewed clinically and histologically examined smokeless tobacco-induced leukoplakias from 45 patients aged 13 to 74 years (20), follow- ing criteria that were previously established by Greer and Poulson (7) as adapted from Axe'll. The vast majority of the mucosal lesions were corrugated, white, and raised. No evaluations for an interrelationship between smokeless tobacco use, smoking, and alcohol use and clinical or histologic tissue changes were attempted. Histologic examinations for specific changes were reported. Dark cell keratinocytes characterized by a strong.affinity for basic dyes and by electron density of their cytoplasm and nucleus and suggested as dedifferentiated precursors of a neoplastic keratiaocyte were found in 17 of 45 cases. However, their presence was unrelated to the clinical degree of the lesion. While they have also been observed in leukoplakias that are associated with smoking (or other causes), the control group of nontobacco- induced hyperkeratoses demonstrated dark cell keratinocytes in only 3 of 45 cases. Chevron keratinization of the epithelial layer representing altered cellular maturation was present in 42 of 45 smokeless tobacco-induced leuko- plakias and in 4 of 45 control leukoplakia cases. Koilocytotic changes , appearing as vacuolated epithelial cells that may obscure the cytoplasm or appear with pyknotic nuclei, which are often associated with inclusion of viral particles in epithelial cells, were present in 27 of 45 smokeless tobacco-induced leukoplakias. In the entire sample of 45 cases, only 1 case of dysplasia (described as occurring in a"long-term" smokeless tobacco user) was identified. Three of the following characteristics had to be present for a lesion to be characterized as dysplastic: • Loss of cellular polarity. • Basal cell hyperplasia. • Altered nuclear/cytoplasmic ratios. • Anaplasia. • Dyskeratosis. • Atypical mitoses. Since the dysplasia case also involved the use of alcohol and smoking, it is not possible to attribute its appearance solely to smokeless tobacco use. In a study of 21 Finnish military recruits aged 17 to 21 years, mucosal lesions corresponded to the site of snuff placement and included the alveolar and labial mucosa to varying degrees (21). The duration and intensity of snuff use for this specific group could not be determined from the study. Epithelial hyperplasia and acanthosis were universally found under the light microscope. Hyperorthokeratinization was noted in 12 cases, hyperparakera- tinization in 9 cases, and Chevron-type keratinization in 1 case. One case of mild epithelial dysplasia was noted that included atypical and increased mitoses and loss of basal cell polarity. The authors concluded that this suggests a positive relation between snuff dipping and malignant changes. 2501258146

Van Wyk biopsied 25 snuff-induced lesions from Bantu smokeless tobacco users whose lesions had existed from a few weeks to 40 years (22). Comparison biopsies were also taken from healthy parts of the mucosa in the users, from healthly mucosa in nonusers, and from other white lesions and squamous carci- nomas. From the biopsies obtained from snuff users, 18 cases of acanthosis; 23 cases of parakeratosis; 5 cases of "keratosis"; and 4 cases with numerous mitotic figures, pleomorphism, hyperchromatism, and an irregular basal cell layer were noted. Additionally, 11 showed a disrupted appearance of the base- ment membrane. Those not associated with inflammation were considered possibly to be premalignant. Epithelium featuring these characteristics has been referred to by some as "disquiet epithelium." Contrarily, the author stated that "the impression is gained that no relationship exists between oral malignancy and the use of snuff." This was based on the widespread use of snuff but the occurrence of only one case of alveolar or sulcular cancer (not in a snuff user) in the hospital during this study. Several investigators have described connective tissue changes i`n snuff- induced lesions. A hyalinized, eosinophilic material that occurs well below the epithelium and around the minor salivary glands and/or in a plane that is genera.ll.y parallel to the epithelial surface has been reported by Pindborg et al. (16), Archard et al. (23), Axell et al. (14), and Greer et al. (20). The exact nature of and underlying explanation for the finding are not clear. Additionally, the role of such a histologic finding in the development or progression of premalignant or malignant lesions has not been identified. Cohort Studies Several investigations have followed persons with oral lesions for sub- sequent health outcomes. Smith reported the 10-year followup results on a group of patients with smokeless tobacco-induced leukoplakias (24). In the original study, oral cytologies were performed on 1,751 patients presenting with leukoplakias out of 15,500 snuff users (6). Results of the oral cytology examination consistently indicated only benign hyperkeratoses.* Biopsies were made of 157 leukoplakic lesions. However, no objective criteria for lesions selected for biopsy were offered. -None of the biopsies showed changes consistent with dyskeratosis or malignancy. These patients were followed with repeat cytology smears for 5.5 years. No additional significant mucosal changes were reported. In a subsequent 4.5-year followup (10 years total followup), periodic biopsies were done on 128 of the 157 patients who had originally received biopsies (24). The authors reported no dyskeratosis or carcinomas in the followup study. The method of followup was not specified. Significant numbers of patients were lost, and the clinical and histologic diagnostic criteria were not fully described. . A prospective study of oral cancer among persons with oral leukoplakia or other possible precancerous lesions was conducted in the Ernakulum district, Kerala State, India, as part of a 10-year followup to a much larger study of *The use of oral cytology for detecting dysplastic changes in leukoplakic lesions is less than satisfactory because of a high rate of false negative findings. The hyperkeratinized nature of leukoplakic lesions renders them resistant to the oral cytology scraping technique. Cellular changes in deeper layers ofthe epithelium would thus likely be missed (25). 25Q125814?

50,915 adults in 5 rural districts of India (26). Among those individuals who had been diagnosed as having a leukoplakia during the original survey, there was a malignant transformation rate of 9.7/1,000 per year for those who only chewed tobacco. For those who both smoked and chewed, the rate was 5/1,000 per year, while no malignancies were reported for individuals with or without tobacco habits who had not had a previous oral lesion. The transfor- mation rates among those with lesions were much higher than rates reported in the United States or European studies. While these results are not directly comparable to United States or European studies since the tobacco chewed in India is a variable mixture of betel leaf, areca nut, slake lime, and coarse tobacco, they suggest that the persons with leukoplakia are at increased risk of oral cancer. Specific clinical morphotypes of leukoplakia demonstrated varying potentials for malignant transformation: homogenous, 2.27 percent; speckled, 21.4 percent; and ulcerated, zero percent. In a small study of English coal miners, 8 of 22 patients with leukoplakia who chewed tobacco were followed for 5 years (27). Five of the eight cases showed no advance in the lesions, and two showed regression. The author does not specify whether these were clinical or histologic determinations or whether the smokeless tobacco habit persisted in all cases. One lesion that had been regarded as benign showed some hyperorthokeratosis and acanthosis of the epithelium but with no more than "minor epithelial atypia." The clinical appearance of this lesion was reported to have regressed initially over an intermediate 2-year period despite continuance of the habit of tobacco chew- ing and smoking. Subsequent followup over a 2-year period indicated that the lesion had progressed to an exophytic squamous cell carcinoma. The site of the lesion was where the patient had held tobacco for 30 years. While the malignant transformation rate in the group of chewing tobacco-associated leukoplakias was 12.5 percent, the small numbers and high dropout rate limit the significance of the finding. Of significance was the unpredictable course of the malignant lesion, initially regressing and then transforming into a squamous cell carcinoma. In a Danish study, 32 patients with snuff-induced leukoplakias from a group of 450 patients with leukoplakia were observed for a median time of 4.1 years (28). Each patient had also used alcohol with 17 percent claiming dail,v use. Thirty-three biopsies demonstrated hyperplastic epithelium with hyper- parakeratosis in 87 percent of the cases; half showed vacuolated cells. One -initial case of epithelial dysplasia was found, and one carcinoma was found to develop from a nondyskeratotic leukoplakia over the followup period. This represents a rate of premalignant or malignant transformation of 6.2 percent for either dysplasia or carcinoma. In comparing the rate of development of dysplasia and carcinoma from snuff-induced leukoplakias to nonsnuff-induced leukoplakias, the authors found no statistically significant differences. However, the rate of transformation in both groups was higher than would be expected in individuals without leukoplakic mucosa. In an earlier report on a small sample of 12 white male, snuff-using leukoplakia patients (use from 20 to 50 years), Pindborg and Renstrup did not find any malignant transformation (15). Biopsies were taken from sites where the snuff was held. All 12 showed unkeratinized hyperplasia of the epithelium with a few deep streaks of parakeratosis and downgrowth and broadening of the rete pegs with the outer layers of cells being vacuolated and large. The authors state that snuff-induced leukoplakias are easily reversible. Based on the limited size of this sample, definitive conclusions could not be made. 2501258148 ' 3-11

Oral Lesions Concomitant With Oral Cancer Three hundred and thirty-three patients with cancers of the buccal cavity and pharynx from the Rober,t Winship Memorial Clinic in Atlanta, Georgia, were compared with three control groups: a group with diseases of the mouth other than cancer or with no diseases; a group with cancer of sites other than the mouth, pharynx, or larynx; and a group without cancer and whose mouths were not examined-see chapter 2 (29). The authors, citing leukoplakia as a precancerous condition, found leukoplakias "more commonly in women with low grade squamous carcinomas arising in the mouth and with multiple cancers. Snuff dipping was frepently associated with leukoplakia and low grade cancer arising in the mouth. In a case-control study in Minnesota of cancers of the alveolar ridge, floor of the mouth, and buccal mucosa, it was noted that leukoplakias and cancers of the mouth were relatedd to the use of snuff or chewing tobacco (4). The most severe leukoplakias were reported among those who used "strong snuff" (no definition was provided) and held the quid at the same site for many years. Patients who quit using smokeless tobacco reportedly had leukoplakias disappear in most instances. A number of patients had multiple primary carcinomas that were also specific to the site of quid place- ment. Cancer lesions were described as having developed slowly over a period of several years, although no evidence of periodic clinical or histologic assessment was provided. McGuirt reported on 76 oral cancer patients, most with carcinomas of the alveolar ridge or buccal mucosa, identified from the tumor registry at the North Carolina Baptist Hospital who had a documented history of heavy smoke- less tobacco use (30). Fifty-seven of these patients used snuff and reported no cigarette, pipe smoking, or alcohol habits. The range of use was from 10 to 75 years. Leukoplakias had previously been excised in 13.9 percent of the cases, and 47.0 percent had associated leukoplakias at the time of surgery. The author cited "panmucosal insult" from smokelesa tobacco use as the cause of multiple lesions and recurrences-a type of field cancerization. From histologic evaluations of oral tissue among 23 Swedish patients with anterior oral vestibular cancer who were snuff users, leukoplakic lesions were noted outside the snuff-associated tumor in 5 (31). Leukoplakia and multiple carcinomas occurred together with the snuff-associated lesion in three cases. Eleven of 19 cases assessed for presence of candida were posi- tive. The temporal relationship between candida and carcinoma was not ascer- tainable; nor was the potential etiologic role of candida. Rosenfeld and Callaway examined data from records at Vanderbilt Ur.iver- sity Hospital, Nashville General Hospital, and the office of Rosenfeld for cases of squamous cell carcinoma arising in the mucous membrane of the anterior two-thirds of the tongue, the floor of the mouth, the gingiva, and the buccal area (32). A total of 525 cases were examined in users and nonusers of smoke- less tobacco-300 occurred on the gingiva and buccal areas. Among women with cancer of the buccal or gingival area, 90 percent had a history of snuff use. While no periodic quantitative or qualitative assessment of the natural his- 2501250149

tory of the cancers is provided, the authors do offer the following clinical impression of snuff-induced lesions in their study: "These carcinomas arising in the inner cheek and gingiva frequently start as leukoplakia. Progressive thickening, cornification, and eventual cauliflower-like ulcerations ensue. All stages in the pro- gressive disease may be seen in microscopic sections from a mere slight increase in the keratin layer, through carcinoma in situ to invasive malignancy." Twenty-five cases of histologically confirmed buccal gingival cancer in female snuff users were identified at the University of Arkansas Medical Center from 1950 to 1959 (33). Eleven occurred at buccal sites, 10 gingival, and 4 buccal and gingival. The patients (ages 44 to 84 years -mean 67.5) had a smokeless tobacco habit between 20 and 50 years. The lesions corresponded to the site of'habitual tobacco placement. Leukoplakia was a concomitant lesion and had been present for many years. Repeat biopsies of lesions were made over long periods in some of the patients. Leukoplakic lesions from other parts of the mouth often showed atypia. An evolution from leukoplakia to pseudoepitheliomatous"hyperplasia to early squamous cell carcinoma was found. Discussion In characterizing the role of smokeless tobacco use in the clinical and histologic course of oral lesions, there are several problems. First, oral leukoplakia should be considered a dynamic changing lesion of the oral mucosa (34). Lesions retain the potential to resolve, remain static, or progress depending on a variety of factors that may be either exogenous (e.g., smoke- less tobacco use) or endogenous (e.g., natural tissue defenses and repair potential). To achieve comparability of results among investigators, a standard system for gauging epithelial dysplasia is needed. Patients then could be followed prospectively to quantify the incidence of dysplastic change, incidence of transformation from a dysplastic state to a cancerous state, or in some cases transformation from an apparent benign to.a cancerous state. But ethical considerations do not allow lesions to be monitored con- tinuously from benign states to moderate and severe dysplasias and carcinoma in situ. The next best alternative would be to provide estimates of risk for malignant transformation based on empirical and clinical observations or at least to quantify descriptively the association that smokeless tobacco-induced lesions have with other lesions or other potential etiologic factors. The body of literature on smokeless tobacco-induced lesions and their potential for malignant transformation allows for the development of a conceptual model of the natural history of smokeless tobacco-iaduced lesions (figure 1). This model is a composite of various prospective, retrospective, cross-sectional, and case studies that relate to smokeless tobacco-iaduced lesions. It depicts progressive changes that may occur in some individuals who are habitual users of smokeless tobacco and potential outcomes that could include death or disfigure- ment for some individuals who use smokeless tobacco for several decades. The data are clear that habitual smokeless tobacco use can produce mucosal lesions 25a1258i5a

(see leukoplakia discussion). It is also clear that where groups of patients with smokeless tobacco-induced leukoplakias have been followed for several years, cases of cancer have been identified. Finally, when considering studies of oral cancers in habitual smokeless tobacco users, there appears to be a consistent finding of leukoplakias either having been previously excised in the area of habitual tobacco placement or being found concurrently and in proximity to oral cancers. In comparing studies on the transformation potential of smokeless tobacco-induced leukoplakias, it is found that different criteria have been used by various investigators in defining dysplastic changes. The number and nature of criteria that are considered and that are considered adequate to classify a case as dysplastic are not consistent. Additionally, the degree of agreement on diagnosis based on histology and clinical history between individuals has been shown to be quite variable. Pindborg, Reibel, and Holmstrup tested the degree to which a group of oral pathologists could agree on diagnoses where nine cases of epithelial dysplasia, carcinoma in situ, or initial squamous cell carcinoma were examined (35). Color photomicrographs and information on the topography of the biopsy were presented. The authors' diagnoses were based on the criteria that are described in the report from the WHO International Collaborating Center for Oral Precancerous Lesions (1). The degree of agreement with the authors' diagnosis for the nine cases ranged between 10 and 78 percent. This could partially explain the range in preva- lence and incidence of malignant transformation that is reported by various investigators. Other contributing factors in comparing studies could include different population groups in terms of age and gender and other confounding variables (e.g., smoking, alcohol use, and type of smokeless tobacco product used). Each of these limitations is suggestive of the type of research that is needed. THE EFFECTS OF SMOKELESS TOBACCO USE ON THE GINGIVA, PERIODONTAL TISSUE, AND SALIVARY GLANDS Background and Definitions Reports of gingivitis, gingival recession, and degenerative salivary gland changes associated with smokeless tobacco use are contained in the literature. As with the previous section on oral leukoplakia, the terms used and the definitions employed to describe gingivitis and gingival, recession vary widely from study to study. Table 4 displays the variations found in the literature. As each study is described in the following narrative, the authors' terms are employed. However, in the discussion portion of this report, the general terms of gingivitis and gingival recession are used. General definitions for these terms and for sialadenitis follow: • Gingivitis-This condition refers to clinically detectable acute or chronic inflammation, either local or general, of the gingiva. 250125e151

• Gingival recession-In general, this condition describes the apical migration of the gingiva with or without clinical evidence of inflam- mation. • Sialadenitis-Inflammation of the salivary glands. Gingival and Periodontal Tissue Studies that assess the relationship between smokeless tobacco use and gingival and periodontal tissue effects are limited. The literature consists of several cross-sectional studies in teenagers and a few case reports. Studies in the United States Three cross-sectional studies have investigated the relationship of gingival and periodontal tissue changes and smokeless tobacco use in teen- agers in the United States (7-9). Offenbacher and Weathers examined the effects of smokeless tobacco use on mucosal pathology, on the presence of gingivitis and gingival recession, and on dental caries status (discussed in the next section) (9). Of the 75 smokeless tobacco users, the authors noted 72.0 percent with gingivitis and 60.0 percent with gingival recession. In those with gingival recession, 6.6 percent presented with recession in direct juxtaposition to the location of the tobacco placement. The authors did not describe how many users of smokeless tobacco had demonstrated combina- tions of these oral conditions. Also, no specific clinical definitions were given for the assessment of gingivitis or gingival recession, although the latter findings were described as "slight to moderate, ranging from 1 to 4 mm apical migration of gingival tissue." The higher prevalence of gingival re- cession among smokeless tobacco users (60 percent) as compared to that found in nonusers (14.1 percent) was found to be statistically significant. There were no statistically significant differences in gingivitis prevalence between smokeless tobacco users (72 percent) and nonusers (77.1 percent). Of 117 adolescent smokeless tobacco users in Denver, Colorado, Greer and Poulson noted that 25.6 percent had tobacco-associated periodontal de- generation (7). As noted earlier, this condition was defined as "site- specific gingival recession with apical migration of the gingiva to or beyond the cementoenamel junction, with or without clinical evidence of inflammation." Concomitant mucosal lesions were noted in 76.6 percent of those who had periodontal degeneration (gingival recession). In a study of rural Colorado teenagers, Poulson, Lindenmuth, and Greer (8) described 26.8 percent of 56 smokeless tobacco users with periodontal degeneration (gingival recession) as defined by Greer and Poulson (7). Eighty-seven percent of these had concomitant mucosal lesions. Several case reports (table 2) describe the occurrence of gingival recession and periodontal tissue destruction in individual smokeless to- bacco/snuff users (10-13). The patients in these case reports were males who ranged in age from 18 to 36 years with varying duration of the smokeless tobacco/snuff habit ranging from 1 to 24 years. Although not universally found, gingival recession was usually noted, and the majority of patients

presented with recession that was specific to the site where the tobacco/ snuff was habitually placed. Periodontal bone loss at the site of snuff placement was described in another patient who used snuff for 13 years (10). In one patient, 3 weeks after cessation of snuff use, there was no regeneration of the lost gingival tissue, although, as noted earlier, the hyperkeratotic areas_had disappeared (12). Studies in Sweden Modeer, Lavstedt, and ARlund studied the oral health effects of smok- ing and snuff use in 232 Swedish school children ages 13 to 14 years (119 boys and 113 girls) (36). Thirteen (11 percent) of the boys used snuff. The children were interviewed regarding their tobacco and toothbrushing habits, and examiners (blind to the interview results) clinically assessed the degree of gingival inflammation, oral hygiene, and the presence of cal- culus (discussed in the next section). Standardized indices were used to assess all oral conditions. Controlling for the presence of dental plaque, gingival inflammation was the only variable that was significantly different between snuff users and nonusers. Snuff use was directly correlated with the degree of gingival inflammation. The gingival inflammation noted was related to the site of smokeless tobacco placement. Discussion The relationship of smokeless tobacco use and the health of gingival and periodontal tissue has received minimal study. Because of the variation in study designs and diagnostic criteria, comparisons between available studies are inappropriate. Thus the effects of smokeless tobacco use on these tissues are not clearly understood. With regard to gingivitis, one cross-sectional study noted no difference between users and nonusers (9). Another study, however, emphasized that there was a significant difference between users and nonusers and that snuff use was directly correlated with the degree of gingival inflammation (36). Gingival recession is a common finding among users of smokeless tobacco/ snuff. In the U.S. cross-sectional studies, gingival recession was found in 25.6 to 60.0 percent of teenage users (7-9). In the two Colorado studies, all the gingival recession was specific to the site of tobacco placement (25.6 (8) and 26.8 percent (8)). In the Georgia study, only 6.6 percent of the gingival recession was in the area of tobacco placement (9). In addi- tion, several case reports have identified gingival recession at the site of habitual tobacco placement (10-13). Between 76.6 (7) and 86.6 (8) percent of smokeless tobacco users who had gingival recession also had concomitant mucosal pathology. These soft tissue changes were found at the site of habitual tobacco placement. 2501258153

Salivary Gland Effects Smokeless tobacco or its components may contribute to degenerative changes and severe damage, such as undifferentiated carcinoma, to the sali- vary glands and excretory ducts of humans and mice (18,20,28,37). In a study that assessed the formation of tobacco-specific nitrosamines from the major tobacco alkaloid nicotine, Hecht et al., reporting from the histologic evalua- tion, noted two undifferentiated carcinomas of the salivary glands in two groups of mice that were given injections of nitrosonornicotine (NNN) in saline or trioctanoin (37). Because of the uncommonness of salivary tumors in strain A mice, Hecht et al. concluded that the tumors were probably a result of systemic administration of NNN. Sialadenitis and degenerative changes in minor salivary glands were found in 16 of 50 habitual snuff dippers with a greater number belonging to the groups that were classified clinically as having the most severe snuff- induced lesions (18) (table 1). The findings f rom this study included a decrease in oxidative enzyme activities and indications of metabolic atypia that were based on enzyme histochemical tests. The salivary glands appeared to manifest more damage than the oral epithelium from snuff use. Variations in degrees of effect may be attributed to the variations in snuff dipping habits and brands of snuff. Iri a recent study by Greer and his colleagues (20) (table 1), 45 smoke- less tobacco users aged 13 to 74 years were clinically and histomorphological ly assessed for the effects of smokeless tobacco on the oral tissues. Of 45 tissue specimens, 18 included salivary gland tissue. Damage in the form of sialadenitis and other degenerative changes in salivary glands was shown in 4 of the 18 specimens. A consistent pattern for chronic sialadenitis was not found among any.of the age groups. The authors did not specify the other degenerative changes. However, four patients ages 21, 25, 50, and 60 years demonstrated either a mild, moderate, or severe salivary gland fibrosis. The most severe salivary gland fibrosis was found in the 21-year-old sub;ect who was considered a"short-term" smokeless tobacco user; a definition for "staort- term user" was not provided. Unlike the findings of Hirsch, Heyden, and Thilander (18), salivary gland fibrosis or changes were not related to the stage (degree) of the clinical lesion. The authors concluded that there is no doubt that salivary gland fibrosis can be shown and that'it is likely to be related to the damage from smokeless tobacco. They also commented that "It is likely that the degree of salivary gland fibrosis and degenerative change, along with sialadenitis, may be a factor that is associated with tobacco brand rather than with a generalized reaction caused by all tobacco." Included among the many questions concerning the effects of smokeless tobacco use on the salivary glands is that of changes on flow and buffering capacity of saliva. In a sample of 48 Finnish snuff users ages 17 to 21 years (mean, 18.9), the resting and stimulated salivary flow was measured (21) (table 1). The subjects refrained from the use of snuff for 1 hour before collection of saliva. The saliva of 10 nonusers was similarly col- lected. The statistically significant findings demonstrated a higher resting salivary flow of snuff users compared to controls. Although the stimulated salivary flow was also higher among the snuff users than the controls, this 2501250154

difference was not statistically significant. Buffering capacity was the same between the two groups. Although these findings offer additional informa- tion regarding the effects of smokeless tobacco on the salivary glands, the clinical significance of these effects has not been systematically assessed, nor have the outcome differences related to the different products. Replica- tion studies of these findings are needed before firm conclusions can be made. In contrast to the effects just cited, Archard et al. were unable to identify lesions or dysfunctions associated with smokeless tobacco use (23) (table 2). These investigators carried out histochemical tests on lesions in the oral cavity that were in close proximity to the salivary glands. These tests revealed no evidence of an inflammatory reaction associated with the glands. . Discussion The interpretation of data within this general area requires caution. Limited evidence suggests a possible relationship between the use of snuff and damage to the salivary glands. Should this be the case, the loss of salivary gland function can result in the decreased production of saliva and the ultimate loss of a protective buffer for the oral epithelium and the teeth against numerous exogenous factors such as infectious agents, including dental caries. THE EFFECTS OF SMOKELESS TOBACCO USE ON TEETH Background and Definitions Thi,s section of the chapter addresses the role of various forms of smokeless tobacco in causing or contributing to diseases or conditions of the teeth. Specific effects that are examined include dental caries, abrasion, erosion, plaque and calculus buildup, and staining. For purposes of discussion, definitions are offered for a number of terms that~ are considered to represent commonly held concepts of diseases and conditions of the teeth as evidenced in the relevant scientific literature. • Dental caries-Clinically detectable cavitation of the coronal or root surfaces of the tooth that is caused by acid demineralization of colonizing bacteria on tooth surfaces. • Abrasion-Clinically evident wear of the coronal portion of teeth either generally or focally that appears excessive for a patient of a given age. This is a mechanical effect that is caused by the action of abrasive substances or objects during normal functioning or by oral habits. • Erosion-Loss of tooth structure that is attributable to a chemical agent. Plaque--Bacterial-laden, proteinaceous material that is continually deposited in the oral cavity through the proliferation of bacterial types. 2501258155

• Calculus-A concretion that forms on the coronal and exposed root surfaces of teeth through the calcification of bacterial plaques. • Staining--An extrinsic stain deposit that results in discoloration on tooth surfaces. Dental Caries Evidence for the effects of smokeless tobacco use on the teeth is available from several cross-sectional studies (table 1), from a limited number of case reports (table 2), and from a limited number of related investigations of the potential for constituents of smokeless tobacco to serve as predisposing or etiologic factors in the development of dental caries. As previously mentioned, Offenbacher and Weathers reported on the oral soft and hard tissue effects of smokeless tobacco use in a study population that comprised 565 males with a mean age of 13.8 years (9). This population typifies the age group that is commonly described as "the cavity-prone years." Although caries rates expressed as decayed, missing, or filled teeth (DMFT) were higher for smokeless tobacco users without gingivitis than for nonusers without gingivitis, these differences were not statistically significant. However, when DMFT scores for smokeless tobacco users with gingivitis were compared with scores from nonusers without gingivitis, a significantly higher caries prevalence was found among users. Among students who used both snuff and chewing tobacco, the DMFT score was 6.56 + 0.71. This score is significantly elevated compared with scores of nonuser gingivitis-free students and the nonuser group that had gingivitis. There was a 2.4-fold increase in disease experience. In this study, the presence of gingivitis was presented as a co- factor with smokeless tobacco use in the increased prevalence of dental caries. This finding has not been reported elsewhere, and the biologic explanation is unclear. The differences that were noted in caries rates could not be accounted for based upon differences in oral hygiene or the frequency of dental visits- two factors that could potentially affect DMFT scores. The examiners had no knowledge from the self-reported survey forms of the history of smokeless tobacco use among the group that was examined; thus, a degree of study "blind- ness" was attained. Absolute "blindness" in these types of surveys is difficult because it is likely that some evidence of smokeless tobacco use (e.g., tobacco residues, stain, odor, and soft tissue effects) is observable. No quantifiable dose-response effect for smokeless tobacco use and dental caries was reported in this study. Dental caries is highly age dependent and no age adjustment was made in the statistical analysis. A cross-sectional study by Greer and Poulson of 1,119 teenage smokeless tobacco users and nonusers from urban Colorado demonstrated neither "tobacco- associated dental caries" nor occlusal or incisal abrasion of the teeth (7). This finding is not surprising, because abrasive effects are cumulative and would likely require a number of years to become evident. The abrasion that has been reported in smokeless tobacco users has been in adults who N have used smokeless tobacco products, generally leaf and plug forms of 0 t.l1 - 3-19 a''

tobacco, for years (10,13). The Greer and Poulson study reported a single case of cervical erosion on the mandibular central incisors. Some case reports have implied a causative role for smokeless tobacco in the development of dental caries (38,39), while others have postulated a potential protective effect from caries (13,40). The presumed mode of protection would be through a greatly increased salivary flow that may provide a buffering action. Additionally, there is evidence that various forms of smokeless tobacco contain fluoride, from a few tenths to several parts per million, which may offer some cariostatic protection (41). At the same time, various types of smokeless tobacco contain up to five dif- ferent forms of caries-promoting sugars (42). Two studies reported that constituents in smokeless tobacco products either cause a proliferation of caries-producing bacteria in vitro or, at the least, do not inhibit bacterial growth in vitro (43,44). The fluoride and sugar contents of smokeless to- bacco vary by product type (41). This may explain the inconsistent and equiv- ocal results obtained by different investigators. Variations in reported caries rates, if truly reflective of the larger population of smokeless tobacco users, may represent the clinical outcome of a number of antagonistic or synergistic factors that operate while smokeless tobacco is used. Other Hard Tissue Effects Plaque, calculus, and staining are extrinsic factors that may be as- sociated with smokeless tobacco use. This is clinically important because dental plaque and calculus that is coated with plaque harbor bacteria that can produce acids and toxins and thus bring about dental caries and diseases of the periodontal structures. The staining of teeth, restorations, and prosthetic appliances have been described as resulting from smokeless tobacco use (13,22,45,46). Van Wyk also reported a constant finding of chronic inflam- mation of tooth pulps that were extracted from oral snuff users (22). He attributed this as being "probably due to the irritation of the snuff over- lying the exposed dentine and cementum." No quantifiable evidence currently documents the risk of smokeless tobacco use compared to nonuse in the develop- ment of plaque, calculus, or staining or the_ relationship of staining to oral disease conditions. CONCLIJS IONS 1. Smokeless tobacco use is responsible for the development of a portion of oral leukoplakias in both teenage and adult users. The degree to which the use of smokeless tobacco affects the oral hard and soft tissues is variable depending on the site of action, type of smokeless tobacco product used, frequency and duration of use, predisposing factors, cofac- tors (such as smoking or concomitant gingival disease), and other factors not yet determined. 2. Dose response effects have been noted by a number of investigators. Longer use of smokeless tobacco results in a higher prevalence of leukoplakic lesions. Oral leukoplakias are commonly found at the site of tobacco placement. 2501258157

3. Some snuff-induced oral leukoplakic lesions have been noted upon continued smokeless tobacco use to undergo transformation to a dysplastic state. A portion of these dysplastic lesions can further develop into carcinomas of either a verrucous or squamous cell variety. 4. Recent studies of the effects of smokeless tobacco use on gingival and periodontal tissues have resulted in equivocal findings. While gingival recession is a common outcome from use, gingivitis may or may not occur. Because longitudinal data are not available, the role of smokeless to- bacco in the development and progression of gingivitis or periodontitis has not been confirmed. 5. Evidence concerning the effects of smokeless tobacco use on the salivary glands is inconclusive. 6. Negative health effects on the teeth from smokeless tobacco use are sus- pected but unconfirmed. Present evidence, albeit sparse, suggests that the combination of smokeless tobacco use in individuals with existing gin- givitis may increase the prevalence of dental caries compared to nonusers without concomitant gingivitis. Reports of tooth abrasion or staining have not been substantiated through controlled studies; only case reports are available. RESEARCH NEEDS The review of the literature for this component of the report has identi- fied the need for research in each of the areas discussed: the oral soft tissues, the periodontium, the salivary glands, and the teeth. Basically, the effects of the various types and forms of smokeless tobacco in all age groups should be investigated. Controlled studies and comparisons between users and nonusers of smokeless tobacco are needed. Established criteria for assessing tissue changes and disease presence should be applied to permit comparability between studies. Studies should include the identification and control of variables that also may affect these tissues. Such variables may include alcohol use, diet, oral hygiene practices, microbial flora changes, and salivary flow rate, composition, and pH. In addition to these variables, consideration should be given to the effects of concurrent disease states. For example, the effects of smokeless tobacco on dental caries in the presence or absence of gingivitis should be investigated. The natural history of smokeless tobacco-induced lesions resulting from continued, intermittent, and discontinued smokeless tobacco use needs investi- gation. Histopathologic evaluations and clinical examinations to determine the natural history of oral leukoplakia/mucosal pathology and salivary gland pathology are desirable to understand completely the extent and severity of smokeless tobacco oral effects. In general, incidence and prevalence studies should be implemented. Prospective study designs should be pursued to assess the temporal relation- ship between smokeless tobacco use and various health effects. In addition, 2501250158

dose-response studies are needed to assess dose in terms of both duration of use (in months and years) and daily exposure (in minutes and hours). REFERENCES 1. World Health Organization Collaborating Centre for Oral Precancerous Lesions. Definition of leukoplakia and related lesions: An aid to studies on oral precancer. Oral Surg. 46: 518-539, 1978. 2. Axell, T., et al. International Seminar on Oral Leukoplakia and Asso- ciated Lesions Related to Tobacco Habits. Community Dent. Oral Epidemiol. 12: 145-154, 1984. 3. International Agency for Research on Cancer. The evaluation of the carcinogenic risk of chemicals to humans: Tobacco habits other than smoking; betel-quid and areca-nut chewing; and some related nitrosamines. IARC Monogr. 37: 113, 1985. 4. Moore, G.E., Bissinger, L.L., and Proehl, E.C. Tobacco and intra-oral cancer. Surg. Forum 3: 685-688, 1952. 5. Peacock, E.E., Jr., et al. The effect of snuff and tobacco on the production of oral carcinoma: An experimental and epidemiological study. Ann. Surg. 151: 542-549, 1960. 6. Smith, J.F., Miacer, H.A., Hopkins, K.P., and Bell, J. Snuff-dipper's lesion. A cytological and pathological study in a large population. Arch. Otolaryngol. 92: 450-456, 1970. 7. Greer, R.O., Jr., and Poulson, T.C. Oral tissue alterations associated with the use of smokeless tobacco by teen-agers. Oral Surg. 56: 275- 284, 1983. 8. Poulson, T.C., et al. A comparison of the use of smokeless tobacco in rural and urban teenagers. CA 34: 248-261, 1984. 9. Offenbacher, S., and Weathers, D.R. Effects of smokeless tobacco on the periodontal, mucosal and caries status of adolescent males. J. Oral Pathol. 14: 169-181, 1985. 10. Christen, A.G., et al. Intraoral leukoplakia, abrasion, periodontal breakdown, and tooth loss in a snuff dipper. J. Am. Dent. Assoc. 98: 584-586, 1979. 11. Christen, A.G., et al. Snuff dipping and tobacco chewing in a group of Texas college athletes. Tex. Dent. J. 97: 6-10, 1979. 12. Hoge, H.W., and Kirkham, D.B. Clinical management and soft tissue reconstruction of periodontal damage resulting from habitual use of snuff. J. Am. Dent. Assoc. 107: 744-745, 1983. 2501258159

13. Zitterbart, P.A., et al. Dental and oral effects observed in a long- term tobacco chewer: Case report. J. Indian Dent. Assoc. 62: 17-18, 1983. 14. Axell, T., Mornstad, H., and Sundstrom, B. The relation of the clinical picture to the histopathology of snuff dipper's lesions in a Swedish population. J. Oral Pathol. 5: 229-236, 1976. 15. Pindborg, J.J., and Renstrup, G. Studies in oral leukoplakias, II. Effect of snuff on oral epithelium. Acta Derm. Venereol. 43: 271-276, 1963. 16. Pindborg, J.J., and Poulsen, H.E. Studies in oral leukoplakias, I. The influence of snuff upon the connective tissue of the oral mucosa. Preliminary report. Acta Pathol. Microbiol. Immunol. Scand. 55: 412-414, 1962. 17. Axell, T. A prevalence study of oral mucosal lesions in an adult Swedish population. Odontol. Rev. (Suppl. 36), 27: 1-103, 1976. 18. Hirsch, J.M., Heyden, G., and Thilander, H. A clinical, histomorphological and histochemical study on snuff-induced lesions of varying severity. J. Oral Pathol. 11: 387-398, 1982. 19. Frithiof, L., et a1. The snuff-induced lesion. Acta Odontol. Scand. 1: 53-64, 1983. 20. Greer, R.O., Poulson, T.C., Boone, M.E., Lindenmuth, J., Crosby, L.K. Smokeless tobacco associated oral changes in the juvenile, adult, and geriatric patients: Clinical and histomorphologic features including light microscopic, immunocytochemical and ultrastructural findings. Gerodontics 2: 3; 1986. 21. Jungell, P., and Malmstrom, M. Snuff -induced lesions in Finnish recruits. Scand. J. Dent. Res. 93: 442-447, 1985. 22. Van Wyk, C.W. The oral lesion caused by snuff. A clinico-pathological study. Medical Proceedings 11: 531-537, 1966. 23. Archard, H.O., and Tarpley, T.M., Jr. Clinicopathologic and histochemical characterization of submucosal deposits in snuff dipper's keratosis. J. Oral Pathol. 1: 3-11, 1972. 24. Smith, J.F. Snuff-dippers lesion. A ten-year follow-up. Arch. Otolaryngol. 101: 276-277, 1975. 25. Brightman, V.J. Laboratory Procedures in Burket's Oral Medicine-Diagno- sis and Treatment. Malcolm A. Lynch (ed). J.B. Lippincott Company, 1977, pp. 723-724. N G.rf a © 3-23

27. Tyldesley, W.R. Tobacco chewing in English coalminers (2). Malignant transformation in a tobacco-induced leukoplakia. Br. J. Oral Surg. 14: 93-94, 1976. 26. Mehta, F.S., Gupta; P.C., and Pindborg, J.J. Chewing and smoking habits in relation to precaacer and oral cancer. J. Cancer Res. Clin. Oncol. 99: 35-39, 1981. 28. Roed-Petersen, B., and Pindborg, J.J. A study of Danish snuff-induced oral leukoplakia. J. Oral Pathol. 2: 301-313, 1973. 29. Vogler, W.R., Lloyd, J.W., and Milmore, B.K. A retrospective study of etiological factors in cancer of the mouth, pharynx, and larynx. Cancer 15: 246-258, 1962. 30. McGuirt, W.F. Snuff dipper's carcinoma. Arch. Otolaryngol. 109: 757- 760, 1983. 31. Sundstrom, B., Mornstad, H., and Axell, T. Oral carcinomas associ- ated with snuff dipping. Some clinical and histological characteristics of 23 tumours in Swedish males. J. Oral Pathol. 11: 245-251, 1982. 32. Rosenfeld, L., and Callaway, J. Snuff dipper's cancer. Am. J. Surg. 106: 840-844, 1963. 33. Landy, J.J., and White, H.J. Buccogingival carcinoma of snuff dippers. Am. Surg. 27: 442-447, 1961. 34. Banoczy, J., and Sugar, L. Progressive and regressive changes in Hungarian oral leukoplakias in the course of longitudinal studies. Community Dent. Oral Epidemiol. 3: 194-197, 1975. 35. Pindborg, J.J., Reibel, J., and Holmstrup, P. Subjectivity in evaluating oral epithelial dysplasia, carcinoma in situ and initial carcinoma. J. Oral Pathol. 14: 698-708, 1985. 36. Modeer, T., et al. Relation between tobacco consumption and oral health in Swedish schoolchildren. Acta Odontol. Scand. 38: 223-227, 1980. 37. Hecht, S.S., et al. Tobacco-specific nitrosamines: Formation from nicotine in vitro and during tobacco curing and carcinogenicity in strain A mice. J. Natl. Cancer Inst. 60: 819-824, 1978. 38. Sitzes, L. On chewing tobacco. ADA News 8: 2, 1981. 39. Croft, L. Smokeless tobacco: A case report. Tex. Dent. J. 99: 15-16, I 1981. 40. Shannon, I.L., and Trodahl, J.N. Sugars and fluoride in chewing tobacco and snuff. Tex. Dent. J. 96: 6-9, 1981. 41. Going, R.E., et al. Sugar and fluoride content of various forms of tobacco. J. Am. Dent. Assoc. 100: 27-33, 1980. 25a12581f 1

42. Hsu, S.C., et al. Sugars present in tobacco extracts. J. Am. Dent. Assoc. 101: 915-918, 1980. 43. Lindemeyer, R.G., et al. In vitro effect of tobacco on the growth of oral cariogenic streptococci. J. Am. Dent. Assoc. 103: 719-722, 1981. 44. Falkler, W.A., and Zimmerman, M.L. Effect of smokeless tobacco' extracts on the growth of streptococcus mutans. Presented at the Annual Meeting of the International Association of Dental Research, Las Vegas, Nevada, March 1985. 45. Christen, A.G. The case against smokeless tobacco: Five facts for the health professional to consider. J. Am. Dent. Assoc. 101: 464-468, 1980. 46. U:S. Department of Health and Human Services. Draft report to the Sur- geon General by the Inspector General on smokeless tobacco. December 20, 1985. 47. Waldron, C.A., and Shafer, W.G. Leukoplakia revisited. A clinicopatho- logic study of 3,256 oral leukoplakias. Cancer 36: 1386-1392, 1975. 48. Waldron, C.A., and Shafer, W.G. Current Int. Dent. J. 10: 350-367, 1960. concepts of leukoplakia. 49. Shafer, W.A., Hine, M.K., and Levy, B.M. Oral pathology, ed. 2. Philadelphia, W.B. Saunders Co., 1969, pp. 80-81, 85. 50. Loe, H., and Silness, J. Peridontal disease in pregnancy. I. Prevalence and severity. Acta Odontol. Scand. 21: 533-551, 1963.

Table 1 Selected Study Summaries for the Noncancerous Oral Health Effects From the Use of Smokeless Tobacco Stud_y AxAll, 1976 Sample Methods Observations Leukoplakia/t'tucosal Pathology • 20,333 Swedes. • Cross-sectional • 51% females, design. 49Z males. • Aged 15 years and older. • Data collected on tobacco habits, medications taken, oral hygiene status and prosthetic status. • Clinical examina- tions utilized diagnosis based on specific clini- cal criteria. • Photographic docu- mentation of all lesions diagnosed as leukoplakia or lichen planus. • Tissue specimens taken uf se:lected cases. • • Statistical analysis conduc:ted: t-tests, E9t8Szt0Sz chi square tests, and if approprfate, Fisher's exact test. Of 1,444 snuff users, 116 (8.0%) had "snuff dipper's lesion" (oral, leukplakia). The prevalence of oral leukoplakia was 3.6% among the total population examined. Comments • It is not clear how many of the snuff users were also tobacco smokers. • Snuff dipper's lesion implies mucosal tissue changes at the site of snuff place- ment.

Table I (continued) Stud~ Sample Methods Observations Comments Greer and Leukoplakia/Mucosal Patho10 9Y Poulson, 1 1983 • 1,119 teenagers in grades 9-12. • Cross-sectional design. • A suggested association between level and dura- tion of smokeless tob- acco use and mucosal lesions (42.7% of smoke- less tobacco users had oral mucosal lesions). • An analysis of the influence of cofactors was not conducted. • 117 (10.5X) smokeless tobacco users: 113 males, 4 females. • Denver, Colorado. w N v fl918SMSZ • Questionnaire administered to determine years of use, frequency of use, brand of tob- acco used, site of application, use of other confound- ing agents, ~and dental care history. • Clinical examina- tion conducted of soft and hard oral tissues. • Lesions graded according to a scale developed by AxAll et al. (1976) cind modi- fied by Greer and Poulson. Gingival and Periodontal • 26% of smokeless tobacco users had site-specific gingival recession. • Users with lesions had longer use and higher daily exposure than users without lesions. • No statistical analyses reported. • Examiners blind to responses on ques- tionnaire. • No comparisons reported between users of smokeless tobacco and nonusers. • Smokeless tobacco-asso- ciated periodontal degeneration defined. • Did not assess the inter- relationship of smokeless tobacco, cigarettes, and alcohol.

Table l (continued) 'tudy Sample Methods Observations Comments Teeth • . . . found no evi- dence of tobacco-associ- ated dental caries." • No evidence of occlusal or incisal abrasion. • One case of cervical erosion. "reer et al., 1986 Go • 45 smokeless tobacco users (43 males and 2 females); 15 subjects in each group known as juveniles, young adults, and geriatric. • Aged 13-74 years. • Denver, Colorado. S910SZ10SZ Iirsch et al., 1982 • 50 male habitual cinuff ci i phers . • Cross-sectional design. • Lesions graded by classification developed by Greer and Poulson, 1983. • Examined only lesions classified according to scheme. • Ilistomorphological methods used on tissue specimens. • No statistlcal anal ysis r.onducted. • Cross-sect I onal des I l;n. Salivary Glands • Of 18 tissue samples with salivary glands, 4 demon- strated sialandenitis and degenerative changes. • A routine pattern of chronic sialandenitts was not shown for any of the three age groups. • Four patients (aged 66, 21, 25, and 50) showed either mild, moderate, or severe salivary gland fibrosis. • Interpretation of histo- morphological and histo- chemi cal retiul ts deirion- o Authors suggest that the degree of salivary gland fibrosis, degenerative change, and sialadenitis may be associated with tobacco brand instead of a general ized response caused by all tobacco. o Dose considerations were made and confnundini; vari ahles cons idered .

Table I (continued) Study Sampl e • 41.3-year mean age (range, 15-84 yea rs ) . • Sweden. Me thods • Subjects classified on a four-degree scale of lesion severity (developed by AxAll et al., 1976); biopsies were taken. • Histomorphological and histochemical methods conducted on subjects' tissue specimens. • Tobacco and alcohol use histories ascertained from a questionnaire. Observations strated that the oral mucosal reaction to snuff Induced hyperplasia in the basal cell layers. Lethal damage was found in surface layers-. Duration of use and daily exposure to smokeless tobacco were shown to affect the severity of the leukoplakia. • Dysplasia could not be predicted by using sug- gested clinical degree of lesion classification. Salivary Glands • Tissue speci- mens from 74% of patients included sali- vary glands. 99l0SZlUSZ • Statistical analysis con- ducted: one-way analysis of variance aod multiple cowpari- sons using the Scheffe method. • The salivary glands and excretory ducts showed degenerative changes of a more severe nature than found in the surface epithelium. • 42% of salivary glands demonstrated staladeni- tis and degenerative changes. • Weak oxidative enzyme activities noted in acinic cells in salivary glands with sialadenttis and del;enerat i ve changes. Comments • Differences in brand of tobacco used were taken into account. • Degenerative changes not specifically defined by authors. • Authors state that vari- ations in degenerative changes of salivary glands may be because of differences in brands of snuff and snuff-dip- ping habits.

Table I (continued) i 'tudy Sample Methods Observations Comments • Some signs of metabolic lungell and -ta l ms t rtim, 1985 • 441 military recruits. • Aged 17-19 years . • Finland • 48 (11X) were snuff users. • Cross-sectional design. • Questionnaire administered to ascertain tobacco product use and drinking habits and frequency of dental care. atypia noted. • Markedly degenerative changes seen in salivary glands associated with the more severely, clini- cally classified lesions. Salivary Clands • Resting salivary flow of snuff users was significantly higher than that of nonusers. • Stimulated salivary flow was higher, but not significantly, among snuff users than among controls. • 18.9-year mean age (range, 17-21 years). • Clinical exam[na- tion conducted. • Biopsies taken of 21 snuff users with lesions. • Resting and stimu- lated (paraffin served as the stlmulator) sali- vary exc r.: t ions measured. • Sliitisllctil aua 1 y5 I s c•A,n- (iurted : I . • Authors Interpret dif- ference in resting sal- ivary flow to be a reac- tion to the presence of the local irritant, snuff. • There was no difference in buffering capacity between the two groups.

Table 1 (continued) Study Sample Methods • 10 nonusers of snuff also measured for salivary excretions. Modeer et al., 1980 • 232 school children: 119 males, 113 females. • 13.5 years mea n age. • 11% of males were regular snuff users. • Sweden. BSMlBSZtU'SZ Offenbacher and Weathers, 1985 . 565 males from 5 schools. • Cross-sectional design. . Interviewed about tobacco product use history and oral hygiene practices. • Standardized dental indices used to measure changes in oral hygiene and periodontal con- ditions. • Dental caries assessed clinically and radiographically. • Statistical analyses conducted: cross tabulations, mul- tiple regression, and Student's t-test. • Cross-sectlonal design. Observations Cingival and Periodontal • The use of snuff demonstrated a signi- ficant relation to gingivitis after controlling for plaque. • Effects of snuff on the gingival tissue included both loca- tion of the snuff and as a predictor of gingivitis in general. Leukoplakia/Mucosal Pathology • Frequency of occurrence of soft tissue patholul;y was significantly Comments • Authors state snuff use may influence gingival tissue directly resulting in gingivitis. • Examiners blind to responses from interview. . Soft tlssue Indices are not described. t

Table 1 (continued) =udy Sample . 13.8-year mean age (range, 10- 17 years). • 75 (13.3%) smokeless tobacco users. • Ceorgia, U.S. Methods • Questionnaire used to obtain history of tobacco product use, dental visits, and social history. • Intraoral examina- tion conducted using some stan- dardized indices. • Statistical analyses included: chi square, odds ratios, kappa coefficient calcula- tibns, and t-tests. • Control group used. Observations elevated in users (pri- marily due to increased prevalence of white mucosal lesions). No attributable risk for mucosal pathology in smokeless tobacco users who were free of gingivitis. Comments Cingival and Periodontal 6910SZIOSZ • No relationship between smokeless tobacco use and the prevalence of gingivitis. • Prevalence of gingival recession significantly elevated In smokeless tobacco users. • A significant attributable risk exists for gingival recession In smokeless tobacco users. • Method of selecting schools for subject ascertainment not described. • Confounding variables considered. • Smokeless tobacco use is viewed as a cofactor with--the presence of gingivitis in promoting gingival recession. No clinical definitions provided for the assess- ment of gingivitis or gingival recession.

Table I (continued) Study Sample Methods Observations Comments Teeth • Smokeless tobacco users with gingivitis had significantly greater caries prevalence compared to nonusers with- out gingivitis. • Prevalence of caries was significantly greater in, users with gingivitis who used both snuff and chewing tobacco compared to non- users with gingivitis or those who were gingivitis free. Peacock et al., 1960 w w w • 1,338 employees of local tex- tile mill. Cross-sectional design. • lnterviewed about • North Carolina. tobacco product use and given an oral examination. • llighly significant rela- tionships between chronic snuff and tobacco use and oral leukoplakia development found for all age groups and for both sexes. Q1-10SZ10sz Poulson et al., 1984 l.eukoplakia/Mucosal Pathology • Examiners blind to interview responses. • 90% of employees had either poorly fitting complete dentures or only few and carious teeth. • Many employees have had the habit since they were 3 years old. . 445 subjects: . Cross-sectional • (lf 56 smokeless • Examiners blind 52% fema les, des Il,n. tobacco users, 35 to responses on 48% males. (63%) had lesions of quest ionna I re. 13

• 56 (12.6X) smoke- less tobacco users (all males). • 16.7 year- median age (range, 14-19 yea rs ) . • Rural Colorado. Table I (continued) • Questionnaire ad- ministered (saine as one used in Greer and Poulson, 1983). • Clinical examina- tion conducted of oral hard and soft tissues. • Lesions graded by classification developed by Greer and Poulson, 1983. the hard or soft tis- sues. • 33 (58.9%) smokeless tobacco users had mucosal alterations. • Mucosal lesions were found in area of quid placement. • Duration of use and length of daily exposure were factors in the development of lesions. • Multiple lesions in the same subject reported. Gingival and Periodontal • Of 56 smokeless tobacco • Periodontal degenera- users, 15 (27%) had site- tion defined. specific gingival recession: 2 users had periodontal • Effects of confounding lesions only; 13 had variables not addressed both mucosal lesions statistically. and periodontal destruc- tion. • Definitions of clinical states provided. • Comparisons to nonusers not reported. • A history of con- founding variables obtained. Effects of variables not addressed statisti- cally.

Table 2 Summary of Selected Case Reports Study Country Number of Users Age Product Used Duration of Use Archard and Tarpley, USA 3 31 Snuff 11 years 1972 42 Snuff 20 years 60 Snuff 50 years Christen, Armstrong, and McDaniel, 1979 USA 1 36 Snuff 13 years Christen, McDaniel, and Doran, 1979 w w USA 14 18-22 Snuff, chewing tobacco 6 months to 9 years ~ Frithiof et al., Sweden 21 31-79 Snuff 10-60 years 1983 Ilobe and Kirkham, USA 1 20 Snuff i year 1983 Pindborg and Poulbon, 1962 lenma rk 7 Not reported Snuff 20-30 years Pindborg and Renstrup, 1963 Denmark 12 39 -83 Snuff 20-50 years Zitterbart, Marl in, and (:hr(sten, 1983 USA l 36 ChewIng tobacc.o 24 years U18SZ1OSZ Findings A homogeneous eosinophilic sub- mucosal deposit above the minor salivary glands did not initiate an inflammatory response nor support the possibility that the deposits were amyloid. Gingival recession, clinical leuko- plakia, periodontal bone loss, and tooth abrasion found where tobacco was habitually placed. 8/14 with clinically detectable gingival recession; 9/14 with clin- ical leukoplakia; 11/14 with eryther atous soft tissue changes where to- bacco or snuff was hahitually held. 21/21 with snuff-induced lesions localized to area where snuff was held; 2/21 with observable•gingival retraction. Cingival recession and hyperkeratosia found where tobacco was habitually placed. 4/7 had whitish mucous membrane with a delicately fol4ed appearance at site of snuff placeafent. 12/12 wlth micous membrane that was "whitlsh, sometimes yellowlsh-brown, dry appearance with a very delicately folded or finely grooved surface." Cinl;ival recession, "snk)keless to- bacco-users leslon,- and abraded occlusal surfaces of posterlor tecth fuund where tobacco waa hahl tual I y held.

Table 3 Study Axdll, 1976 Variations in Terms Used and Definitions Provided for Leukoplakia/Mucosal Pathology Associated With Smokeless Tobacco Use by Studies Cited Snuff-dipper's lesion A four-category classification scheme based on tissue color, wrinkling, and thickening was used. Christen, Armstrong, Clinical and McDaniel, 1979 leukoplakia w ~ w rn Christen, McDaniel, and Doran, 1979 i Frithiof et al., 1983 l.eukoplakia Snuff-induced lesion Creer and Poulson, 1983 Hirsch, Ileyden, and Tliilander, 1982 "Implies only the clinical feature of a white patch or plaque on the oral mucosa which will not rub off and which cannot be characterized clinically or histologically as any other specific disease." "Implies only the clinical feature of a white plaque on the mucosa..." "Tissue changes in the oral mucosa" that are due to snuff use. Oral mucosal Tl)ese lesions were defined by a lesions (alterations) modification of a clinical associated with the grading method developed by use of smokeless Axdll et a1., 1976. tobacco Snuff-incluced These Iesions were def Ined by I es I ons t he g rad[ n}; me t hod deve l olied by A0 11 1! t a l., 1976. The authors believe that this is a well-defined irritation that ex- cludes it from the diagnosis of leukoplakia. The authors cite the W110 1978 and Waldron and Shafer 1975 refer- ences (1,47). The authors cite the Waldron and Shafer 1960 reference (48). The authors cite the WlIO 1978 reference for the definition of "leukoplakia" and state that "since the snuff-induced lesion, with its typical clinical pattern and its spe- cific etiology, obviously constitutes a definite diagnostic entity, the term 'leukoplakia' is avoided..." ln addition, lesions were classified by their texture, contour, and color.

Table 3 (continued) Study Hoge and Kirkham, 1983 Moore, Bissinger, and Proehl, 1952 Offenbacher and Weathers, 1985 w i w v Peacock, Greenberg, and Brawley, 1960 Pindborg and Poulson, 1962 Pindborg and Renstrup, 1963 h1_1$Sz 1asz Term(s) Used Ilyperkeratotic- appearing tissue Oral leukoplakia Mucosal pathology, soft tissue pathology Leukoplakia Leukoplakla Snuff-i nduced leukoplakia Definition(s) Provided No definition is provided, although the authors discuss the "formation of a hyperkeratotic zone in the region of the 'snuff pouch' where the tobacco is habitually held." No definition provided. No definitions provided. "A pearly white plaque on the mucous membrane which could not be scraped off with a tongue blade." No definition provided. No definition provided. Comments The authors cite a Shafer, Iline, and Levy 1969 reference (49). The pathological findings Identified by the investigators included morsicatio, ulcer, keratotic/leuko- plakia, vesiculobullous, petechiae, abscess, erythema, mucocele, and pericoronitis. The investigators described the mucous membrane as having a slightly whitish, delicately folded appearance. The investigators described the leuko- plakias as "slightly whitish, some- times yellowish-brown, dry appearance with a very delicately folded or finely grooved-surface."

Table 3 (continued) Study ~ferm(s) Used ,~- Poulson, Lindenmuth, and Greer, 1984 Oral mucosal lesions (alterations) associated with the use of smokeless tobacco Zitterbart, Marlin, and Christen, 1983 Generalized smokeless tobacco-users lesion Definition(s) Provided The clinical appearance of these lesions were defined by a gradLng method developed by Creer and Poulson, 1983. No definition provided. Comments Alterations in texture, color, and contour of the mucosal lesions also were identified. The lesion was described clinically as "peculiarly wrinkled and thickened." 7tLOSGtQS[.

Christen, Armstrong, and McDaniel, 1979 Christen, McDaniel, and Doran, 1979 Greer and Poulson, 1983 w ~ w ~ Hoge and Kirkham, 1983 Modeer, Lavstedt, and Ahlund, 1980 Offenbacher and Weathers, 1985 Table 4 Variations in Terms Used and Definitions Provided for Gingivitis and Gingival Recession, by Studies Cited Definition(s) Provided Gingival recession, periodontal pocket, and loss of alveolar bone Clinically detectable gingival recession Tobacco-associated periodontal degen- eration and perio- dontal lesions Cingival recession Cingivitis/gingival inflammation Gingivitis Cingival recession No definitions provided. "Defined as site-specific gingival recession with apical migration of the gingiva to or beyond the cementoenamel junction, with or without clinical evidence of inflammation." No definition provided. Estimated on the basis of the Cingival Index of LtSe and Silness, 1963 (50). No definition provided. No definition provided. Comments The tissue changes were described in general by the authors. The authors defined the recession as having "exposed approximately 5 mm of labial root surface" and having destroyed the "entire functioning border of keratinized gingiva." The gingival recession was "considered slight to moderate, ranging in 1-4 mm 91-1ssMs?

Table 4 (continued) StudY Poulson, l.indenmuth, Tobacco-associated and Greer, 1984 periodontal degener- ation (other terms Include "periodontal deterioration," and "localized perio- dontal degeneration associated with the site of tobacco placement") Zitterbart, Marlin, and Christen, 1983 Term(s) Used Definition(s) Provided Comments "Defined as site-specific gingival recession with apical migration of the gingiva to or beyond the cementoenamel junction, with or without clinical evidence of inflammation." Gingivitis No definition provided. Gingival recession No definition provided. The clinical findings were described for each tooth site involved. LL `QSG 10J[.

Figure 1. r A Conceptual Natural History of Oral Mucosal Changes Associated with the Use of Smokeless Tobacco Diagnostic Level Oral Tissue Status H EALTH Smokeless Tobacco Habit Oral Lesions CLINICAL Leukoplakias Erythroplakias Probability Moderate / Probability High Dysplastic Changes Probability High HISTOLOGIC CANCER Smokeless Tobacco Exposure Time I Months to Years Probability Low,,-,," ~~Probability High 1 Verrucous Carcinoma I ~f Sauamous Call Carcinoma QCT ~ ~ • Locally Invasive • Highly Invasive i ~ • Unlikely Metastasis .." Years • Likely Metastasis N 1 CLINICAL and f Death Possible + Death Highly Lr' OaI HISTOLOGIC or Loss of Tissue Possible or Potential and Function * for Disfigurement * I Potential for in Sur Recurrence vivors * 'These factors deoend upon stage of diagnosis, form of treatment and continuation of habit(s).

CHAPTER 4 NICOTINE EXPOSURE: PHARMACOKINETICS, ADDICTION, AND OTHER PHYSIOLOGIC EFFECTS

CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1 PHARMACOKLNETICS OF :1ICOTINE , , , , , , , , , 4-1 Levels of Nicotine in Smokeless Tobacco . . . . . . . . . . . . . . . 4-1 Absorption of Nicotine . . . . . . . . . . . . . . . . . . . . . . . . 4-1 Distribution of Nicotine . . . . . . . . . . . . . . . . . . . . . . . 4-2 Nicotine Elimination . . . . . . . . . . . . . . . . . . . . . . . . 4-2 Nicotine and Cotinine Levels in Users of Smokeless Tobacco ...... 4-2 Time Course of Nicotine Turnover During Daily Tobacco Use . . . . . . 4-3 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-3 NICOTINE ADDICTION ASSOCIATED WITH SMOKELESS TOBACCO USE ........ 4-5 Background and Definitions . . . . . . . . . . . . . . . . . . . . . . 4-5 Commonalities Between Tobacco Use and Other Addictive Substances . . . . . . . . . . . . . . . . . . . . . . . 4-7 Experimental Studies of the Abuse Liability and Dependence Potential of Nicotine . . . . . . . . . . , . . . . . 4-13 Evidence That Orally Delivered Nicotine~(Including Via Smokeless Tobacco) Has a Liability for Abuse and a Potential to Produce Dependence . . . . . . . . . . . . . . . . . . 4-19 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-20 PHYSIOLOGIC AND PATHOGENIC EFFECTS OF NICOTINE AND SMOKELESS TOBACCO . . 4-29 Physiologic Effects of Nicotine . . . . . . . . . . . . . . . . . . . 4-29 Nicotine, Smokeless Tobacco, and Human Diseases . . . . . . . . . . . 4-30 Nonnicotine-Related Adverse Metabolic Consequences . . . . . . . . . . 4-32 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-33 CONCLUS IONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-36 RES EARCH NEEDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-37 i

INTRODUCTION This chapter examines the consequences of exposure to nicotine from smokeless tobacco. It draws from the vast literature on the effects of nicotine delivered via smoking and intravenously and includes recent evidence of the effects of orally delivered nicotine. The first section describes the pharmacokinetics of nicotine, including absorption, distribution, and elimination. The data presented.indicdte that nicotine is present in smokeless tobacco in significant amounts and that users attain blood levels of nicotine similar to those produced by cigarette smoking. The second section reviews the established evidence that nicotine is an addictive and dependence-producing substance, having a number of important characteristics in common with prototypic addictive and dependence-producing substances, as well as substantial experimental evidence of its abuse liability and dependence potential. Given the nicotine content of smokeless tobacco, its ability to produce high and sustained blood levels of nicotine, and the well-established data implicating nicotine as an addictive substance, one may deduce that smokeless tobacco is capable of producing addiction in users. In addition, very recent studies provide direct confirmation that nicotine delivered orally from smokeless tobacco and nicotine chewing gum is addictive, producing abuse liability and dependence potential. The final section of the chapter reviews the multisystem physiologic effects of nicotine and examines the evidence pertaining to the potential contributory role of nicotine in the causation of several diseases. PHARAIACOKINETICS OF NICOTINE Levels of Nicotine in Smokeless Tobacco Tobacco is a plant product, and therefore differences exist iii nicotine content among and within different strains of tobacco. Nicotine content among smokeless tobacco products also differs: moist snuff contains 4.56 to 15.1 mg nicotine per gram (1); plug tobacco has been measured to contain 17.2 mg per gram (2). Assuming a daily consumption of 10 grams of smokeless tobacco, the habitual user can be exposed to roughly 130 to 250 mg nicotine per day, of which varying amounts may be absorbed. By comparison, cigarette tobacco averages 15 mg nicotine per gram or 9 mg nicotine per cigarette (3). A person who smokes a pack of cigarettes per day therefore can be exposed to 180 mg nicotine per day. AbsorDtion of Nicotine Nicotine is a weak base (pRa 7.9). In its ionized form, as in the acidic environment of most cigarette smoke, nicotine crosses membranes poorly. As a consequence, there is virtually no buccal absorption of nicotine from cigarette smoke. In contrast, smokeless tobacco products are buffered to an alkaline pH that facilitates absorption. Z501Z5B1S1

The rate of absorption of nicotine from smokeless tobacco depends on the product and the route of administration. With fine-ground nasal snuff, blood levels of nicotine rise almost as fast as those that are observed after cigarette smoking (4). The rate of nicotine absorption with the use of oral snuff (and presumably chewing tobacco) is more gradual (5). People who use oral smokeless tobacco, particularly those who chew tobacco, generate large amounts of saliva, some of which is expectorated and some of which is swallowed. Due to first pass metabolism in the liver follow- ing absorption from the intestines, the bioavailability of swallowed nicotine is approximately 30 percent (6). By changing how much is chewed, how much is held inside the mouth, and how much saliva is expectorated or swallowed, the user of smokeless tobacco has considerable control over the dose of nicotine that is absorbed. Distribution of Nicotine Smoking is a unique form of drug administration in that entry into the circulation is through the pulmonary rather than the portal or systemic venous circulations. The lag time between smoking and the appearance of nicotine in the brain is even shorter than after intravenous injection. Nicotine enters the brain quickly, but then brain levels decline rapidly as it is distributed to other body tissues. The rapid brain uptake of nicotine from smoking allows easy puff-to-puff titration of desired nicotine effects and partly may explain the highly addictive nature of cigarette smoking. In contrast, the concentrations of nicotine that enter the brain from smokeless tobacco use are likely to be lower (6), and the pharmacologic effects may differ. The rate of exposure to psychoactive drugs is an important determinant of their effects. Thus there could be differences in the effects of nicotine that is taken by smoking compared to using smokeless tobacco, even with the same average body concentrations of nicotine. Nicotine Elimination Nicotine is rapidly and extensively metabolized primarily in the liver but also to a small extent in the lung and kidney. Renal excretion depends on urinary pH and urine flow and accounts for 2 to 35 percent of total elimina- tion (7,8). The half-life of nicotine averages 2 hours, although there is considerable individual variability that ranges from 1 to 4 hours (9). The major metabolites of nicotine are cotinine and nicotine-N-oxide. Neither metabolite appears to be pharmacologically active (8). Because of its long half-life, cotinine is commonly used as a marker of nicotine intake in survey and cessation studies. It should be recognized, however, that first pass metabolism of swallowed nicotine may result in cotinine levels that are disproportionately higher than nicotine levels with the use of smokeless tobacco compared to the use of cigarettes. Nicotine and Cotinine Levels in Users of Smokeless Tobacco Blood or plasma concentrations of nicotine in cigarette smokers who were sampled in the afternoon generally ranged from 10 to 50 ng/ml (10). The increment in blood nicotine concentration after a single cigarette is smoked ranges from 5 to 30 ng/ml, depending on how the cigarette is smoked (11,12). 25a1258102

In users of moist oral snuff or chewing tobacco, the levels of nicotine increase on average from 2.9 to 21.6 ng/ml during 8 hours of repeated use (1). In habitual users of nasal snuff, blood levels of nicotine increased on average by 12.6 ng/ml after a single dose of snuff, and levels averaged 36 ng/ml after multiple doses (4). Similarly, blood cotinine concentrations averaged 197 ng/ml and 411 ng/ml in groups of oral and nasal tobacco users, respectively, compared to an average cotinine level of 300 ng/ml for cigarette smokers described in many studies (1,4). These comparisons indicate that the intake of nicotine and nicotine levels in habitual users of smQkeless tobacco are similar to those that are observed in habitual cigarette smokers. Time Course of Nicotine Turnover During Daily Tobacco Use Tobacco use is commonly-considered to be a process of intermittent dosing of nicotine, which in turn is rapidly eliminated from the body. Smoking produces considerable variations from highest to lowest blood nicotine levels from one cigarette to the next cigarette. However, consistent with a half-life of 2 hours, nicotine accumulates over 6 to 8 hours of regular smoking, and nicotine levels persist overnight, even as the smoker sleeps (13). The same accumula- tion is probable with repeated smokeless tobacco use. Thus as with the smoker, the smokeless tobacco user may be exposed to nicotine for 24 hours each day. References 1. Hoffmann, D., Harley, N.H., Fisenne, I., Adams, J.D., and Brunnemann, K.D. Carcinogenic agents in snuff. JNCI 76: 435-437, 1986. 2. Hoffmann, D., Hecht, S.S., Ornaf, R.M., Wynder, E.L., and Tso, T.C. Chemical studies on tobacco smoke. XLII. Nitrosonornicotine: Presence in tobacco, formation and carcinogenicity. In: E.A. Walker, P. Bogovski, and L. Griciute (eds.). Environmental N-Nitroso Compounds. Analysis and Formation (IkRC Scientific Publications ao. 14~). Lyon, Internationa:. Agency for Research on Cancer, 1976, pp. 307-320. 3. Benowitz, N.L., Hall, S.M., Herning, R.I., Jacob, P., III, Jones, R.T., and Osman, A-L. Smokers of low-yield cigarettes do not consume less nicotine. N. Engl. J. Med. 309: 139-142, 1983. 4. Russell, M.A.H., Jarvis, K.J., Devitt, G., and Feyerabend, C. Nicotine intake by snuff users. Br. Med. J. 283: 814-817, 1981. 5. Russell, M.A.H., Jarvis, M., West, R.J., and Feyerabend, C. Buccal absorption of nicotine from smokeless tobacco sachets. Lancet 8468: 1370, 1985. 6. Jenner, P., Gorrod, J.W., and Beckett, A.H. The absorption of nicotine- 1'-Pl-oxide and its reduction in the gastrointestinal tract in man. Xenobiotica 3: 341-349, 1973. 7. Beckett, A.H., Gorrod, J.W., and Jenner, P. A possible relation between pKa and lipid solubility and the amounts excreted in urine of some tobacco alkaloids given to man. J. Pharm. Pharmacol. 24: 115-120, 1972. 2501250183 4-3

8. Benowitz, N.L., Kuyt, F., Jacob, P., Jones, R.T., and Osman, A-L. Cotinine disposition and effects. Clia. Pharmacol. Ther. 309: 139-142, 1983. 9. Benowitz, N.L., Jacob, P., III, Jones, R.T., and Rosenberg, J. Inter- individual variability in the metabolism and cardiovascular effects of nicotine in man. J. Pharmacol. Exp. Ther. 221: 368-372, 1982. 10. Russell, M.A.H., Jarvis, M., Iyer, R., and Feyerabend, C. Relationship of nicotine yield of cigarettes to blood nicotine level concentration in smokers. Br. Med. J. 280: 972-976, 1980. 11. Armitage, A.K., Dollery, C.T., George, C.F., Houseman, T.H., Lewis, B.J., and Turner, D.M. Absorption and metabolism of nicotine from cigarettes. Br. Med. J. 4: 313-316, 1975. 12. Herning, R.I., Jones, R.T., Benowitz, N.L., and Mines, A.H. How a ciga- rette is smoked determines nicotine blood levels. Clin. Pharmacol. Ther. 33: 84-90, 1983. 13. Benowitz, N.L., Kuyt, F., and Jacob, P., III. Circadian blood nicotine concentrations during cigarette smoking. C1in. Pharmacol. Ther. 32: 758-764, 1982. -

NICOTINE ADDICTION ASSOCIATED WITH SMOKELESS TOBACCO USE Background and Definitions Clinical observations and data, historical anecdotes, and sworn testi- mony all support the conclusion that some users of smokeless tobacco are unable to abstain permanently from smokeless tobacco, even when ill health is apparent (1). Such observations suggest that smokeless tobacco use.can become a form of drug addiction or dependence.* This section of the report will evaluate the scientific evidence that smokeless tobacco is an addictive substance whose use results in drug depen- dence. Drug dependence as used in this review is defined in accordance with the World Health Organization's Expert Committee on Drug Dependence (2) and other recognized sources (3). Drug dependence is substance-seeking behavior that is controlled by the activity of a constituent drug in the central nervous system and displaces other behavior such that drug seeking assumes greater priority. Tolerance and physiologic withdrawal may or may not be present (2,3), and the severity of dependence may vary considerably among individuals. The scientific standard for classifying a drug as likely to cause addic- tioa or dependence is based on the degree to which "abuse liability" and "physical dependence potential" are present. Both terms are accepted terminol- ogy of the Committee on Problems of Drug Dependence and the Addiction Research Center (ARC) of the National Institute on Drug Abuse (4,5)** and are commonly accepted to refer to drugs whose actions are mediated by the central nervous system. Abuse liability refers to drug effects that contribute to compulsive self-administration, often in the face of excessive financial cost, physical and social dysfunction, and the exclusion of more socially acceptable behaviors (5,6). Physical Dependence potential (also referred to as physiological *The terms "addiction and dependence" will be used almost interchangeably throughout this section. While many argue the value of one of these terms over the other, it is important to note that in the context of this chapter they address the question of whether nicotine resulting f rom smoking o r smokeless tobacco use leads an individual to lose voluntary control over his or her use of tobacco products (i.e., does the drug cause either dependence or addiction). **The Committee on Problems of Drug Dependence is an internationally comprised body of researchers who provide advisory information to organizations, including NIDA, the World Health Organization, the Drug Enforcement Administration, and the Pharmaceutical industry, regarding the understanding of drug dependence and the identification of dependence-producing drugs. The ARC is the intramural research laboratory of the National Institute on Drug Abuse, which has as a portion of its mandated responsibility the task of assessing the abuse liabil- ity and physical dependence potential of substances. For nearly 50 years, the ARC has been the largest research facility in the United States devoted to the problem of drug abuse and addiction. 2501250185

W20 dependence potential) pertains to the direct physiologic effects that are produced by the repeated administration of a drug that results in neuroadapta- tion (3,4). Neuroadaptation is characterized by demonstrated tolerance to the effects of the drug and the occurrenCe of physiologic withdrawal signs following the termination of drug administration. Physiologic or physical dependence, as evidenced by physiologic and behavioral rebound (withdrawal) effects, is neither necessary nor sufficient to define drug dependence (3,5). Nevertheless, the process of drug dependence and abuse entails physical components, including physical interactions between drug and tissue in the central nervous system (specific receptors in the case of some drugs such as nicotine and opioids) that are critical.* Three lines of evidence are important to assess the abuse liability and physical dependence potential of smokeless tobacco use. The first involves inference from the systematic comparison of tobacco use (including smokeless forms) to the use of prototypic dependence-producing drugs (e.g., alcohol, morphine, and cocaine) to determine whether the patterns of tobacco use, as well as the behavioral and physiologic effects of such use, are similar to those of the prototypic dependence-producing drugs. This issue is discussed below in the section entitled "Commonalities Between Tobacco Use and Other Dependence-Producing Substances." The second line of evidence emerges from recent studies in which nicotine was evaluated using the same methods and criteria that have been used to eval- uate any substance that is suspected of causing abuse and physical dependence. This deductive approach evaluates whether nicotine meets rigorous experimental criteria as a drug that has substantive liability for abuse and physical de- pendence potential. This issue is discussed in the section entitled "Experi- mental Studies of the Abuse Liability and Dependence Potential of Nicotine." The third line of evidence comes from recently completed studies that involve direct assessments of the abuse liability and dependence potential of orally given nicotine. Examination of these studies provides indications of whether the consumption of nicotine through oral forms of administration delivers pharmacologically active quantities of nicotine to the bloodstream and whether smokeless tobacco itself meets specific criteria for abuse liabil- ity and dependence potential. This issue is discussed in the section entitled "Evidence That Orally Delivered Nicotine (Including Smokeless Tobacco) Has a Liability for Abuse and a Potential to Produce Dependence." Taken together, the first and second lines of evidence support the conclusion that smokeless tobacco contains an addictive substance. The third line of evidence suggests that delivery of the addictive substance (nicotine) in the form of smokeless tobacco does not alter its addictive properties. *A concept that is central to many discussions of drug dependence is that the substance produces damage or debilitation. This aspect of tobacco dependence will not be addressed here because extensive data already exist indicating the actual toxicity of tobacco and there is widespread recognition even by tobacco users that the substance is harmful. 25Q1258186

Commonalities Between Tobacco Use and Other Addictive Substances The assertion that tobacco use can occur as a form of drug addiction rests firmly on the observed commonalities between the use and effects of tobacco and the use and effects of addictive substances such as alcohol, opium, and coca. Systematic reviews of these commonalities have been published (7-11), and the major points that tobacco and addictive substances have in common are as follows: • A centrally (CNS) active substance (drug) is delivered. • Discriminative (subjective) effects are centrally mediated. • The substance (drug) is a reinforcer for animals. . The patterns of acquisition and maintenance of substance ingestion are orderly. • The patterns of_self-administration of the substance are orderly. • The patterns of self-administration of the substance vary as a function of the'dose that is consumed. • Tolerance to the behavioral and physiologic effects of the substance develops with repeated use (neuroadaptation). • Therapeutic effects may be produced by the substance. • The treatment of addiction resulting from the substance (drug) involves similar strategies. The evidence concerning tobacco and these factors is presented in the fol;owin; subsections. Tobacco Use Delivers a Centrally Active Substance-Nicotine The fundamental commonality between tobaco use and the use of known addictive substances is the delivery of a chemical to the central ner-rous system. The primary agent in tobacco, nicotine, is delivered to the central nervous system in all commonly used forms of tobacco (12). The fact that cigarette smokers will substitute smokeless tobacco, when cigarettes are not available or when the use of combustibles is restricted, certainly suggests that different forms of tobacco use produce acceptably similar effects for the user (13). Discriminative Effects of Nicotine Are Centrally Mediated Nicotine, like other drugs of abuse, produces dose-related effects in animals which can be attenuated by centrally acting antagonists (14-16). When the animals confuse these effects with other drugs (i.e., effects par- tially generalize to other drugs of abuse), it is more likely to be a drug like amphetamine rather than a sedative-like drug (17). These findings are also consistent with data derived from studies with humans in which the

dose-related effects of intravenously given nicotine were attenuated by mecamylamine pretreatment (18). Nicotine Is a Reinforcer for Animals Most drugs that are abused by humans are voluntarily self-administered when they are made available to animals in laboratory studies; in other* words, the drug serves as a reinforcer or a reward (19,20). Such findings confirm that the physiologic effects of the drug in the central nervous system are sufficient for the substance to control behavior by virtue of its reinforcing effects. Definitive studies that were undertaken in•the early 1980's support this statement. As seen in table 1, nicotine has now been shown to function as a reinforcer for five nonhuman animal species and under a variety of conditions (21,22). Furthermore, its functional behavioral ef- fects are similar to those engendered when other drugs of abuse (e.g., cocaine) serve as reinforcers. Patterns of Acquisition and Maintenance of Tobacco Use Are Orderly The use of tobacco, like that of prototypic addictive substances, is often initiated due to peer influences (23). The contribution of social support to the initiation of tobacco use may be even greater than with illicit drugs, because family members, other social models, and advertising often tolerate, approve, or promote tobacco use while disapproving the use of some nonprescription drugs (24). Also, as is the case with addictive drugs, an accelerated pattern of development of tobacco use has been observed, which is followed by relatively stable drug intake. Initially, the level of consump- tion increases gradually from the first day of use until some point, perhaps several years later, when it becomes relatively stable over time. Although many factors can operate to produce such a biphasic pattern of intake, it is genera]1 y assumed that tolerance and learning factors account Lor the gradual acceleration and that a level of.optimum drug effect combined with toxicity and adverse effects at higher doses takes over to produce the stabilization phenomenon. A preliminary survey, conducted at Johns Hopkins University, indicates that nicotine, whether administered as cigarette smoke or smokeless tobacco, does not differ from other drugs in this regard. That is, tobacco users tend to begin smoking a few cigarettes a day or consume a portion of a container of smokeless tobacco each day and gradually increase consumption levels over a period of months or even years before they stabilize the amount they finally use (personal communication, J.E. Henningfield). Patterns of Tobacco Self-Administration Are Orderlv Daily patterns of cigarette smoking are orderly. Addicted smokers tend to smoke their first cigarette within 30 minutes of waking from a night of sleep and find it difficult to abstain from tobacco use for more than a few hours (25). If smoking behavior is relatively unconstrained, regular patterns develop that closely resemble those of psychomotor stimulant self-administration in animals (20). Similar orderly patterns of tobacco self-administration are evident with cigarette smoking by humans. Several studies have demonstrated that across successive puffs on a cigarette, puff duration decreases and interpuff intervals tend to increase (26,27,28,29), although these changes 25a1258108

are multifactorially determined (30). Anecdotal reports by smokeless tobacco users suggest that while consumption patterns are necessarily different (e.g., some keep a plug in their mouth almost continually during their waking hours) they are no less regular or orderly. Tobacco Self -Administration Varies as a Function of Nicotine Dose The effective dose of a substance may be varied by changing the qi.~antity of drug per unit (the unit dose), by pretreating the individual (animal or human) with either an agonist or antagonist, or by altering the rate of elimination of the substance. Studies that involve these three manipulations have been done extensively with other drugs and more recently with nicotine. The results across study, drug, and species are remarkably similar. For general reviews of human and animal studies see Griffiths, Bigelow, and Henningfield (20) and Henningfield, Lukas, and Bigelow (31). See Gritz (32) and Henningfield (33) for recent reviews of the nicotine-specific literature. Over a wide range:of dose levels, frequency of self-administration is inversely related to dose but drug intake is directly related to dose, reflecting par- tial compensatory changes (26,32). Pretreatment with other agonists (or forms of nicotine) reduces drug taking, e.g., decreases cigarette smoking, (34) and reduces preferred nicotine concentration of tobacco smoke (35). Pretreatment with antagonists initially increases drug self-administration. For example, the centrally and peripherally acting ganglionic blocker, mecamylamine, but not the peripherally acting blocker, pentolinium, increases subsequent smoking rates and increases preferred nicotine concentrations of tobacco smoke (36,37). In addition, altering the elimination rate of nicotine alters the amount of nicotine that is self-administered in the form of tobacco smoke (38). There has been debate over the degree to which smokers regulate their nicotine intake, i.e., the "titration" hypothesis. It is now generally agreed that smokers do not precisely titrate their nicotine intake any more than animals titrate their intake of reinforcing drugs (except under extremely limited conditions) or humans titrate their intake of other reinforcing drugs (20). However, when dose manipulations are observed and objective,.sensitive dependent variables are measured in both animals and humans (26,32,33), most of the studies demonstrate an increase in smoking as cigarette nicotine con- tent falls below accustomed levels and a decrease in smoking when cigarette nicotine content is unusually high (32). Kozlowski and his coworkers describe these findings in terms of a "boundry" model of dose compensation (39). Tolerance of Nicotine Develops With Repeated Use (Neuroadavtation) The administration of most drugs of abuse results in neuroadaptation as measured by tolerance to the repeated administration of the drug and a subsequent rebound (withdrawal) when drug administration is terminated (3). Tolerance to drug effects is determined either by the diminished response to repeated doses of a drug or the requirement of increasing doses to achieve the same drug effect. Tolerance to the behavioral and physiologic effects of nicotine has been studied for decades (33). As is the case with other drugs of abuse, a variety of mechanisms accounts for tolerance to many of nicotine's effects, including metabolic (40), behavioral (41-43), and physiologic tolerance (44-46). More recently, studies have shown that the effects of nicotine that are suspected to be critical to the addiction process also show tolerance with repeated dosing (47,48). 2501250109 4-9

Physiologic dependence on-drugs is determined by showing that termina- tion of drug administration produces a syndrome of effects that is generally opposite to those produced by drug administration. This syndrome is reversible, at least in its early stages, by administration of the drug. Prolonged drug abstinence (detoxification) results in ultimate return to baseline (normal) values of behavioral and physiologic functions. It is now clear that repeated tobacco administration produces physiologic dependence that is specifically due to nicotine administration. Recent data that confirm this fact are reviewed in the section on Dependence Potential of Nicotine. Nicotine Produces Therapeutic Effects Most drugs of abuse have specific therapeutic applications; nicotine is no exception (48-50). The degree to which the therapeutic effects of nicotine depend upon the individual's history of nicotine use, as opposed to the possibility that nicotine is efficacious for preexisting conditions, remains to be investigated. Similar issues are true for other drugs of abuse as well. Pomerleau and his coworkers (51) have studied a variety of mechanisms •by which the possibly weak, initial reinforcing effects of nicotine can be greatly strengthened by subtle effects on mood, cognition, and normal physio- logic and behavioral functioning. For instance, as will be described below, nicotine may produce small, but important enhancement of work performance. These effects appear to be mediated by the effects of nicotine on hormonal release and regulation. The following is a brief summary of some of the effects of nicotine, considered therapeutic by tobacco users, that have been investigated. Several studies have shown that nicotine enhances performance on a variety of cognitive tasks that involve speed, reaction time, vigilance, and concen- tration (52-55). These effects are strongest in cigarette smokers who are deprived of cigarettes. However, such performance enhancement was also evident after the administration of nicotine to nonsmokers and was produced by increasing the nicotine dose in persons who were already smoking. Nicotine may also be a useful mood regulator by virtue of its release of norepinephrine from the adrenal medulla (56). Norepinephrine release is also stimulated by excitement, exercise, sex, antidepressant drugs, and other drugs of abuse, suggesting that cigarette smoking may function pharmacologically to alleviate, boredom and stress. Finally, as an anoretic (57-60), nicotine appears to function in three ways: by decreasing the efficiency with which food is metabolized (61,62); by reducing the appetite for foods that contain simple carbohydrates (sweets) (63); and by reducing the eating that may occur in times of stress (64). Nicotine may also function as an anxiolytic by reducing responsiveness to stressful stimuli and enhancing mood (56). In addition, nicotine reduces aggressive responses in experimental situations (65). A well-documented therapeutic role for nicotine as a drug is evident in the treatment of tobacco abstinence for many individuals following depen- dent patterns of tobacco use, e.g., as assessed by the Fagerstrom Tolerance Questionnaire (25). This test provides both scientific and practical evidence of the role of nicotine in tobacco dependence. It is well established that abstinence from tobacco in heavy cigarette smokers produces signs and symptoms of rebound that can be reversed by resumed tobacco use N tJ'1 O_ tV LJ1 CQ ~ ~ O

and at least partially reversed by other forms of nicotine administration (66). For example, nicotine gum treatment for cigarette smoking is effica- cious although a variety of factors limit success rates (34).* This drug substitution strategy is analogous to those obtained when intravenous opioid users are treated with other opioids given via other routes. For example, methadone administration may reverse signs and symptoms of opioid withdrawal, while leaving the patient feeling partially treated yet likely to relapse if not provided with an adjunctive behavioral treatment (67). Although the euphoriant properties of drugs can stand apart from collat- eral therapeutic actions (as is the case with morphine, amphetamine, and alcohol), attention to such drug effects may enhance the efficacy of treat- ment. Because nicotine, in the form of tobacco, is widely available, is rela- tively inexpensive, and is in a convenient form for precise dose regulation, it provides an ideal means of self-medication. These effects may contribute to the abuse liability of tobacco and are of demons•trable significance in the treatment of tobacco addiction (51). Similar Strategies Are Involved in the Treatment of Tobacco Addiction and Other Forms of Drug Addiction If tobacco use is a form of drug addiction, then strategies of treatment of other forms of drug addiction should be applicable. Most available informa- tion and existing strategies for treatments of tobacco use are based on non- pharmacologic approaches. Such approaches have been no more useful in the treatment of tobacco dependence than in the treatment of dependence on opioids, stimulants, sedatives, or alcohol. On the contrary, experience in the treat- ment of drug addiction disorders makes clear the importance of addressing the pharmacologic components of the addiction (67). This conclusion is strengthened by the observation that persons being treated for opioid addiction regard tobacco to be as necessary as methadone (68) and that persons successfully treated for other kinds of drug addiction are unable to give up tobacco (69). This provides the support for the fundamental premise that tobacco addiction generally constitutes an independent health-impairing disorder. Specific treatment implications relating to cigarette smoking as a form of drug abuse are considered below. To the extent that tobacco use is similar to other forms of drug abuse, treatment strategies that are used for drug abusers may be applied to the treatment of cigarette smoking. Although it is not'the purpose of this chapter to describe in detail the treatment for cigarette smoking, a few com- monalities, as well as differences, are worth mentioning. Four basic pharma- cologic treatments for drug abuse provide the advantage of licit administration of an agent controlled by a certified clinician. These involve substitution therapy (e.g., methadone for opiate dependence) in which a more manageable form of the drug is provided according to a prearranged maintenance protocol; blockade therapy (e.g., naltrexone for opiate dependence) in which the effects *These therapeutic effects are produced by nicotine chewing gum, an orally administered form of nicotine that is approved by the Food and Drug Adminis- tration (FDA). The gum is obtainable in the United States by prescription only and is commonly used by physicians to help individuals quit smoking. 4-11

of the abused drug are blocked by pretreatment with an antagonist; and nonspecific supportive therapy in'which the patient is treated symptomatically, exemplified by the temporary use of benzodiazepines during alcohol detoxification (67). All three approaches have been used in the treatment of cigarette smoking with varying degrees of success (48). A fourth strategy of pretreating the patient with a drug that results in adverse side effects when the subsequent abused drug is taken (e.g., treatment of alcoholism with disulfiram) has not been systematically explored with tobacco. The most recent, widely used treatment for cigarette smoking, and the first of those recognized as efficacious by the FDA, is modeled directly after the treatment of heroin addiction by methadone substitution. This treatment is nicotine gum substitution (70). It is a practical application of the postulate that tobacco use is basically a form of drug addiction on nicotine. This recognition is especially relevant here, because smokeless tobacco is an ora1l form of nicotine. All of the relevant therapeutic data support the premise that compulsive tobacco use entails nicotine addiction, which in the form of tobacco exposes the user to health hazards, and that therapeutic strategies paralleling those for other forms of drug abuse are effective in treatment. Differences appear to be principally related to the social tolerance of tobacco addiction, relative to other forms of drug addiction, which contribute to greater difficulty in treating this form of drug. abuse. Summary of Commonalities Between Tobacco and Prototypic Addictive Drugs The preceding review has shown that tobacco shares many points in common with prototypic addictive drugs. These similarities provide a strong concep- tual basis for th-e categorization of tobacco as an addictive drug. The behavioral process is orderly, tobacco self-administration results in the delivery of a centrally active drug (nicotine), and the drug appears to be the major determinant in the control of the compulsive behavior of tobacco self-administration. These findings are consistent with those expected with animal and human subjects, as determined across a broad range of studies of drugs of abuse (20). In summary, tobacco, opium, and coca produce different effects but share a number of important similarities. Whereas large doses of opioids can produce a debilitating sedation, high doses of coca alkaloids (cocaine HCI) produce levels of behavioral excitation that are not normally produced by tobacco; but the intake of all of these substances leads to compulsive use. Compulsive use and the other commonalities described in the preceding subsec- tions provide compelling evidence that tobacco use can be a form of drug dependence or addiction. The next major question is what element(s) of to- bacco are critical to controlling the behavior of the user. The conceptual leap from habitual behavior to drug abuse and addiction can be made only on the basis of evidence that a specific psychoactive drug is critical to the behavior. The next section on the abuse liability and dependence potential of nicotine will address this question. 2501258192

Lf! s~. Mrc y ~~~ r ~w s7tt~i.:;au"~S"!i Experimental Studies of the Abuse Liability and Physical Dependence Potential of Nicotine The comparison of tobacco to prototypic addictive drugs is the basis for concluding that compulsive tobacco use is a form of drug dependence behavior in which nicotine plays an important role. To test this hypothesis further, it should be possible to show that nicotine is an abusable substance even in the absence of the many stimuli associated with cigarette smoking. This can be done by evaluating nicotine in accordance with methods and criteria that have been used to assess any substance that is suspected of causing abuse and physical dependence. One-half century of research at the NIDA Addiction .Research Center, and research in other laboratories, has produced valid and reliable experimental methods to evaluate a substance's potential to cause abuse and to produce physical dependence. The methods are empirically based on generally accepted examples of drug addiction, most notably opioid dependence (e.g., morphine) and, to a lesser degree, psychomotor stimulant dependence (e.g., cocaine) and sedative dependence (e.g., barbiturates and alcohol). These methods encompass standards for assessing the two dimensions of drug addiction-abuse liability and physical dependence potential. The evidence that is related to the abuse liability and physical dependence potential of nicotine is presented below. Abuse Liability of Nicotine Abuse liability refers to drug effects that contribute to compulsive self- administration, often in the face of excessive financial cost, physical and social dysfunction, and the exclusion of more socially acceptable behaviors (5,6). In other words, it entails those effects of a substance that contri- bute to diminution of voluntary control over the use of the substance by the individual. Objective methods to assess abuse liability are available and have been used to assess diverse agents (5). These methods have been readily adapted to studies of nicotine abuse liability, with consideration given to the fact that nicotine has more rapid effects than many other drugs of abuse. The hypothesis is that nicotine is,psychoactive and serves as a euphoriant and reinforcer. Psychoactivity and euphoria are determined by assessing the pharmaco.dynamic subjective effects of single doses of the drug ("single-dose" or "abuse liability" studies) and are validated by observed behavioral and physiologic responses. Reinforcing efficacy is determined by assessing the ability of the drug to strengthen and maintain orderly patterns of behavior when the subject is permitted access to the drug (i.e., the prototypic "self- administration" study). Pharmacod amic Effects of Nicotine. In human studies of nicotine-related psychoactivity, volunteers are given a range of doses of the test compound and placebo under double-blind conditions. Persons with histories of drug abuse are used because they can accurately discriminate compounds with a potential for abuse and can compare the effects of the compounds to those of abuse drugs (5). In one study, three doses of nicotine were given both intravenously and in the form of tobacco smoke under controlled conditions (71). Nicotine produced a similar profile of effects (figure 1). Self-reported (subjective), 2501250193 4-13 .

observer-reported (behavioral), and physiologic variables were measured before, during, and after drug administration. In brief, nicotine was shown to be psychoactive, as evidenced by the reliable discrimination of nicotine from placebo. Self-reported effects of nicotine peaked within 1 minute after administration (by either route) and dissipated within a few minutes: peak and duration of response were directly related to the dose. The two hallmark indicators of euphoria in such studies are the Liking Scale (Single Dose Questionnaire) and the Morphine Benzedrine Group (MBG) Scale (Addiction Research Center Inventory jARCI]) (5). Responses on the 5-point Liking Scale, which asked how much the drug was liked (0 - "not at all," 4 s"an awful lot") are presented in figure 2. Nicotine produced responses on the Liking Scale similar to those of morphine and d-amphetamine. MBG scale scores of the ARCI were consistent with the Liking Scale data, confirming that nicotine, given by both routes of administration, was a euphoriant. In another comparison between drugs, subjects more frequently identified nicotine injections as cocaine. Similar results for intravenous and inhaled nicotine were also obtained on several physiologic measures, including pupil diameter, blood pressure, and skin temperature. These data confirmed that nicotine, given in either tobacco smoke or intravenously, was the critical pharmacologic compound accounting for these effects of tobacco smoke. A subsequent study showed that nicotine's subjective and physiologic effects could be partially blocked by pretreating the subjects with the antagonist mecamylamine (18). Results of studies with animals also indicate that nicotine produces discriminable effects, and the data suggest that animals identify nicotine as being more similar to cocaine than to placebo or pentobarbital, but not identical to cocaine (17). Self -Administration of Nicotine. The second abuse liability dimension uses the "self -administration" procedure to examine the conditions under which a subject will voluntarily take the drug. Self-administration studies determine whether the drug serves as a biologically effective, positive reinforcer (or reward). Variants of these strategies are conducted in both animal and human subjects, thereby providing a means of establishing the biologic generality of the phenomena, while controlling the possible confounding influence of personality, social, or cultural variables. A high degree of concordance between findings from animal and human studies has been established over a wide range of drugs (20). Therefore, this section focuses on the results of studies using human volunteers. The methods developed in animal studies can be used to assess whether the pharmacologic activity of a drug maintains self-administration parallel- ing drug seeking and drug taking by individuals in the natural environment or "real world." The strategy is particularly useful in studies of nicotine, because it precludes confounding by other stimuli that are associated with tobacco smoke inhalation (e.g., the tobacco brand, smell of the smoke, and lighting-up rituals). In one such study, tobacco-deprived volunteers were tested during 3-hour sessions in which 90 presses on a lever resulted in either a nicotine or placebo injection (72). All six subjects voluntarily self-administered nico- 2501 258194

tine (figure 3). Patterns of self-administration (injections) were similar to those observed when human subjects smoke cigarettes and when rhesus monkeys ~ take intravenous amphetamine injections in comparable experimental situations (20). One subject, who lacked a history of drug abuse, exhibited an acquisition pattern of nicotine self-administration that developed gradually over several sessions. The pattern was a prototypic example of drug abuse development. Double-blind substitution of saline for nicotine resulted in cessation of the self-injection behavior of subject KO (figure 3). Subjects who were given access to both nicotine and placebo concurrently (by pressing alternate levers) chose nicotine, confirming that nicotine had come to serve as a posi- tive reinforcer (73). These data indicate that the pharmacologic activity of nicotine was critical to the maintenance of the behavior. _ ''. Nicotine self-administration has been studied in a variety of nonhuman ° species under a variety of experimental conditions (74). As noted eaYlier, ~ recent results confirm that nicotine can function as an effective reinforcer although the conditions under which it serves as a reinforcer for animals are more restricted than those for morphine or cocaine (21). Nicotine self- administration via cigarette smoke or smokeless tobacco may provide ideal confluences of conditions for the establishment and maintenance of nicotine dependence in humans (33) with the presence of immediate and abundant peripheral taste and olfactory stimuli (75). Implications of Pharmacodynamic and Self-Administration Studies. The results of the pharmacodynamic and self-administration studies provide direct evidence that nicotine itself, and apart from its being presented in combination with all of the orosensory properties of tobacco smoke, is an abusable drug. That is, nicotine meets the criteria of being psychoactive: it serves as a euphoriant and as a reinforcer. These findings strongly sugges t that nicotine parallels other drugs (e.g., morphine in opium use, cocaine in coca lzaf use, and ethanol in alcoholic beverage consumption) in its ability to maintain self - administration. The findings are of sufficient strength that the relevant pub- lic health implications have already been incorporated into issues of public health policy by the former Director of the National Institute on Drug Abuse, Dr. W. Pollin (76), the U.S. Public Health Service (77), and the former Secretary of the Department of Health and Human Services, Mrs. M. Heckler (78). Physical Dependence Potential of Nicotine Physical dependence potential (also referred to as physiological depen- dence potential) pertains to the direct physiologic effects that are produced by the repeated administration of a drug that results in aeuroadaptat'_on (3,4). Neuroadaptation is characterized by demonstrated tolerance to the ef- fects of the drug and the occurrence of physiologic withdrawal signs following the termination of drug administration. Physical dependence potential studies are conducted according to standard- ized tests, using methods such as the substitution approach in which an active drug is removed and replaced with either a placebo or another form of the drug (5). Although many studies on the effects of tobacco abstinence on mood, behavior, and physiologic functions have been conducted, until recently 2501258195

the classic "direct addiction" or "substitution" methodologies had not been used to study the physical dependence potential of nicotine (79). The absence of such studies and the fact that many critical markers of tobacco abstinence are not overt or easily measured (e.g., change in affect, EEG, and cognitive performance impairment) have led to questions about the severity of the tobacco withdrawal syndrome (33). However, as shown below, abstinence from chronic tobacco or oral nicotine use is followed by a syndrome of behavioral and physiologic changes that are orderly, replicable, specific to nicotine, and of functional consequence in relapse to tobacco following abstinence. The apparent absence of withdrawal symptoms among some people is not inconsistent with the finding that nicotine has the potential to produce physical dependence. As is true for users of opiates (e.g., heroin), the mag- nitude of the withdrawal syndrome is related to a variety of factors such as 'dosage and individual predispositions (80). Definition of Tobacco Withdrawal. There are abundant data indicating neuroadaptation to tobacco use, showing that this adaptation is at least partially nicotine specific and that termination of chronic tobacco use produces a behavioral and physiologic rebound or withdrawal syndrome (33). This has been stated in the Diagnostic and Statistical Manual (DSM) of the American Psychiatric Association (APA) as follows (81): Tobacco Withdrawal (APA, DSM, III, 1980). The essential feature is a characteristic withdrawal syndrome due to recent cessation of or reduction in tobacco use that has been at least moderate in duration and amount. The syndrome includes craving for tobacco, irritability, anxiety, difficulty concentrating, restlessness, headache, drowsiness, and gastrointestinal disturbances. It is assumed that this syndrome is caused by nicotine withdrawal, since nicotine is the major pharmacologically active ingredient in tobacco. Withdrawal does not occur with all smokers; but in many heavy cigarette smokers, changes in mood and performance that are prob- ably related to withdrawal can be detected within two hours after the last cigarette. The sense of craving appears to reach a peak within the first 24 hours after the last cigarette, thereafter gradually declining over a few days to several weeks. In any given case it is difficult to distinguish between a withdrawal effect and the emergence of psychological traits that were suppressed, t~ controlled, or altered by the effects of nicotine. U1 O ~ This definition by the American Psychiatric Association represents a Ln reasonable consensus from various reviews of the literature on cigarette Co smoking and physiologic dependence on tobacco (3,13,32,82,83). It is based -i on experimental data and clinical observations from cigarette smoking treat- ~ ment studies demonstrating that certain signs and symptoms are of unusually high prevalence during the first few days of abstinence. Decreased heart rate and blood pressure have been studied experimentally (84), as well as changes in cortical EEG potentials (85,86), changes in urine catecholamine excretion (87), and weight gai:n (57). Other possible concomitants of tobacco withdrawal 'reported clinically include headaches, gastrointestinal disturbances,

insomnia, and fatigue (82,87). A variety of behavioral effects occurs when tobacco or nicotine administration is abruptly terminated in human and animal subjects, including increased irritability, aggressiveness, and anxiety; performance also is impaired in various psychomotor and learning tests such as simulated driving, vigilance, and paired-associate learning (88-90). Self-reported desire to smoke cigarettes ("craving") increases sharply for about 1 day following abstinence, then gradually declines over the course of about 1 week to a lesser level (91). Most of these signs and symptoms qf withdrawal subside over 1 to 2 weeks; however, some former tobacco users report that the desire to smoke may recur for many years and may be evoked by specific environmental stimuli that were previously associated with smoking, such as after meals or in selected social situations. This, too, parallels the powerful conditioning phenomena that are reported to be associated with other drugs of abuse (92). Evidence of Tobacco Withdrawal Symptoms. There is compelling evidence that acute tobacco abstinence produces a rebound (withdrawal) syndrome. This evidence comes from studies of two laboratories in which increases in low- frequency-EEG bands and decreases in cortical activity were observed during the first day of tobacco abstinence (85,86). These effects were immediately reversed when the subjects were allowed to smoke two cigarettes. In a study of self-reported withdrawal symptomatology, 40 participants completed four 25-item questionnaire forms daily for 2 weeks (93). Subjects were divided into two groups: totally abstinent and partially abstinent whose smoking levels were maintained at an average of 60 percent. Four symp- tom clusters emerged: (1) drowsiness in both groups declined over the first week and then increased over the second week, forming a U-shaped function; (2) physical symptoms (e.g., headaches and gastrointestinal disturbances) in both groups declined rapidly the first week and then remained stable across the second week; (3) psychological symptoms (e.g., anxiety and irritability) in both groups paralleled physig-al symptoms; and (4) craving symptoms in the totally abstinent group closely paralleled physical and psychological symptoms, whereas craving levels of the partially abstinent subjects remained elevated across the 2 weeks. The finding that partial abstinence is accompanied by persistent craving symptomatology is similar to the results of studies on the treatment of illicit opioid dependence with methadone. In these studies, low-dose methadone maintenance is associated with a persistent opioid craving (94). An important series of studies on the dependence potential of nicotine has recently been completed at the University of Minnesota (95,96,97). The goals of these studies were to determine reliable and valid indicators of tobacco withdrawal by examining physical, subjective, and behavioral reactions to tobacco deprivation. The first three studies of this series evaluated the dependence potential of tobacco and established a reliable battery of measures. In a residential study, 27 smokers resided for 7 days on a research ward (95). Following baseline, they were assigned to abstain from smoking or to continue smoking for 4 days. Physiologic, subjective, and behavioral measures were obtained and analyzed. The second study was conducted on a nonresiden- tial basis to assess tobacco withdrawal in the nonlaboratory environment (96). In this study, signs and symptoms of tobacco withdrawal were measured in 100 smokers. Following baseline measurements, subjects were randomly 2501250197 4-17

assigned to either nicotine or placebo gum, to be chewed at each subject's own rate. The subjects returned on three different occasions for assessment. The third study assessed the reliability of the tobacco withdrawal syndrome within subjects (97). This study employed a modified, within-subject experi- mental design; baseline smoking, tobacco deprivation, return to baseline smoking, and tobacco deprivation were assessed in each subject. The results of all three studies demonstrated that the syndrome of with- drawal that occurs reliably and consistently in chronic smokers-after tobacco deprivation includes decreased heart rate, increased caloric intake/eating, an increased number of awakenings during sleep, an increased desire to smoke cigarettes, and increased confusion. Other changes that were found, but not consistently, included increased irritability and decreased vigor. A prospec- tive examination of data from both residential and nonresidential studies revealed that there were no statistically significant differences between men and women in either number or severity of tobacco withdrawal symptoms (98). A subsequent study was designed to assess the relationship between to- .bacco withdrawal symptoms and pre- and post-cigarette blood nicotine levels, pre-cigarette cotinine levels, change in nicotine level from pre- to post- cigarette, half-life of nicotine, and total smoke exposure (99). Twenty sub- jects were required to smoke cigarettes for 3 days using a portable recorder that allowed measurements of smoking topography in a nonlaboratory environ- ment. Blood samples were drawn to determine blood nicotine and cotinine levels. Subjects abstained from cigarettes for the next 4 days. A battery of tests to measure tobacco withdrawal symptoms was administered. In general, results showed an inconsistent relationship between measures of nicotine intake and tobacco withdrawal. The most consistent finding was the relationship of the desire to smoke cigarettes to blood nicotine and cotinine levels and change in nicotine from pre- to post-cigarette; that is, the higher the nico- tine and cotinine level and "nicotine boost," the greater the desire for cig- arettes during abstinence. The three initial studies that were conducted at the University of Minnesota (95,96,97) systematically examined the physiologic dependence pro- duced by chronic tobacco use. This work,represents a major advance in fur- thering the understanding of tobacco dependence. The NIDA Addiction Research Center is also nearing the completion of a series of studies on the physical dependence potential of tobacco and the degree to which oral nicotine treats the abstinence syndrome. Preliminary data analysis confirms the findings from the Minnesota studies. Implications of Physical Dependence Potential Studies. These recent studies confirm and extend the findings of earlier investigations that demon- strated that nicotine had the potential to produce physiologic dependence. It is now known that the syndrome is orderly and is due to the administration and withdrawal of nicotine. The overt signs are more subtle than those marking opioid and sedative withdrawal, but these signs are not necessarily less important to the individual. For instance, withdrawal effects such as mood changes, performance deficits, and weight gain may be of considerable importance to the normal functioning of the individual. It is anticipated that just as detoxification and treatment of opioid and sedative dependence have benefited from improved understanding of these syndromes of withdrawal, so also may detoxification and treatment of tobacco withdrawal benefit.

Evidence That Orally Delivered Nicotine (Including Via Smokeless Tobacco) Has a Liability for Abuse and a Potential to Produce Physical Dependence As previously indicated, moist snuff contains as much as 15.1 mg nicotine per gram; plug tobacco contains 17.2 mg per gram (100,101). Lower-nicotine- containing brands exist. However, marketing efforts encourage (and users demonstrate) graduation to the higher-n.icotine-containing products (1). These levels of nicotine are substantial, since the relative potency of. nicotine is 5 to 10 times greater than that of cocaine in producing discri- minable subjective effects (1 to 2 mg of nicotine given intravenously, orally, or inhaled produces reliable behavioral and physiologic effects). Two studies have confirmed.that typical patterns of smokeless tobacco use result in the delivery of quantities of nicotine that produce plasma nicotine elevations comparable to those produced when cigarettes are smoked (102,100). These studies also found that smokeless tobacco use reflected several of the indices of abuse liability and physical dependence potential. Smokeless tobacco users self-administered substantial quantities of nicotine; the patterns of smokeless tobacco use were orderly and stable; and subjective and behavioral effects may be produced from such use. More recently, a new form of smokeless tobacco, moist brown tobacco in tea bag-like pouches, was also shown to deliver pharmacologically active quantities of nicotine to the central nervous system (104). Reinforcing Properties of Nicotine in the Form of Chewing Gum There is growing evidence that nicotine is reinforcing and has the potential to produce dependence even when absorbed through the buccal mucosa (and therefore more slowly) via chewing gum (nicotine polacrilex). One recently completed study involved the self -administration of either a nicotine- or placebo-containing chewing gum by smokers who had quit smoking (105). When given a choice between placebo and nicotine chewing gum, sub- jects preferred nicotine to placebo and self -administered the nicotine gum throughout each day.* These data are particularly compelling, because nico- tine, in the form of the nicotine polacrilex, is in an ion-bound complex. In this preparation, the nicotine is released and absorbed slowly compared to the nicotine in smokeless tobacco; and the polacrilex form of nicotine administration appears to be of relatively low abuse liability. This study also demonstrated that instructions by a physician can alter patterns of gum use and preference (105). These data, which suggest that instructions can modulate the self-administration of orally delivered nicotine, are in keeping with the well-known fact that physicians control their patients' use of narcotics, sedatives, and stimulants. Physical Deoendence Potential of Smokeless Tobacco Hatsukami and coworkers, at the University of Minnesota, studied neuro- adaptation (physiologic dependence) in smokeless tobacco users (106). All 16 subjects in the study used moist snuff and no other nicotine-delivering product. Measures of mood, feeling, behavior, and physiologic function were *Self-administration took place at an average rate of 7.4 pieces compared to an average of 1.2 pieces of placebo gum per day. 2501250199 4-19

compared at baseline and during abstinence. Subjects showed significant signs and symptoms of nicotine withdrawal as measured by decreased resting pulse, attenuated orthostatic pulse changes, and increases in tobacco seeking ("craving"), eating, sleep disruptions, and confusion. A study with nicotine gum showed orally delivered nicotine may cause physical dependence (107). The subjects that were tested had been treated for tobacco dependence with nicotine gum that they used on a daily basis for at least 1 month. Eight subjects were then tested over the course of 4. weeks. They were given nicotine-containing gum during the first and fourth weeks; during the second and third weeks, they received nicotine gum for 1 week and placebo gum for the other. During the week that placebo gum was presented, seven subjects showed signs and symptoms of withdrawal, and two subjects relapsed to smoking or nicotine-containing gum. This study confirms that orally given nicotine has the potential to produce physical dependence. These findings were most recently confirmed by another study that showed development of physical dependence to nicotine gum in patients treated for tobacco dependence (108). References 1. Connolly, G.N., Winn", D.M., Hecht, S.S., Henningfield, J.E., Hoffman, D., and Walker, B. Science public policy and the re-emergence of smokeless tobacco. N. Engl. J. Med. (in press). 2. World Health Organization. Technical Report Series, No. 407. Geneva, Switzerland, 1969. 3. Jaffe, J.H. Drug addiction and drug abuse. In: A.G. Gilman, L.S. Goodman, T.W. Rall, and F. Murad (eds.). Goodman and Gilman's Pharmaco- logical Basis of Therapeutics. New York, Macmillan, 1985, pp. 532-581. 4. Brady, J.V., and Lukas, S.E. (eds.). The Committee on Problems of Drug Dependence, Inc. Testing drugs for physical dependence potential and abuse liability (NIDA Research Monograph 52). Washington, D.C., U.S. Govt. Printing Office, 1984. , 5. Jasinski, D.R., Johnson, R.E., and Henningfield, J.E. Abuse liability assessment in human subjects. Trends in Pharmacological Sciences 5: 196-200, 1984. 6. Jasinski, D.R. Assessment of the abuse potentiality of morphine-i.ike drugs (methods used in man). In: W.R. Martin (ed.). Handbook of Experi- mental Pharmacology, Vol. 45. Drug Addiction I. Berlin, West Germany, Springer-Verlag, 1977, pp. 197=258. smoking habit. In: W.A. Hunt (ed.). Learning Mechanisms in Smoking. Chicago, Aldine, 1970, pp. 155- 190. 7. Jarvik, M. The role of nicotine in the rJ 8. Russell, M.A.H. Cigarette smoking: National history of a dependence r-1 disorder. Br. J. Med. Psychol. 44: 1-16, 1971. Q 4-20

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91. Shiffman, S.M., and Jarvik, M.E. Smoking withdrawal symptoms in two weeks of abstinence. Psychopharmacology 50: 35-39, 1976. 92. Grabowski, J., and O'Brien, C.P. Conditioning factors in drug dependence: An overview. In: N. Mello (ed.). Advances in Substance Abuse: Behavioral and Biological Research, Vol. 2. Greenwich, Connecticut, JAI Press, 1981, pp. 69-121. 93. Shiffman, S.M., and Jarvik, M.E. Withdrawal symptoms: First week is the hardest. World Smoking Health 5: 15-21, 1980. 94. Jasinski, D.R. Opiate withdrawal syndrome: Acute and protracted aspects. Ann. N.Y. Acad. Sci. 362: 183-186, 1981. 95. Hatsukami, D.K., Hughes, J.R., Pickens, R.W., and Svikis, D. Tobacco withdrawal symptoms: An experimental analysis. Psychopharmacology 84: 231-236, 1984. 96. Hughes, J.R., and Hatsukami, D. Signs and symptoms of tobacco withdrawal. Arch. Gen. Psychiatry (in press). 97. Hatsukami, D.K., Hughes, J.R., and Pickens, R.W. Characteristics of tobacco abstinence: Physiological and subjective effects. In: J. Grabowski 4nd S.M. Hall (eds.). Pharmacological Adjuncts in Smoking Cessation (NIDA Research Monograph 53). Washington, D.C., U.S. Govt. Printing Office, 1985. 98. Svikis, D.S., Hatsukami, D.K., Hughes, J.R., Carroll, K.M., and Pickens, R.W. Sex differences in tobacco withdrawal syndrome. Addict. Behav. (in press). 99. Hatsukami, D.K., Hughes, J.R., and Pickens, R.W. Blood nicotine, smok- ing exposure and tobacco withdrawal syndrome. Addict. Behav. (in press). 100. Hoffmann, D., Harley, N.H., Fisenne, I., Adams, J.D., and Brunftemann, K.D. Carcinogenic agents in snuff. JNCI 76: 435-437, 1986. 101. Hoffmann, D., Hecht, S.S., Ornaf, R.M., Wynder, E.L., and Tso, T.C. Chemical studies on tobacco smoke. XLII. Nitrosonornicotine: Presence in tobacco, formation and carcinogenicity. In:' E.A. Walker, P. Bogovski, and L. Griciute (eds.). Environmental N-Nit=oso Compounds. Analysis and Formation (IARC Scientific Publications No. 14). Lyon, International Agency for Research on Cancer, 1976, pp. 307-320. 102. Gritz, E.R., Baier-Weiss, V., Benowitz, N.L., Van Vunakis, H., and Jarvik, M.E. Plasma nicotine and cotinine concentrations in habituaL smokeless tobacco users. C1in. Pharmacol. Ther. 30: 201-209, 1981. 103. Russell, M.A.H., Jarvis, M.J., Devitt, G., and Feyerabend C. Nicotine intake by snuff users. Br. Med. J. 283: 814-817, 1981. 104. Russell, M.A.H., Jarvis, M., West, R.J., and Feyerabend, C. Buccal absorption of nicotine from smokeless tobacco sachets. Lancet 8468: 1370, 1985. 4-27

~..,~. F !,~ ~. ~P1d~ 105. Hughes, J.R., Pickens, R.W., Spring, W., and Keenan, R.M. Instructions control whether nicotine will serve as a reinforcer. J. Pharmacol. Exp. Ther. (in press). 106. Hatsukami, D.K., Gust, S.W., and Keenan, R. Physiological and subjective changes from smokeless tobacco withdrawal. Manuscript submitted to JAMA, 1986. 107. Hughes, J.R., Hatsukami, D., and Skoog, K.P. Physical dependence on nicotine gum: A placebo substitution trial. Paper presented at the Committee on Problems of Drug Dependence Meeting, Baltimore, Maryland, 1984. 108. West, R.J., and Russell, M.A. Effects of withdrawal from long-term nicotine gum use. Psychol. Med. 15: 891-893, 1985.

PHYSIOLOGIC AND PATHOGENIC EFFECTS OF NICOTINE AND SMOKELESS TOBACCO The user of smokeless tobacco is systematically exposed to significant amounts of nicotine, a potent multisystem pharmacologic agent. This chapter addresses the physiologic effects of nicotine upon the cardiovascular, nervous, and endocrine systems and the possible roles of nicotine in the pathogenesis of a variety of diseases. Nicotine is described in pharmacology textbooks as a stimulant of auto- nomic ganglia and skeletal neuromuscular junctions (i.e., nicotinic muscarinic receptors). However, in vivo the actions of nicotine are far more complex depending on the dose, target organ, prevalent autonomic tone, and previous exposure history (tolerance) (1,2). For purposes of this review, the focus is on the effects of nicotine in humans. Where human data are lacking and animal studies provide important information about physiologic effects, those studies are also discussed. Most data on the actions of nicotine in humans derive from studies of the effects of cigarette smoking, comparing cigarettes with and without nicotine, and studies of the effects of intravenous nicotine. These studies provide the basis for our understanding of the human pharmacology of nicotine. However, as noted previously, actions of nicotine from smokeless tobacco and nicotine via inhalation or intravenous infusion may differ. Physiologic Effects of Nicotine Cardiovascular System The predominant cardiovascular actions of nicotine result from activation of the sympathetic nervous system. Smoking a cigarette increases the heart rate (10 to 20 BPM), blood pressure (5 to 10 mmHg), cardiac stroke volume and output, and coronary blood flow (3-5). Smoking may have different effects in smokers with coronary heart disease. It may reduce left ventricular contractility and car- diac output (6), effects that are believed to be related to myocardial ischemia due to smoking-mediated tachycardia and the effects of carbon monoxide. Coronary blood flow may also decrease after smoking, which possibly is related to a nicotine-mediated increase in coronary vascular resistance (7,8). Smok- ing, or nicotine intake, causes cutaneous vasoconstriction that is associated with a decrease in skin temperature, systemic venoconstriction, and increased muscle blood flow (9-11). Smoking results in increased circulating concentrations of norepinephrine, consistent with neural adrenergic stimulation, and epinephrine, indicating adrenal medullary stimulation (3). Circulating free fatty acids, glycerol, and lactate concentrations increase. Cardiovascular and metabolic effects are prevented by combined alpha and beta adrenergic blockade, which indicates that the cardiovascular effects of cigarette smoking are mediated by activation of the sympathetic nervous system. Smoking-induced reduction in skin blood flow also can be antagonized by a vascular vasopressin antagonist, which sug- gests a role for vasopressin in mediating some cardiovascular responses (12). The cardiovascular effects of oral snuff have been examined systematically in only one study (13). Changes in heart rate and blood pressure that are 2501250209 4-29

. ~. .,:...,.,-:; •.~., .. . ., similar in magnitude to those of cigarette smoking were observed. However, the time course appears to be slower than the response to cigarette smoking, with maximum effects observed at 5 to 10 minutes after a dose of oral tobacco. Similar findings, along with increased myocardial contractility and coronary, femoral, and renal blood flow, were also noted in anesthetized dogs after the administration of oral tobacco (13). Thus it appears that single doses of smokeless tobacco can produce hemodynamic effects that are similar to those of cigarette smoking. Whether such changes are sustained throughout the day with repeated daily doses remains to be established. Central Nervous System Although smokers give different explanations for why they smoke, most agree that smoking produces arousal, particularly with the first few cigarettes of the day, as well as relaxation, especially in stressful situations (14). Desynchronization, decreased alpha and theta activity, and increased alpha frequency that is consistent with arousal are the usual electroencephalographic responses to cigarette smoking (15,16). These effects are blocked by mecamy- lamine, a centrally active nicotinic receptor antagonist, which indicates a role for nicotinic cholinergic receptor activation (17). Tobacco abstinence is associated with effects that are opposite those of smoking, namely, increased alpha power and reduced alpha frequency (15,18). Endocrine System Cigarette smoking and nicotine have been reported to increase circulating levels of catecholamines, vasopressin, growth hormone, cortisol, ACTH, and endorphins (3,19,21). Nicotine inhibits the synthesis of prostacyclin in rabbit aorta and human peripheral veins and the hypoxia-induced release of prostacyclin from rabbit hearts (22). Cigarette smoking has been reported to decrease the urinary excretion of prostacyclin metabolites in humans, which supports the prediction from animal studies (23). Prostacyclin has antiaggregatory and vasodilating actions that are believed to play a homeostatic role ia preventing vascular thrombosis. Nicotine, Smokeless Tobacco, and Human Diseases As attested to in the Surgeon General's reports since 1964, smoking is a major risk factor for coronary and peripheral vascular disease, cancer, chronic obstructive lung disease, peptic ulcer disease, and reproductive disturbances, including prematurity. Tobacco smoke is a complex mixture of chemicals, including carbon monoxide, many of which are believed to contribute to human disease. Smokeless tobacco likewise exposes users to a number of chemicals, particularly nicotine. Nicotine may play a contributory or supportive role in the pathogenesis of many smoking-related diseases. That nicotine causes human disease de novo has not been proven; however, its potential ~ health consequences deserve serious consideration. More direct data on its p causal role are needed. ~ ta1 ~ rQ ... C

Coronary and Peripheral Vascular Disease Nicotine may contribute to atherosclerotic disease by actions on lipid metabolism, coagulation, and hemodynamic effects. Compared to nonsmokers, cigarette smokers have elevated levels of low density (LDL) and very low density lipoproteins (VLDL) and reduced levels of high density lipoproteins (HDL). This profile is associated with an increased risk of atherosclerosis (24). It is hypothesized that nicotine, by releasing free fatty acids,. increases the synthesis of triglycerides and VLDL by the liver, which in turn results in decreased HDL production. In most studies, the blood of smokers is shown to coagulate more easily (25), platelets are found to be more reactive, and platelet survival is shortened when compared to nonsmokers (26). Thrombosis is believed to play that promotes the growth of vascular endothelial cells that contribute to the atherosclerotic plaque. •The importance of nicotine as a determinant of platelet hyperreactivity is supported by a study that shows an apparent re- lationship between nicotine concentrations after smoking different brands of cigarettes and platelet aggregation response (27). Nicotine may affect platelets by releasing epinephrine, which is known to enhance platelet reac- tivity; by inhibiting prostacyclin, an antiaggregatory hormone that is secreted by endothelial cells; or perhaps directly. Finally, by increasing the heart rate and cardiac output, nicotine increases blood turbulence and may promote endothelial injury. Although several potential mechanisms for promoting atherogenesis have been considered, nicotine has not yet been demonstrated to accelerate atherosclerosis in experimental animals. Nicotine may play a role in causing acute coronary events. Myocardial infarction can occur with one or more of three precipitants: thrombosis, excessive oxygen and substrate demand, and coronary spasm. Nicotine can promote thrombosis as discussed previously. Nicotine increases the heart rate and blood pressure and, therefore, myocardial oxygen consumption. Cor- onary blood flow increases in a healthy person to meet the increased demand. In the presence of coronary heart disease, ischemia may develop and myocar- dial dysfunction may occur. Nicotine may induce coronary spasm by sympatho- mimetic actions or by the inhibition of prostacyclin. Coronary spasm has recently been reported to occur during cigarette smoking (28). 31.l of the above may contribute to the precipitation of acute myocardial infarction in a person with preexisting coronary atherosclerosis. Cigarette smoke exposure decreases the ventricular fibrillation threshold after'experimental myocardial infarction in dogs (29). How much of this effect is due to nicotine and how much is due to carbon monoxide have not been established. Sudden cardiac death in smokers might result from ischemia, as discussed above, combined with the arrhythmogenic effect of increased circulating catecholamines. Hvpertension Cigarette smoking has not been associated with an increased prevalence of hypertension. However, a recent preliminary report suggested higher blood pressure in young men who used smokeless tobacco compared to cigarette smokers or nonsmokers (30). Smokers who have essential hypertension experience an 25012513211 4-31

accelerated progression of vascular and renal disease. Nicotine may con- tribute to such a process by producing vasoconstriction or enhancing coagula- tion. There also may be other interactions with hypertensive disease. For example, a patient with a pheochromocytoma developed paroxysmal hypertension and angina pectoris following the use of oral snuff (31). In a controlled situation, blood pressure was recorded to increase from 110/70 to 300/103 with a heart rate increase from 70 to 110 within 10 minutes after the use of oral snuff. Rechallenge after surgery for the pheochromocytoma revealed only the usual blood pressure increase. Peptic Ulcer Disease Smoking is strongly related to the prevalence of peptic ulcer disease, and failure to stop smoking is the major predictor of failure to respond to ulcer therapy (32). Smoking decreases pancreatic fluid and bicarbonate secretion that result in greater and more prolonged acidity of gastric fluid of the duodenal bulb (33). Similar effects after the infusion of nicotine have been reported in animals (34). The swallowing of tobacco juice that contains large concentrations of nicotine may conceivably have local effects and therefore elicit added concern for the use of smokeless tobacco. r Pregnancy Smoking is a major risk factor for low birth weight and, consequently, fetal morbidity and mortality (35). Tobacco smoke may influence the fetus either through alterations in maternal physiology that limit the nutrient flow to the fetus or by the transplacental passage of smoke components that have direct effects on the fetus. The factors that are considered most likely to affect the fetus are carbon monoxide and nicotine. Carbon monoxide inhalation has been shown to increase carboxyhemoglobin in both maternal and fetal blood that possibly limits oxygen supply to the fetus (36). However, while newborn infants of smoking mothers have higher concentrations of carbosynemoglobin than do neonates of nonsmokers, there are only trivial differences in hemoglobin concentrations, hematocrit, and various characteristics of hemoglobin (37). Thus it is difficult to explain an adverse effect that is based on chronic hypoxia due to carbon monoxide in tobacco smoke. It is more likely that nicotine is important in causing adverse effects. The effects of nicotine on the fetus may include a reduction of uterine blood flow or a direct effect on fetal function (38,39). The presence of nicotine and its principal metabolites has been demonstrated in the umbilical cord blood and urine of newborn infants of smoking mothers, as well as in amniotic fluid, indicating transplacental passage (40). • Nonnicotine-Related Adverse Metabolic Consequences Certain brands of chewing tobacco and snuff contain glycyrrhizinic acid, which is also an ingredient of licorice. Glycyrrhizinic acid has potent mineralocorticoid hormone activity that can result in potassium wasting. Two patients who were heavy users of oral smokeless tobacco developed severe hypokalemia with muscle weakness (and in one case, evidence of muscle break- down) that apparently was due to the ingestion of large amounts of this substance (41). Smokeless tobacco also contains large amounts of sodium (42) that, if swallowed, may aggravate hypertension or cardiac failure. 2501258212

References 1. Comroe, J.H. The pharmacological actions of nicotine. Ann. N.Y. Acad. Sci. 90: 48-51, 1960. 2. Su, C. Actions of nicotine and smoking on circulation. Pharmacol. Ther. 17: 129-141, 1982. 3. Cryer, P.E., Haymond, M.W., Santiago, J.V., and Shah, S.D.' Norepinephrine and epinephrine release and adrenergic mediation of smoking-associated hemodynamic and metabolic events. N. Engl. J. Med. 295: 573-577, 1976. 4. Irving, D.W., and Yamamoto, T. Cigarette smoking and cardiac output. Br. Heart J. 25: 126-132, 1963. 5. Bargeron, L.M., Ehmke, D., Gonlubol, F., Castellanos, A., Siegel, A., and Bing, R.J. Effect of cigarette smoking on coronary blood flow and myocardial metabolism. Circulation 15: 251-257, 1957. 6. Pentecost, B., and Shillingford, J. The acute effects of smoking on myocardial performance in patients with coronary arterial disease. Br. Heart J. 26: 422-429, 1964. 7. Klein, L.W., Ambrose, J., Pichard, A., Holt, J., Gorlin, R., and Teichholz, L.E. Acute effects of cigarette smoking on coronary vascular dynamics. Circulation (Abst.) 68: 165, 1983. 8. Reddy, C.V.R., Khan, R.G., Feit, A., Chowdry, I.H., and El Sherif, N. Effects of cigarette smoking on coronary hemodynamics in coronary artery disease. Circulation (Abst.) 68: 165, 1983. 9. Freund, J., and Ward, C.. The acute effect of cigarette smoking on the digital circulation in health and disease. Ann. N.Y. Acad. Sci. 90: 85-101, 1960. 10. Eckstein, J.W., and Horseley, A.W. Responses of the peripheral veins man to the intravenous administration of nicotine. Ann. N.Y. Acad. Sci. 90: 133-137, 1960. 11. Rottenstein, H., Peirce, G., Russ, E., Felder, D., and Montgomery, H. Influence of nicotine on the blood flow of resting skeletal muscle and of the digits in normal subjects. Ann. N.Y. Acad. Sci. 90: 102-113, 1960. in 12. Waeber, G., Schaller, M., Nussberger, J., Bussien, J., Hofbauer, K.G., and Brunner, H.R. Skin blood flow reduction induced by cigarette smoking: Role of vasopressin. Am. J. Physiol. 247: H895-H901, 1984. 13. Squires, W.G., Branton, T.A., Zinkgraf, S., Bonds, D., Hartung, G.H., Murray, T., Jackson, A.S., and Miller, R.R. Hemodynamic effects of oral smokeless tobacco in dogs and young adults. Prev. Med. 13: 195-206, 1984. - 2501250213

14. Henningfield, J.E. Behavioral pharmacology of cigarette smoking. In: T. Thompson, T.B. Dews, and J.E. Barrett, (eds.). Advances in Behavioral Pharmacology, Vol. IV. New York, Academic Press, 1984, pp. 131-210. 15. Herning, R.I., Jones, R.T., and Bachman, J. EEG changes during tobacco withdrawal. Psychophysiology 20: 507-512, 1983. 16. Knott, V.J., and Venables, P.H. EEG alpha correlates of nonsmokers, smokers and smoking deprivation. Psychopharmacology 14: 150-156, 1977. 17. Domino, E.F. Behavioral, electrophysiological, endocrine, and skeletal muscle actions of nicotine and tobacco smoking. In: A. Remond and C. Izard (eds.). Electrophysiological Effects of Nicotine. Amsterdam, Elsevier/North Holland Biomedical Press, 1979, pp. 133-146. 18. Ulett, J., and Itil, T. Quantitative electroencephalogram in smoking and smoking deprivation. Science 164: 969-970, 1969. 19. Sandberg, H., Roman, L., Zavodnick, J., and Kupers, N. The effect of smoking on serum somatotropin, immunoreactive insulin and blood glucose levels of young adult males. J. Pharmacol. Exp. Ther. 184: 787-791, 1973. 20. Winternitz, W.W., and Quillen, D. Acute hormonal response to cigarette smoking. J. Clin. Pharmacol. 17: 389-397, 1977. 21. Pomerleau, O.F., Fertig, J.B., Seyler, L.E., and Jaffe, J. Neuroendocrine reactivity to nicotine in smokers. Psychopharmacology 81: 61-67, 1983. 22. Wennmalm, A. Nicotine inhibits hypoxia- and arachidonate-induced release of prostacyclin-like activity in rabbit hearts. Br. J. Pharmacol. 69: 545-549, 1980. 23. Nadler, J.L., Velasco, J.S., and Horton, R. Cigarette smoking•inhibits prostacyclin formation. Lancet 1: 1248-1250, 1983. 24. Brischetto, C.S., Connor, W.E., Connor, S.L., and Matarazzo, J.D. Plasma lipid and lipoprotein profiles of cigarette smokers from randomly selected families: Enhancement of hyperlipidemia and depression of high-density lipoprotein. Am. J. Cardiol. 52: 675-680, 1983. 25. Billimoria, J.D., Pozner, H., Metselaar, B., Best, F.W., and James, D.C.O. Effect of cigarette smoking on lipids, lipoproteins, blood coagulation, fibrinolysis and cellular components of human blood. Atherosclerosis, 21: 61-76, 1975. 26. Mustard, J.F., and Murphy, E.A. Effect of smoking on blood coagulation and platelet survival in man. Br. Med. J. 1: 846-849, 1963. 27. Renaud, S., Blache, D., Dumont, E., Thevenon, C., and Wissendanger, T. Platelet function after cigarette smoking in relation to nicotine and carbon monoxide. Clin. Pharmacol. Ther. 36: 389-395, 1984. 4-34

28. Maouad, J., Fernandez, F., Barrillon, A., Gerbaux, A., and Gay, J. Diffuse or segmental narrowing (spasm) of the coronary arteries during smoking demonstrated on angiography. Am. J. Cardiol. 53: 354-355, 1984. 29. Bellet, S., DeGuzman, N.T., Kostis, J.B., Roman, L., and Fleischmann, D. The effect of inhalation of cigarette smoke on ventricular fibrillation theshold in normal dogs and dogs with acute myocardial infarction. Am. Heart J. 83: 67-76, 1976. 30. Schroeder, K.L.,*and Chen, M.S. Smokeless tobacco and blood pressure. N. Engl. J. Med. 312: 919, 1985. 31. McPhaul, M., Punzi, H.A., Sandy, A., Borganelli, M., Rude, R., and Kaplan, N.M. Snuff-induced hypertension in pheochromocytoma. JAMA 252: 2860-2862, 1984. 32. Korman, M:G., Shaw, R.G., Hansky, J., Schmidt, G.T., and Stern, A.I. Influence of smoking on healing rate of duodenal ulcer in response to cimetidine or high-dose antacid. Gastroenterology 80: 1451-1453, 1981. 33. Murthy, S.N.S., Dinoso, V.P., Clearfield, H.R., and Chey, W.Y. Simulta- neous measurement of basal pancreatic, gastric acid secretion, plasma gastrin, and secretin during smoking. Gastroenterology 73: 758-761, 1977. 34. Konturek, S.J., Dale, J., Jacobson, E.D., and Johnson, L.R. Mechanisms of nicotine-induced inhibition.of pancreatic secretion of bicarbonate in the dog. Gastroenterology 62: 425-429, 1972. 35. Abel, E.L. Smoking during pregnancy: A review of effects on growth and development of offspring. Hum. Biol. 52: 593-625, 1980. 36. Longo, L.D. The biological effects of carbon monoxide on the pregnant woman, fetus and newborn infant. Am. J. Obstet. Gynecol. 129: 69, 1977. 37. Bureau, M.A., Shapcott, D., Berthiaume, Y., Monette, J., Blovin, D., Blanchard, P., and Begin, R. A study of P50,2,3-diphosphoglycerate, total hemoglobin, hematocrit and type F hemoglobin in fetal blood. Pediatrics 72: 22, 1984. 38. Ayromlooi, J., Desiderio, D., and Tobias, M. Effect of nicotine sulfate on the hemodynamics and acid base balance of chronically instrumented pregnant sheep. Dev. Pharmacol. Ther. 3: 205-213, 1981. 39. Resnik, R., Brink, G.W., and Wilkes, M. Catecholamine-mediated reduction in uterine blood flow after nicotine infusion in the pregnant ewe. J. Clin. Invest. 63: 1133-1136, 1979. 40. Hibberd, A.R., O'Connor, V., and Gorrod, J.W. Detection of nicotine, nicotine-l'-N-oxide and cotinine in maternal and fetal body fluids. In: J.W. Gorrod (ed.). Biological Oxidation of Nitrogen. Amsterdam, Elsevier, 1978, pp. 353-361. 41. Valeriano, J., Tucker, P., and Kattah, J. An unusual cause of hypokalemic muscle weakness. Neurology 33: 1242-1243, 1983. 2501258215 4-35

42. Hampson, N.B. Smokeless is not saltless. N. Engl. J. Med. 312: 919, 1985. CONCLUSIONS 1. The use of smokeless tobacco products can lead to nicotine dependence or addiction. 2. An examination of the pharmacokinetics of nicotine (i.e., nicotine absorp- tion, distribution, and elimination) resulting from smoking and smokeless tobacco use indicates that the magnitude of nicotine exposure is similar for both. 3. Despite the complexities of tobacco smoke self-administration, systematic analysis has confirmed that the resulting addiction is similar to that produced and maintained by other addictive drugs in both humans and animals. Animals can learn to discriminate nicotine from other substances because of its effects on the central nervous system. These effects are related to the dose and rate of administration, as is also the case with other drugs of abuse. 4. It has been shown that nicotine functions as a reinforcer under a variety of'conditions. It has been confirmed that nicotine can function in all of the capacities that characterize a drug with a liability to widespread abuse. Additionally, as is the case with most other drugs of abuse, nicotine produces effects in the user that are considered desirable to the user. These effects are caused by the nicotine and not simply by the vehicle of delivery (tobacco or tobacco smoke). 5. Nicotine is similar on all critical measures to prototypic drugs of abuse such as morphine and cocaine. The methods and criteria used to establish these similarities are identical to those used for other drugs suspected of having the potential to produce abuse and physiologic dependence. Specifically, nicotine is psychoactive, producing transient dose-related changes in mood and feeling. It is,a euphoriant that produces dose-related increases in scores on standard measures of euphoria. It is a reinforcer (or reward) in both human and animal intravenous self -administration paradigms, functioning as do other drugs of abuse. Additionally, nicotine through smoking produces the same effects, and it causes neuroadaptation leading to tolerance and physiologic dependence. Taken together, these results confirm the hypothesis that the role of nicotine in the compulsive use of tobacco is the same as the role of morphine in the compulsive use of opium derivatives or of cocaine in the compulsive use of coca derivatives. 6. The evidence that smokeless tobacco is addicting includes the pharmaco- logic role of nicotine dose in regulating tobacco intake; the commoaalities between nicotine and other prototypic dependence-producing substances; the abuse liability and dependence potential of nicotine; and the direct, albeit limited at present, evidence that orally delivered nicotine retains the characteristics of an addictive drug. 2501258216 4-36

7. Several other characteristics of tobacco products in general, including smokeless tobacco, may function to enhance further the number of persons who are afflicted by nicotine dependence: nicotine-delivering products are widely available and relatively inexpensive; and the self -administra- tion of such products is legal, relatively well tolerated by society, and produces minimal disruption to cognitive and behavioral performance. Nicotine produces a variety of individual-specific therapeutic actions such as mood and performance enhancement; and the brief effects of nico- tine ensure that conditioning occurs, because the behavior is associated with numerous concomitant environmental stimuli. 8. All commonly marketed and consumed smokeless tobacco products contain substantial quantities of nicotine; the nicotine is delivered to the central nervous system in addicting quantities when used in the fashion that each form is commonly used (or as recommended in smokeless tobacco marketing campaigns). 9. Since the exposure to nicotine from smokeless tobacco is similar in magnitude to nicotine exposure from cigarette smoking, the health consequences of smoking that are caused by nicotine also would be expected to be hazards of smokeless tobacco use. Areas of particular concern in which nicotine may play a contributory or supportive role in the pathogenesis of disease include coronary artery and peripheral vascular disease, hypertension, peptic ulcer disease, and fetal mortality and morbidity. RESEARCH NEEDS Available data clearly support the view that nicotine produces behavioral and physiologic dependence and has effects on all critical dimensions exemplified by a drug with a profile of high abuse liability. Nevertheless, the resolution of several questions is essential. These questions revolve around the relation- ships between the several forms of tobacco use. They parallel and have com- monalities with important issues in other forms of drug abuse (e.g., cocaine). There are several major research areas that could provide data of potential public health significance. The first area of research is the relationship between the rate of nicotine administration and abuse liability. Existing data suggest that the slowest commercially available nicotine-releasing preparation, nicotine gum, has a lower abuse liability than the fastest commercially available nicotine- releasing preparation, cigarettes. These facts further suggest the possibility that there might be quantifiable differences in abuse liability among tobacco product forms. The second area of research importance involves the relationship between the initiation of one form of tobacco use, e.g., smokeless tobacco, and the use of other forms of tobacco, e.g., cigarettes. The relationships between common forms of tobacco use, the extent to which they are interchangeable, and the possibility that the use of one form of tobacco leads to the use of another need examination. A third area of specific importance relates to the extent to which to- bacco use, with its implicit acceptance, encourages other drug use. A related 2501258217 4-37

question is the extent to which exposure to drug effects, both neurologic and behavioral, modifies subsequent drug responses or establishes the conditions for other equally harmful drugs to become reinforcers. These issues follow from the observations that cigarette use is a major that regular tobacco use generally leads to other forms of drug addiction. A fourth area of research is prevention and treatment. Recent surveys indicate that youth attribute negligible risk to smokeless tobacco products, suggesting the possible need for education-based prevention approaches. Regarding treatment, it.is plausible that nicotine gum treatment could be of even greater relative utility for smokeless tobacco users than for cigarette smokers because of the more similar pharmacokinetic profiles of smokeless tobacco- and gum-delivered nicotine compared to cigarette smoke-delivered nicotine. The absorption and distribution characteristics of nicotine with the use of smokeless tobacco may differ from those of cigarette smoking. The pharma- codynamic and pharmacologic consequences of such differences may be important but require additional future research. Further studies to define more precisely the role of nicotine and of smokeless tobacco in the causation of diseases other than those that involve the oral cavity are clearly needed. Specifically, research is needed to: • Determine nicotine blood levels and time course in various popula- tions of smokeless tobacco users, including established users. • Determine the cardiovascular, hormonal, and metabolic effects of smokeless tobacco when used in a regular fashion throughout the day. • Determine the influence of the rate of absorption of nicotine on the effects from smoking cigarettes and the use of smokeless tobacco. • Using experimental studies, determine the effects of smokeless tobacco in users of different ages and high-risk status (i.e., patients with hypertension, coronary heart disease, peripheral vascular disease, and peptic ulcer). • Using epidemiologic studies, determine the risk potential of the regular use of smokeless tobacco on the development of diseases such as coronary heart disease, peptic ulcer, and complications of pregnancy.

Table 1 Summary of Reports in Which Nicotine was Available Under Intravenous Drug Self-Administration Procedures Reinforcement Study Species Schedule Main Finding Comment Deneau and Inoki (1967) Rhesus Monkey Fixed-ratio 1 (FR 1). Several doses of nicotine were tested. Two monkeys initiated self-administration (S-A); the others required a priming procedure. Yanagita, Rhesus Monkey Experiment 1: FR 1. Sever- Ando, Oinuma, al doses of nicotine and and Ishida lefetamine and saline were (1974) tested. Experiment 2: FR 1. Sever- al doses of nicotine were continuously available for at least 4 weeks. Experiment 3: Progressive ratio (PR) procedures. Two doses of nicotine and saline and three doses of cocaine were tested. Lang, Latiff, Hooded F'k 1. Nicotine and saline McQueen, and Rat were tested in food-sated Singer (1977) and food-deprived rats. Nicotine did not serve as a reinforcer when compared to saline or lefetamine. Stable rates of nicotine S-A occurred in most subjects but were not clearly related to dose. Currently accepted criteria to assess reinforcing efficacy were not achieved. No direct test of reinforcing efficacy was done. At 0.2 mg/kg nicotine, response rates slightly exceeded those maintained by saline or the lowest cocaine dose (0.03 mg/kg). In food-deprived (but not food-sated) rats, nicotine was a reinforcer when compared to saline. Singer, llooded Rat, Concurrent ((Ftt 1: Nicotine) Food satiation decreased Simpson, and (Nlxed-time i min: food rate of nicotine S-A, Lang (1978) pellet)] in food-deprived however, nicotine was a 61Z8SZlOSZ rats. Subsequently, the reinforcer in both rats were fuod-sated. conditions. Nicotine was margin- ally reinforcing when compared to cocaine. Results were similar to those obtained when rats were similarly tested with ethanol.

Table 1 (continued) Reinforcement Study Species Schedule Main Finding Comment Griffiths, Baboon Brady, and Bradford (1979) Hanson, Albino Rat Ivester, and Moreton (1979) Latiff, Smith, Hooded Rat and Lang (1980) Smith and Hooded Lang (1980) Rat Goldberg, Squirrel Spealman, and Monkey Goldberg (1981) 0zz8Szt0Sz FR 160 followed by 3-hr timeout. Several doses of nicotine and saline were substituted for cocaine. FR 1. Several doses of nicotine and saline were tested. CONC[(FR 1: injection) (FT 1 min: food pellet)]. Several doses of nicotine and saline were tested. FR 1. One dose of nicotine and saline were tested. Second, order schedule FI I or2aein (FR10: stimulus) followed by 3-min timeout. One dose of nicotine and saline was tested. Number of nicotine Caffeine, ephedrine, injections per day did and a variety of not exceed that of saline. other similarly test- ed stimulants did serve as reinforcers relative to saline in this paradigm. Mecamylamine (centrally Group data suggest acting antagonist) but that nicotine was a not pentolinium (peri- reinforcer; however, pherally acting antagonist) there was no clear altered S-A behavior. dose-effect curve. Nicotine was a reinforcer relative to saline. Urine pll manipulations had mild effects on rate of S-A only during initial exposure to nicotine. Rate of S-A was inversely related to dose during initial exposure to nicotine but not after nico- tine S-A was estab- lished. Nicotine was established as a reinforcer both with and without a concurrent food delivery schedule in food-deprived but not food-sated rats. Nicotine maintained high rates of responding. Rates decreased marked- ly when (1) saline replaced nicotine, (2) the brief stimuli were omitted, and (3) subjects were pretreated with mecamylamine. Demonstrated the importance of ancil- lary environmental stimuli in maintain- ing high rates of responding.

Table 1 (continued) Study Species Reinforcement Schedule Main Finding Comment Ator and Baboon Griffiths (1981) Dougherty, Rhesus Miller, Todd, Monkey and Kosten- bauder (1981) Goldberg and Squirrel Spealman Monkey (1982) Singer, Long-Evans Wallace, and Rat Hall (1982) i''~z8Sz10Sz FR 2 followed by 15-sec Nicotine was marginally timeout. Several doses of reinforcing compared to nicotine and saline and saline across a narrow cocaine were tested. dose range. FI 16 and second order Nicotine maintained Fl 1 min (FR 4: stimulus). higher rates of S-A than Several doses of nicotine saline under the FI and and saline were tested. second order schedules but was only a marginally effective reinforcer when continuously available. FI 5 min. Several doses Nicotine and cocaine of nicotine and cocaine were qualitatively similar and saline were tested. reinforcers when compared to saline. Cocaine main- tained higher rates of responding in one of two monkeys. Mecamylamine pretreatment reduced rates of nicotine S-A. CONC'[(FR 1: nicotine) A group of rats with (FT 1 min: food pellet)]. 6-011DA lesions in the One dose of nicotine was nucleus accumbens S-A tested. nicotine at lower rates than a sham-lesioned group. Initial dose-response curve was inverted U- shaped, and final dose-response curve was flat. (From abstract of study). Establishment of nicotine as a rein- forcer required several months using procedures that typ- ically require only a few days to estab- lish cocaine or codeine as reinforc- ers. This study also showed that nicotine could serve as a punisher similar to electric shock. Extended the range of scheduled-induced behaviors that are --inhibited by such lesions.

Table 1 (continued) Reinforcement Study Species Schedule Spealman and Squirrel Second order FI 1, 2, or 5 Goldberg Monkey min (FR 10 stimulus) and (1982) FI 5 min schedules were tested. Several doses of nicotine and cocaine and saline were tested. Risner and Goldberg (1983) Beagle Dog FR 15 followed by 4 min timeout. Several doses of nicotine, cocaine, and saline were tested. Pro- gressive ratio schedule was used. Henningfield, Human Miyasato, and Jasinski (1983) Goldberg and Henningfield (1983) Human and Squirrel Monkey ZZZBsztosz FR 10 followed by 1 min timeout. Several doses of nicotine and saline were tested. FR 10 followed by 1 min timeout'. Several doses of nicotine and saline were tested. Main Finding Comment Nicotine and cocaine Nicotine•s reinforc- maintained similar ing efficacy was patterns of responding comparable to that on the schedules. of cocaine. Nicotine, but not co- caine S-A, decreased to saline-like rates when animals were pretreated with mecamylamine. Nicotine and cocaine Cocaine maintained maintained qualitatively substantially greater similar patterns of response rates than responding and were nicotine. reinforcers relative to saline. Mecamylamine pretreatment reduced nicotine but not cocaine S-A. Number of nicotine Nicotine produced injections generally subjective effects exceeded number of saline similar to those injections and were in- produced by intra- versely related to nico- venous cocaine and tine dose. Post- session had both reinforcing cigarette smoking was and punishing suppressed by nicotine. effects. Patterns of responding were qualitatively similar In both species. Number of nicotine Injections exceeded number of saline injec- tions in 3 of 4 human and 3 of 4 monkey subjects. In both the human and monkey subjects, there was evidence that nicotine func- tioned with both reinforcing and punishing properties.

1.0 5 SELF-REPORT Srrsears d drua 2.0 0 10 0~ ~ . OA 1.0 GROUP MEANS: 3 MINUTES POST ORUG 10 r . 30r I SELf-R£-ORT: FtM " 5 .3 0.0 L l 0.0 tAr t.0 i SELf-REPCRT• Rom.O h.rnq 0 04 C85ERVM rb U,Q,q. euM to en" 10 2 1 L 404 1 4y1 r+s. 77 w / p rv 2l1N IO rv 5 0.0 08SErNED: Onq .H.d 3.0 I •IN lWCrr COSERVED- R.hm46 tSrv 08.SE1tYED' SiQns of Eruq . 0.0 2.0 10 SE1.f-REPo4T osn ro wr*aq 0.0 L 1 2.21" 41M 14114 10rv vaer. 'S IV I Srv 231M : CIY Figure 1. This EiRure is a summary of the data from a study of the liabilitv of nicotine delivered as tobacco smoke (filled symbols-IN) or intravenous injections (open s,ymbols-IV). Dose is presented on the horizontal axes. ~_ven with a controlled smoking procedure, nicotine dose administration via cigarette smoke is more variable (producing flatter dose-response functions) than when given intravenously. Also, important effects of nicotine are covert though reliable and orderly (e.g., relaxed feelings, symptom scores). The finding that a low dose of tobacco smoke was more effective in reducing desire to smoke than a low dose of intravenous nicotine is consistent with the fact that satisfaction from smoking is also due to stimuli provided by the cigarette and the smoke. 2501258223 4-43

2 N9 W M 0 0 U rn W J Q 2 V N 2 Y J 0 Q W 0. 2 a W 2 1 MORPHINE (SC) /-AlAPHETAIiiNE (SC) . ~• / P 7.5 IS 30 PEN70sARSITAI cPO, ~ ~• 0 . 120 240 SUPRENORPHINE (SL) I 2 NI t0v'NE .. . P£NTAZOC)NE (!Al) PiT / L, 40+ 30T ~ p-l-TNC (PO) . x ~_L • 1. .75 1.5 3 P 5 10 20 CtIlOR01AZEPOXIDE (PO) ZOMEP1 RAC (PO) . • •--/~ P 50 100 200 P 200 400 e00 DRUG DOSE (mq) Figure 2. This figure presents data from a series of abuse liability studies conducted at the Addiction Research Center. The findings that Liking Scale scores are directly related to dose and exceed placebo values are important in identifying dependence-producing drugs. Intravenous nicotine produced the same elevated dose-response function as highly addictive narcotics (e.g., morphine) and a prototypic stiraulant (d-amphetamine). These data are also consistent with the lower abuse liability of chlordiazepoxide and almost negligible abuse liability of zomepirac. Adrainistration of intravenous cocaine results in a function similar to that shown for intravenous nicotine, except that the cocaine dose levels must be increased by a factor of 5 to 10. 4-44

I.V. NICOTINE INJECTIONS SUBJECT ;kg/kg BE-1 , , , , , , , 27 KO1 I ~ , , , 27 SK11„ KUl I I L 1 t 22 PE111 1 il 1 1 , 1 I, , , r 1 1 I i ~ 1 8 LA, „ l,„ 1 l 1,, , , , l I I I „ 1 8 KF „,,,„ ,i , , , „,,i ,, , „ ,, , 13 ` 3 HOURS Figure 3. This figure shows the patterns of nicotine self-administration that occurred when volunteer cigarette smokers were given the opportunity to take injections of nicotine, but not smoke cigarettes, during 3-hour tests. The amount of nicotine available was roughly comparable to that obtained by smoking cigarettes. The subjects smoked Less following sessions in which they took nicotine than following sessions in which only saline (the placebo) was available. 4-45