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1 / t ( I r 02:21:97 09:33 V212 835 1111 D&P NY Chronic Inhalation Studies in Mice. 11. Effects of Long-Term Exposure to 2R1 Cigarette Smoke on (C578L/Cum X C3H/AnfCum)F, Mice t•L' Carol J. Henry •-s and Richard E. Kourt'•a•7 ABSTRACT-Standardlred exposure eond'nions with Kentucky .reference 2R1 ctgarettes were used to espose 2463 (C57HIJCum X C3H/AntCum)Ft Iemale mice (nose only) to fresh, wtwle cigarette amohe. In addition. 1,014 mice were sham.epose0, and N9 m:ee were neld as snelf eontrots. The protowl entailed expos- ing mice to smoke for snam-eaposure) on a aaily eaais, 5 daysJ week, for 110 weeks snd otacerving remaining mice until Oeath. A large number of animals was us.a so that tne smoke generation and animat-notoinq systems eould ae testeo and e.aluated and yet provide signiliunt numbers ol animala for eaposure to cigarette smoke tor a mapr portion of their lifetirne. Deposition of smoke particulates was esomated to be at+aut 125-200 µ9 total Deniculate rnatter/lung/day. The only lung cancers observed were diapnoa.d as alveolar adenocarcinomas (AAC). A total of 19 of 973 amokF eaposed mies aro 7 of 651 snam-espoaea mice were observed with AAC. The difference between tne amoke- and aham-eaposed grouQs waa not statiatiully aipniticant at P<oS, but the data sup- pested that tne tumen oecurred with a shorter latency in the smok rexoosed group (Par.101. The data were analyZed by various methods, Including analyais of subsets of tne poputation of ani- mala. A significant inereaae In the inoidenee of lung eaneer was observed in one subset: noN.er, thia difference was not found In the population as a wnole or as a result of any other analyses. Under tneae exposure conditions, 211111 cigarette smoke would seem to have weak carcinogenic activity in mouse lung tissue. Other changes associated with satoke exposure were increased Incldenee of oiqmenteC alveolar macropnage accumulation. otitis media. and head and neck fibrosanxmaa. However. the ineidenee of nepnrltla. nematopoietic cancers (e.g., leukemiaa. lympnosar- eomas. ano ret:cvlum een sareomas), and pulmonary congestion was significantly higher in the sham-eaposed animala.-JNCI 18e5: 77:203-212. The experimental design of studies to evaluate the long-term effects of exposure to cigarette smoke in an animal model system must attempt to simulate human exposures. The tziteria used to design such studies should be based on those reported to be important in cigarette smoke-auaeiated human diseases Those fae- tors include high exposure to fresh mainstream cigarette smoke on a daily basis for a major portion of the life- time of the individual (2. 3). Even under these eondi- tions, however. resultant smoke-associated diseasea have been observed in only a small fraction of the exposed individuals. Our laboratories have been involved in the development of animal model systems and inhalation equipment to assess the biologicsl effeets of acute and chronic inhalation of cigarette smoke. In the develop- ment of the mouse modcl system. two approaches were taken: 1) quantitation of short-term effects known to be tt,Juu: C:r associated with smoke exposure and 2) quantitacion of long-term effects of model chemicals on eareinogenesis of the respiratory tract (4-6). The expesimental condi- tions in this chronic smoke inhalation study were designed to maximize the responses known to be in- volve:d from the short-term effects and to observe the results of long-term exposure to cigarette smoke. Mice were chosen as the animal model for these stud- ies and, in parucular, (C578VCum X C3H/AngCumlFl mice. This choice was based on several facton: a) avail- ability of large numbers of animals; b) susceptibility to the induction of lung tancer (squamous cell carcinoma. A1C, poorly differentiated carcinoma. etc.) by model chemical areinogetu (4, 6), which was linked to;eneti- ally determined differences in hydrocarbon metabolism capacity (7); c) availability of animals that were free of common adventidotu agents, in particular. Scndai virus and pneumonia virus of tnice; d) case and cost effective- ness of handling and manipulating on a daily basis; c) cotuideiable badtgtound known on longevity and spon- tancous expression of disease (4); J) smoke partieulate deposition characterized with nose-only exposure equip- ment (S): g) certain eharaeserised short-term effects such as induction of atyt hydt«arbon hydroxylase (7), induc- tion of sister chrottatid exchange (1. 9), inerease in per- AsauvttTtows useo: AAC>=alreolar adenossednomatr): ACNoalre- olar twmpresring noduler. ANCNaeaiveolar nonmmpRSUins noduies: COHbasorbox.heenoalobin; PAMA=piasnenud alveolar maeropnas e aecumulationl.t TPMptotal panicvlate maua. t Recci.ed August 19. 19a5: revised Janusry 27. 1486: accepted February K 19a6 rSupported by oontraet Cf'R-00s0 %.ith The Council for Tobacco Reaearch-U,S,A„ Irc t PRsented in pan at the?Airteenth ItttQnauonal f.aeeer Constru, Seauk. WA. Seqemher a.1S, 1982 (/). The interpretationt snd opinions are thoae ofthe authors and not neceturily thote of The Council tor Tot.xm Roeare3a "Xiaobioloaial Aa.oeiates Ine.. 5221 River Rd.. Itetheada, -%tD 20111111, a.iddrrn neprinr nrpuuu to Dr. Henry at her prerenr addrear: ICF Incorporated Inurnational Square, It150 KSt., N.W, Washington. DC 2000ti al4esent.ddreuctnternatiorul siaeehnok><tes. Ine, 2275 Winchet:a A•e., New Ha.en, CT 06511. . t We thank Dr. Laortard tiillupa and Dr. William Hall for pathololy support: Ata. Deniee Avery, Mr. David Daruie, and Mt. H. Doyle.%tul• linaa (or tecAninl support; Mr. Robett Kranko lot computer oro aramminjr. and Ma. Cynthia Whittield for typina the manateript, 203 jNta. VOL 77. No. I. tUt.r taet CTR Ve N ...r' 4SM' '711

02: 21; 97 09:33 $212 835 1111 D&P NY 204 Henry and Kourl , centage of cells undergoing DNA synthesis (10. 11). and inhibition of lung DNI+ repair capacity (12); and h) similarity to the mouse strain used by the National Toxi- cology Program for the Chemical Bioassay Program. The susceptibility of these mice to the long-term effects of cigarette smoke was not known. However, the average life-span of the animals was approximately 124 weeks. and diseases that could interfere with interpretation of the effects of cigarette smoke (i.e.. lung cancsr) were known to occur very late in life (mean time of 112 wk of age) and at low frequency (GT.G) (4). The smoke generation and exposure equipment used in these studies has been described in detail elsewhere (5, 13). This equipment was designed for standard'ued. documented inhalation exposures of large numbers of mice to fresh, whole cigarette smoke. Dosimetry studies demonstrated that 80-90% of the smoke puticulates that were deposited onto smoke-exposed mice were found in the respiratory tract and that animal-to-animal variation was less than 20% (5). In this study, standardized exposure conditions with Kentucky reference 2R1 cigarettes (high tar and high nicotine) were used to expose 2.053 (CS7BLCum X C3H/AnfCum)FI female mice to whole dgarette smoke, 1.014 mice were sham-exposed. and 449 mice were held as shelf controls. In addition, 710 mice wen included for co-treatment with benzo[a]pyrene by intratracheal inoc- ulation, followed by smoke exposure or sham<xposurc. The results from these latter studies will be reported sep- arately (Henry CJ. Kouri REt Submitted forpublintion). MATERIALS AND METHODS Data colfection.-An Experiment Information Man- agement System on a Hewlett Packard 3000 computer pro, •ided means for recordingand collating the observa- tions and data for each animal over the course of this 3-vear study. Animals were zandomized. given individ- ual identification car tags (National Band and Tag, Knoxville, TN), and assigned to a specific experimental group and treatrnent schedule. This information was entered into the experimental data base. interpreted by an edit program. and added to the existing data base via an update program. As the experiment proceeded. ob- servations. inventories, monthly body weights, and the levels of TPM generated each day were obtained and added to the data base. An automacic-tare Sartorius bal- ance was interfaced with a Hewlett Packard 2644 eom- puter terminal to collect and record animal weights. At the time of the animals' death or saaifice. autopsy forms were prepared to describe any gross pathological obser- ••ations and to assign specific histopathology numbers to each animal. The computer system supported the diagnoses using the Systematized Nomenclature of Pathology codes for the interpretation and reporting of the microscopic results. elnimals.-Mice were purchased from Cumberland View Farms (Clinton. TN) at 4-6 weeks of age. Females from the first filial hybrid generation from CS7BVCum JNq. VOl. 77. NO. I. JULY 1N6 lm 003 X C3H/AnfCum mice (i.e., BC3F1/Cum) were usr+ Females were chosen beause they were not aggrest with their cage mates during long-term experiments a.. because their smaller body size reduced the need for neck restraints of different sizes. These neck restiaints were used for smoke exposure and for shamtxposure. Upon arrival, mice were tested serologimlly for adven- titious agents, inoculated ip with 0.1 ml Sendai virus vaccine (M.A. Bioproducts, Walkersville. mD: (I, it)). and quarantined for a minimum of 3 weeks prior to treatment. The serological testing and animal husbandrv procedures have been descibed previously (4. 13). Smoke generation and monitoring.-Cigatette smoke was generated on the SEM II, a large-capacity, dynamic, intermittent smoke exposure system in which the smoRe is routed to the animal containment system as a contin- uously flowing stream for nose-only exposure of mice (5,13,15). Mice were restrained in stocklike holden, and up to 480 mice were exposed nose only to the same smoke-exposure regimen (13). Two SEM 11 machines were required for the large numbers of animals in these studies. A separate sham-exposure machine was also used (1)). Three animal contaitunent racks were used. one for each smoking machine and one for the sham• exposure machine. Mice were loaded into the neci;- rutraint holders and transported on specially designed carts for loading onto the exposure modules. The animal-holding system was described elsewhere (13). The mice were restrained approximately 2.5 hoursitlay. ,-k standardized smoke-exposure regimen was used. The cigarette was puffed once per minute. generating average SS-ml puff during a 2-second period. For tht atudies, a nominal 10% percent (vol/vol) smoke aerosol was produced. The exposure cycle alternated 20 seconds of smoke with 40 seconds of air/minute for 6-8 consecu- tive minutes (6-8 puffs/cigarette). Preliminary studies showed that these numbers of puffs approached a maxi- mum tolerated dose for this cigarette; however, this regi- men also resulted in smoking about the first one-half of the cigarette and resulted in delivery of approximately one-third of the potential TPM of the 2R1 cigarette. After an 8-minute.rest during which air was provided. the smoke exposure cycle was repeated. Five such expo- sure cycles (cigarette e4uiva)ents) with an air rest be- tween each cycle were given each day. To acclimate the mice to this exposure regimen. the length of exposure was gradually increased over the lst month of the study. Smoke• and airflow-monitoring devices were devel- oped to provide documentation of smoke particulate exposure levels and to provide safety systems for acci- dental toxic exposures or machine malfunctions (13). The on-line smoke monitor provided daily puff-by-puff documentation of the smoke particulate levels (13). Flow sensors were added to the exposure equipment during the lst year of the study and were designed to provide auxiliary air to the exposure modules if neces- sary. Detai)s of the design and use have been given else- where (13). University of Kentucky 2R1 cigarettes •+ere used for all exposures. These cigsrettes are standard 85-mm no CTR HN 043 7 1 61,21 3 ' )

02: :1~ 97 09:5a '$212 835 1111 D&r NY Z003 Chronic Clqar.tt. Smoka Inhalatlon Studi.s I filtered experimental reference cigarettes and have been characterized under standard Federal Trade Commission orocedures at Oak Ridge National Laboratories, Oak Ridge. TN. Under these conditions. the 2RI cigarette delivered approximately 44 mg TPM. 2.4 mg nieotine, and 21 ml of carbon monoxide (16). Cigarettes were removed from frozen storage and conditioned at 70-75'F and 60% relative humidity for at leut 18 hours before use. Animaf wciShts.-AII mice were weighed at monthly 'intervals at approximately the same tiine of day. 2 days aftcr cages were changed. Smoke- and sham<xposed animals were weighed at least 2 hours after exposure. Data were recorded for each animal, and the me:tn and standard deviation were calculated for each group at monthly intervals. TPbf deposieion and COHb fevefs-As determined from parallel radioactive dosimctry experiments, smoke particulate deposition was determined to be between approximately 125 and 200 µg TPM/day/mottse lung for this exposure regimen using 6-8 puffs/cigarette. [See Henry et al. (5) (or a description of the methods and results.] COHb levels were determined at monthly intervals. Blood from the retro-orbital sinus was collected from 3-5 rnicergroup. Natelson blood•co)lecting tubes (175 µl; Sherwood Medical Industries. Inc., St. Louis. MO) were prepared shortly before use by rinsing L'te tubes with a drop of sodium heparin (10.000 U/ml: Abbott Liboratories. Baltimore. MD). After blood collection, the tubes were sealed with Critocaps, stored on iee. and analyzed within 30 minutes using an IL-CO-Oximeter (IL-282: Instrumentation Laboratory. Ine, Lexington, `tA). ,Veeropsy.-;'vIice were observed twice daily for evi• dence of illness or respiratory distress. Dates and cireum- stances of death were recorded for all mice. \onauto- lyred tissues from mice found dead and those killed when moribund were examined microscopically. Lungs were fixed in situ with approximately 1.5 ml of 10% buf- (ered Formalin by infusion via the trachea. Lungs were lit,,ated at the trachea, and the thoneie viscera were removed as a single unit and examined grossly. Lung. trachea, esophagus. and thoracic lymph nodes were see- tioned (6 umi as a unit at 3 levels. using a frontal plane of sWion. During the lst year, respirztory tissues (lungs, larynx. trachea, nasal cavity, and middle ear) and abnormal tissues wcre examined microscopically from a random sampling of 13% of the animals that died Dur- ing the 2d and 3d years, respitatory tissues and any abnormal tissues from all animals were fixed and stained. Respiratorv tract tissues from 96% of the ani• mals were examined miaoscopieally.'In addition. 16 other tissues from major organs were examined miao- scoptcall.• irom a random sampling of 10% of the ani- inals. These tissues included salivary glands. cervical lymph nodes. spleen. liver. kidneys, adrenal glands. uri- nary bladder. ovaries, uterus, heart, stomaeh. large and small incestincs. pancreas, and thymus. Tissues were examined microscopically from a total of 987. 659. and 369 mice in the smoke-exposed, shamtxposed, and shclf-control animals, respectively. Morphological crite,io.-A brief description of the lesions observed In these studies is presented below. AAC occurred as discrete grayish-white, firirt masses, located in the peripheral portions of the lung, and were often multiple. Some tumors occasionally showed pleu• nl invasion and metastasis. Adenomu are not included in this category. Pulmonary adenomas were scored in two categories: ACN and ANCN. This classification scheme was devised as part of other on-going studies to determine the possi. ble progressive nature of adenomatous lesions to carci. nomas. Details of the chemical inducibility. time course. and transplantability of the ACN and ANCN will be reported elsewhere: however, ACN, as well as AAC. grew into tumon after transplantation into newborn BC3Ft%• Currt mice in about 50% of the ases (Henry CJ, Billups LH. Hall WC, et al.: Manusaipt in preparation). Histo- logially. ACN and ANCN were differentiated on the basis of size and extent of compression of the surround- ing lung parenchyma. ACN were rounded masses of hyperplasdc alveoto- genic cells, about 2-3 mm in diameter, that did eom- press the surrounding parenchyma. Nodules located ir. the most peripheral portions of the lung tended to invade the pleura. ANC.~f were similar to ACN. but they did not compress the surrounding parenchyma. These nodules were barely visible grouiy. Mitoses were oo• served oecasionally. Congestion in the lung was observed a; di)ated pul- monary vefas and capillaries, which were engorged with blood. The congestion could have been the result of an active process resulting in lung irritation, a passive pro- cess resulting in reduced flow of blood from the lung. or an inadvertent delay in necropsy after dcath. Head and neck fibrosarcomss were malignant neo- plasms of fibrous tissues. They were unencapsulated anc often showed scattered yellow areas of necrosis or red- dish foei of hemorrhage. Manv of these observed in this study were highly aggressive. met;static tumors, com- posed of cells with large spindle-shaped nuclei and scan: cytoplasm. Once established. these tumors grew rapidly. invaded veins. and metastasized to other tissues, includ• ing the lung. Hematopoictic eincen were grouped together as can cers of the hemic and lymphatic systems. They ineludet lymphosareomas, reticulum cell sarcomas, and lympho cytic leukemias, iVephritis fnduded all inflammatory lesions of th, kidney. glomeruli, and renal pelvis. The in(lammator lesion esa be caused by adventidous agents. toreigt materials. or other processes. Otitis externa is an inflammation of the external ear It is usually considered an incidental finding in th mouse. Otitis media is an inflamrrtati.on of the middle ear ant was relatively severe. Usually of bacterial origin, it cat be due to ascending infections along the eustachia: tube. The eusuchian tube, lined by ciliated cpitheliurr JNCS. Vot. 77. NO. t. JULY 19t C~'R twI>~>~ 0,~',~~` ~, ~~

I 02:21; 97 09:35 $212 835 1111 D&P N1 206 Henry and Kourl can be a portal of'entry for inhaled material. Extension of this lesion into the brain can result in death of the mouse. PAMA was observed as clusters of alveolar foam cells or pulmonary macrophages in the alvcolar spaces. The cells contained brown pigment and were often large and vacuolated. Data analysis.-An attempt was made to determine the cause of death of each animal. The major reasons for death were either conditions that randomly removed the animals from the study (i.e.. smoke-related or sham• exposure-related. hotder-related. or documented airflow or smoke flow factors) or diseases that likely led to the death of the animal. This tabulation allowed us to ana- lyre the data by two methods (1. 17, 18). The first method assumed that the lesion caused the death of the animal. and thus an "actuarial" table was constructed that compared the numbers of animals that died carrying that specific lesion to the total number of animals alive at the beginning of that interval. For the actuarial analysis. the total numbers of animals at risk either were those that were strictly defined to have died as a result of their lesions or were those generally defined as the total number of animals that had histo- pathologiml examinations. The second method assumed that the lesion did not lead to the death of the animal. In this way. the "inci- dence" of a particular lesion at a given interval was eal- culated by determining the number of animals found with a specific lesion compared to the total numbers of animals that dted during that intervaL The numbers of animals in this interval were either those animals that were strictly defined to have been taken off test ran- domly or the total number of animals that died during that interval. Statistical analyses were determined according to the method of Mantel and Haenszel (19) and Katilan and Meier (20). The procedure is briefly stated as follows. For 2 groups to be compared, the number of aniraals with a given lesion and the number of animals that were at risk for that lesion for each interval were used to con- struct 2X2 contingency tables. The number of lesions expected and the variance of this number could then be determined for cach interval. The sum of the expected values was treated as an approximately normal random variable with known mean and variance. The chi-square statistic. corrected or uncorrected for continuity, w: then used to determine the level of significance of tt: difference between the expected number of lesions ar._ the ob'served number of lesions for 2 experimcnui groups over any given interval. RESULTS Smok. Generatlon and Monitoring Data from the on-line smoke monitor showed that an average of 0.62 g TPM/day was delivered to the mice. Thus an animal that survived for the complete exposure time of 110 weeks was exposed to approximately 339 g TPM. Data from previously reported dosimetry studies (5) showed that following exposure to 6-8 puffs of 2R: cigarette smoke, about 125-200 pg TP.1ii would be de• posited in the respiratory tract of these mice during one exposure session. Multiplyins the daily amount depo;- ited by the total exposure time suggested that up to 100 mg TPM could have been deposited per mouse lung over the course of this study. COHb levels were also determined at mont.hlv int:r- vals. The mean COHb levels were 17.1 :: 4.2 iSD:. 1.4% d: 0.4 (SD). and 1.8% = 0.4 (SD) for the smol•e- exposed. sham-exposed. and shelf-control mice. respec- tively. Toxlelty of Treatment Survival.-A large number of animals were used i• these studies so that this smoke generation and anima holding system could be tested and evaluated and still allow for significant numbers of animals to be expose~' to cigarette smoke for a sipifiant percentage of their lifetime. Table I summarizes the disposition of mice during this 3-year study. During the lst year. almost 50 e of the smoke-exposed and 30% of the sham-exposed mice died of exposure-equipment-related problems. One major problem was an inability to monitor when or if air was flowing through the modules. At the end of the lst year. flow sensors (see "Materials and Methods" for de!cription) were added, and these safety systems resulted in enhanced survival of mice in both the smokc- and sham-exposed groups. ) 1 1 ) 1 TaetE 1.-Dispoation of ani.noL drrin4 ehrvsie inAatatien e,12R1 e{pnreRe e+noke' No. of animala Treatment Nlo. of Year I Year2 Year3 Total with animals tested Mieroseopic evaluation No necropsy or evaluation Mictoaeopie evaluation No neeropsy or evaluation Miereseopie evaluation Ne neeropsy or evaluation miervseopie evaluation 2R1 smone 2.053 15+ 975 427 27 406 7 98T Sham 1.014 ?i 325 311 10 2746 S e39 Shelf 449 3 1i' 129 +0' 237 21' 369 ' Conditions torsmoke exDosureef 10% 2R1 eigarette amoke. with2o.ee of smoke alternatint; with 60sec of air for a period of 6-e min. Five such expoeures were riven efch day. Sham<xposure was the aarne without smoke. ' Includes 2 animals that died in the 4th yr. '[ncludu animals removed from tat for collaborative studiea. )!ta. vot_ 77. Y0. 1. JULY 19e6 ) ,) i. r f R i f f'i 4-A4...1i f`x 14

i I 02:21~9i 09:J5 '$:1: 835 1111 D&P NY The surviving fraction of mice as a function of time on test is presented in teat-figure I. 'Ihese curves were corrected (or those animals that were determined to be andomly removed from the study (i.e.. thry died of expostue-cquipment-related factors). The survival curves of the smoke- and sham-exposed groups decreased more rapidly than those of the shelf-control animals. indicat- ing that the daily smoke exposure or sham<xposure caused a detectable level of morbidity and mortality. After 80 weeks on test, however, the shelf-control group was observed to die at a more rapid rate than the smoke- or sham-erposed mice. This was also the time when the shell-control animals were losing body weight (see next section and text-fig. 2). Body weighrs.-Body weight analyses of the smoke- exoosed, sharn-exposed, and untreated shelf-control ani- mals are presented in text-figure 2. The shelf-control mice gained weight until 30-60 weeks and then gradu- ally declined in weight during old age. The rate of weight gain of the shelf-control animals was signIfi- antly greater (P:S.05) than that of the smoke- and sham- erposed mice. At about 80 weeks on test, the shelf- control mice had an average weight of SO g. No differ- ence between the mean body weighu of the smoke- and sham-exposed mice was found over the course of the study. Smoke- and sham-exposed mice gained weight slowly until about 60 weeks on test. after which their weight gradually declined. The average maximum weight of BC3FllCum mice in the smoke- or sham- exposed groups was 32 g, which was s.een at about 60 weeks on test. 11006 Chronic Cipar.tl. Smok. Inhalatlon Studl.s Clinical sigru.-Mice were observed during and im- mediately after dafly exposetre to smoke and sham- treatmenL For the smoke-exposed animals, while a czr- tain amount of adaptation occurred, the animals never accepted smoke exposure without some agitation or struggling in the holder. They occasionally demonstrated . some shallow breathing or gasping during expostue. Immediately after exposure. mice in the smoke-exposed groups were often lethargic, ataxic, and hypothermic. The sham-exposed animals also demonstrated agitation and struggling while restnined in the holders. but generally they apperued normal after exposure. After 20-30 weeks of exposure. certain animals were observed to have reddened skin and worn-away hair around the neck area that fits into the stocklike holder. In 30-40% of the cases, these itTitated areas progressed to open sores as exposures eontinued. Periodically, during the 2d year of the study the tnice were rested and not exposed. Such rcsts enabled the neck cuts to heal. The length of the rt:sts varied from 1 day to I week. Polycar- bonate inserts for the stocklike restraints were found to cushion the neck slot and could be effective in limiting such neck abrasions. It is recommended that such inserts be used for any long-term studies where animals of dif- ferent ages and/or sizes may be used. Mlstopatholoqic Obs.rratlons The shelf-control mice died carr.ying a similar spec• trum of diseases to that reported previously for this hybrid strain of mice (4); i.e., about 60% died of neoplas- SMOKEEXPOSURE STOPPED WEEKS ON TEST TtCr.rtcurlt t.-Sur-i•inR tnction nf mtcc presentad u a function of time on test. Mice tncludcd in this snatvui -ere thore that died or -erc killtd when monCund Inonnndom dcatht; ue te:t (or dctinitionl. PC1. VOL 77. NO. l. JULY lset C T)"`b / Il ~ t -7 37 ` 1r E-,t+

t, 0:/:1/97 09:35 $21: 855 1111 D&P \T 20s Henry and Kourl so 1 11 1111111 ~6ill~iP~l~~ ~III : ~~~! ~ 20 10 I~~ ~ ~It~I1 She1t Sham Sm9ke 0 , ~ , 20 to 60 ao too tzo 140 WEEKS O*t TEST TLtT.rtct'tt[ :.-N(ean Sodr weishu of EC3Ft/Cum mice in the smokeetqtosed sh4m-exposcd. and shelf•conzrol ssoups as a(unctton o( time tesa_ One standan: ecnation ol the mean is indiated by the Oart. tic diseases and 10% died of nonneoplastic causes. The major neoplastic diseases observed were hematopoictic tumors (31 0), sarcomas (39G), fibrosarcomas (9%). lung adenocarcinomas (4%), liver carcinomas (!!6), and marn- mary carcinomas (3%). The major nonneoplastic dis- eases observed were congestion-pneumonia (8%), neph• ritis-cystitis (SS1, and conditions where no major disease was found (13a. In general. the same spectrum of diseases was observed in the smoke- and sham-exposed mice. The distribution of lesions and the latency of occurrence, however, seemed to be influenced by smoke exposure or shittt- exposure. Table 2 summarizes the histologic lesions observed in the smoke-exposed and sham-exposed mice. In addition. it prescnts the number of mice observed with a lesion, the total number of mice whose tissues were evaluated for that lesion, the highest lewe) of signifi• cance (i.e.. "P"-value) observed between the smoke• expused and sham-exposed groups. and the time during the study when this difference occurred. The only lung cancers observed were diagnosed as AAC-no squamotu cell carcinomas or poorly differen- ttated carcinomas were found. A total of 19 of 978 smoke-exposed mice and 7 of 651 sham-exposed mice JNQ. VOL 77. MO. I. JULY 19b6 were observed with AAC. The difference between the smoke- and sham-exposed gv.;tps was not statistically significant at PS05. but there s.•ere suggestions that the tumors occurred with a shorter latenq• in the smoke- exposed group. At 813 days following initiation of the study. the numbers of AAC in smoke•exposed animals were =rrater than those in sham-exposed mice at P=.10. Atternpt,s were made to analyze these data in different wavs- Fint. the number of animals that were found with alveologenic adenomatous lesions. thought to be prccur- sors of AAC, was denoted. These adenomatous lesions were tetmed "ANCN" and "ACN" (zrr "Materials and Methods"). Analysis of the incidence of these lesions. both individually and grouped in various ways (see "Materials and Methods" and table 2). indicated that at no time was the incidence in smoke-exposed mice higher than that in the sham-exposed animals at a level of PSOS. Again. there was the suggestion of a shorter latency for expression of either AAC or ACN by 813 days of treatment (P=.09). Another method of analysis took advantage of the fact that during the conduct of the study an attempt was made to determine the cause of death of each animal (res ) ) CTR NN 043"Y>"16

0:::1%97 09:36 '$212 835 1111 D&P NY Chronio Cl9arsti. Srrsoka Inhalation Studl.e TL eLE .^.--S-ry o/ ireidexe oJlesienf iw BCJFt/Crm w:ice dur+n0 tone+s*+w es7wrr-t te !R! eipyreofe am.ie No. of anim als Lesion Smoke-ezposed Sham.zpasd liighot level of sis'nificanee' Present TotaJ Present Total Respirstory tricc' AAC 19 971 7 631 Smoke > sham: P=.10 at 113 days AAC + ACN 49 978 28 661 Smoke > sham: P-.09 at 813 days ANCN 4- ACN 38 97B 21 651 Smoke > sham: P-.39 at 7S7 days AAC + ACN r AYCN 69 978 38 651 Smoke > sham; Pi.18 at 813 days PAMA 158 978 1 651 Seeoke» sham Congestion 189 978 1S8 551 Sham > smeke; PS.0S at 412 days Rhinitis 14 1502 8 319 Smoke > sham: P-.S2 at 869 days Otitis media 66 579 35 371 Smoke > sham: PS.OS at 421 days Otitis eaerna 140 579 T8 371 Smoke > sham: Pa.l4 at 661 days Nonrespiratory tract Nephritis 27 179 26 110 Sham > smekos P=.16 at 953 days Head-netk Ctbrosar. 29 987 8 659 Smoke > sham: P5.05 at M 1 days comss Hematepoietie cancers 12S 987 126 659 Sham > smoke: P5.05 at 729 days " The highest level of silrnificanee for the difference between the smoke- and sham+zposed wouos and the time at which the differenc, occurred are gi~en. These dacs were determined from ehi-aquare analysis of each of the lesions or erouos of lesioeu ata-wk Interrsu ever tiu eouru of the study (up to 1.120 days on tett). ' Respiratory tract tissues included lung. larynz, tnckea. nacal oavity, and middle ear (ue "blaterials and Methods'1. "tilaterials and Methods"). Thus the total ntunbers of animals that died could be divided into those that died of factors unrelated to their laions (i.e.. random factors) and those that likely died as a result of their lesions (i.e.. nonrandom factors). Assuming that the animals died as a result of these tumots yields the analysis shown in table 3. In this actuarial analysis, the total number of animals at risk was 304 and 379 in the smoke- and sham- exposed groups. respectively. Analysis of this subset of the population showed that there was no difference in the incidence or latency of A.4C between the smoke- and sham-exposcd mice. Table 4 presents the results of the analysis of another subset of the population. where only thtsse animals that were determined to be randomly taken off the test were studied. In this case the numbers of animals found nr- rying lung tumors were compared to the numbers of animals that were observed not carrying the tumor. In this analysis. a total of 7 animals were found with AAC, and all were in smoke-exposed animals. The final inc- dence of 7A.iC of 4 74 smoke-exposed animals is sienifi- cantly higher than 0AAC of 272 sham-exposed anirttals (chi-square statistic = 4.18: P=.04). The month-by- month cumulative, probabilitl•. however, never reached a level of significance of MOS (see table 4). Similar analyses were done on all the lesions listed in table 2. The respiratory tract lesions found to be signifi• cantly higher in smoke•exposed mice were PAMA and otitis media. PAMA was found only in the smoke- exposed animals. and approximately 35% of all mice that died during the last 4 months of smoke exposure had this lesion. For some unknown reason, pulmonary congestion was found to bc higher in the sham-exposed animals than in the smoke-exposed animals. Certain other nonrespiratory traa lesions were also analyzed by the methods described above. Table 2 shows that the incidence of head-neck fibrosarcomas was st3 nificantly higher in the smoke-exposed 'animals than ii the sham-exposed animals. Analysis by actuaria methods also showed significant dillerenees (data nc shown). However, the incidence of nephtitis and hemt copoietic talncers (i.a, leukemias. lymphosareornas. an reticulum cell sarcomas) was significantly higher in th sham-exposed animals than in the smoke-exposed mic: This latter observation would seem to be an example c competing risks, when the oceutsena of a specific lesio was altered by the simultaneous expression of anotte disease procea: that removed the animal from the studt G1SCUSS1oN . The results of this study suggest that 2R1 cigaret smoke has weak carcinogenic activity in mouse lurr tissue. No bronchogenie squamous cell eareinontas we observed in any of these animals. AAC, as well as ot2t alveologenic lesions. were observed in smoke-es:pose animals, and the incidence was higher in the smok exposed aaitssals than in the sham-exposed anitna: The difference in incidence of these AAC and oth alveologenic lesions. however, never reached the level significance of MOS. The data suggested that the I !ency for the oecurratce of these tumors is shorter in t: smoke-exposed animals (Ps.10; see table 2). Various methods of analyses were performed on the dats, including incorporation of other alveologer lesions with the lung cancers and analysis of subsets the population of animals that were categorized as cause of death (i.e.. random vs. nonrnndom). In tener these analyses yielded no significant differences betwe the smoke- and sham-exposed groups. However. : analysis of one subset of the population, those anim taken off test randomly, did yicid an interesting obser jwa. VOL 77. Yo. t. JULY CTR MN

I 02:1i97 09 : J6 '$212 935 1111. DSP NY 210 Hsnry and KOuti / TaBI.E S.-ActuariaW nttaiyris of BGFilCtiwt tnice dYi*4 a f+m0 caaerr during loap-ttrwt ezyvrre ta !Rl csqaretv smektt' No. of animals Days en tot Smeke-ezpoe.ed Shamexpo.ed p-valuee tumort At risk' tDmW At risk 1-28 0 504 0 379 1.0 29-66 0 603 0 377 1.0 57-84 0 502 0 377 1.0 85-112 0 501 0 377 1.0 113-140 0 S01 0 377 1.0 141-168 0 501 0 37T 1.0 169-196 0 501 0 377 1.0 197-224 0 600 0 37 i 1.0 225-252 0 499 0 377 1.0 253-280 0 499 0 377 1.0 281-308 0 499 0 377 1.0 309-336 0 498 0 3 i. 1.0 337-364 0 495 0 377 1.0 365-392 0 493 0 376 1.0 393-420 0 490 0 375 1.0 421-448 0 483 0 9T0 1.0 449-476 0 479 1 369 .90 (16) 477-504 0 476 0 365 .90 (.26) 306-632 0 470 0 361 .90(26) 533-560 0 465 0 3S6 .90(.26) b61-588 1 454 0 346 1.0 (.86) 589-616 0 445 0 339 1.0 (.86) 617-644 0 427 0 327 1.0 (.86) 645-872 0 404 0 306 1.0 (.86) 673-700 0 394 0 291 1.0 (.86) TO1-728 1 366 1 277 1.0 (.79) 729-756 0 343 0 257 1.0 (.79) ?57-784 1 813 0 !33 1.0 (.39) 78S-812 1 294 0 204 .96 (.65) 813.840 3 264 1 179 .67 (.45) 841-868 0 Y29 2 156 1.0 (.99) 869-896 0 190 0 124 1.0 (.99) 897-924 0 161 0 98 1.0 (.99) 925-962 1 128 1 71 1.0 (.88) 933.980 2 94 0 52 1.0 (.84) 981-1.008 1 66 0 39 39(.70) 1.009-1.036 0 43 0 114 ::9 (.70) 1.f13"-1.064 . 1 1 i 0 7 .79 (.61) 1.065-1.092 0 8 0 6 .-69 (.61) 1.093-1.120 0 3 1 2 .9-0 (.79) 12 7 ' 11iet in this analysis were only those whose deatht were assumed to be related to their leaione. (.er nonrandotn. All lung cancers were AAC. '`o. of animals found dead with tumor. ' No. of animals at risk in a subset of the population that was siit•e at the beginning of the interval. See text for definition of the popuiation subset. ' Level of sieniGeance for the differences between the expected and observed No. o( tumors was detertnined from the chi-satare ststistic corrected for continuity. The ehi-square statietie uneer- reeted (or continuity is siven in DarentAew. tion. The final incidence of 7 lung cancers (all AAC) in 47 i smoke-eaposed mice compared to 0 lung cancers in 272 sham-exposed mice was significantly di((ersnt (see table 3). In contrast. cu(nulative chi-square analysis at 4-w•eek intervals was not significantly different. The most likely reason for disagreement between the two sta. tisticnl tests concerns the population dynamics of how JVC7. vOL :7. NO. 1. JULY 19l6 10009 the animals were taken off test. In our opinion, the fact that the two tests did not agree tends to lessen th• impact of the conclusion that smoke-exposed animals i the subset population had a significantly higher inci- denee of lung tumors thae the sham-exposed animals. Exposure to 2R1 cigarette smoke resulted in signifi- cantly increastd incidence and decreased latency of PAMA and otitis media. PAMA were observed at a rela- tively low incidence, with the (itst occurrence after 309 days on cest- During the (ast 4 months of smoke expo- sure. the incidence of PAMA reached 35%. su=gesting that macrophage accumulation was in response to a T.it.E 4.-OceurmteeoJ(uayeeneer ie BGF,,'Cuet ntier rowdom/y tekaw off te.t dur+nD iono-tmn ssponAre to sRl cigarette rntaker' No. of snimals Stnoke-szpeud Shamespesed Days on test Tumor- y~r, Tumor- u;no Dearin2 yearinQ besrins y.arins P•value` 1-28 0 8 0 3 1.0 29-66 0 16 0 7 1.0 5T-84 0 7 0 3 1.0 85-112 0 16 0 0 1.0 113-140 0 6 0 2 1.0 141-168 0 7 0 3 1.0 169-196 0 10 0 2 1.0 19T-224 0 14 0 2 1.0 225-252 0 10 0 6 1.0 253-280 0 13 0 5 1.0 281-808 0 4 0 4 1.0 309.386 0 5 0 7 1.0 537-364 0 22 0 28 1.0 355-392 0 26 0 28 1.0 898-420 0 21 0 19 1.0 421-448 1 19 0 1S 1.0 (.42) 449-476 0' 20 0 21 1.0 (.421 477-504 0 16 0 18 1.0 (.,21 605-532 0 23 - 0 16 1.0 (.42) 533-560 0 26 0 12 1.0 (.421 561-S88 0 19 0 12 1.4 (.42) 689-616 1 11 0 18 .48 (.16) 617-644 0 11 0 7 .i8 (.16) 645-672 2 23 0 13 .20 (.081 673-700 1 30 0 6 .18 (.07) 701-728 0 20 0 a .16 (.07) 729-756 2 31 0 2 .17 (.07) 757-784 0 20 0 4 .17 (.071 785-812 0 1 0 0 .17 (.071 813-8a0 0 0 0 0 .17 (.07) 841-858 0 0 0 0 .17 (.071 869-896 0 0 0 0 .17 (.07) 897-924 0 0 0 0 .17(.07) 925-952 0 0 0 0 .17(.07) 953-980 0 0 0 0 .17(.07) 981-1,008 0 0 0 0 .171.071 1.009-1.036 0 12 0 6 .17 (.07) 7 W7 0 272 •Mice in this analysis were only thoae.hose deatha were assumed to be unrelated to their lesions. i.e.. randotn All lung cancers were AAC. ' Level of e(enificance for the differences between the esoected and obseeved No. of tumors was determined from the ehi-square statistic eorreeted for wntinuity. The chi-square statistic untor- racted for continuity is given iw parewthea... CTR 11N 04,3t 18 ) 1 ) ) ) .~

I 0 0:~21/97 09:57 V212 835 1111 D&P \T cumulative dose of smoke. These data regarding PAMA give biologial evidencc that the smoke paruttilates •cached thc alveolar regions of the lung. Further evi- dence that the smoke particulates reached other anms of the respiratory tract can be seen from the increased incidence and decreased latency of otitis media. 'Me eu- stachian tube with its ciliated epithelium was likely a portal of entry for these inhaled materials and would seem to have been impacted by smoke exposure. Exposure to 2R1 cigarette smoke also resulted in significantly increased incidence and deaeased latency of head and neck fibrosarcomas. The mechanism by which these tumors develop is unclear. Whether such factors as localized deposition of smoke particulates in this area and/or the relative severity of neck cuts or abrasions caused by chronic restraint played a role in the formation of these tumors is not known at this time- In nts. chronic exposure to cigarette smoke has been asso- ciatrd with inceued incidence of tumors of the forelegs (21). In these studies the rats were exposed to smoke in whole-body tubes. It was suggested that these tumors resulted from the combination of smoke particulate deposition on the forelegs and the presence of abrasions on the forelegs caused by the method of restraint The dose of dgarette smoke given in this study was limited by the inhermt toxicity of this particular cigarette smoke for this strain of tnioe- Following expo- sure to 6-8 puffs of 2R1 cigarette smoke, the dose was estimated to be 125-200 µg TPM/lung/day. Previous studies with another cigarette type (high tar and low nicotine) have shown that up to 1 mg TPM/lung/day was given (J). Additionally, the exposure regiasett uti- lired about the first one-half of the 2RI cigarette. It is not known whether the first one-half of the cigarette has different biological activity compared to that of the other one-half. Thus the smoke exposure conditions used in this study (low dose and first one-half of the cigarette) may not have been optimal to detect signifi- cant biological or carcinogenic activity. In drawing conclusions from this study, several alter- natives are possible. The first is that the mouse may not be a good model for long-term human cigarette smoke inhalation studies. Mice are obligate nose breathers. have a turbinate structure different from that of humans. have a shorter life-span. do not actively inhale smoke as do humans. and are more sensitive to certain smoke- associated chemicals (e.g., nicotine) that limit the dose of smoke that can be given. All of these factors tend to mitigate against rodents as an implicit model for human inhalation studies. On the other hand, the mouse appears to be a good model for the study of the effects of smoke inhalation as noted by the following: Lung and kidney aryl hydrocarbon hydroxylases are inaeased in smoke-exposed mice (6, 7. 10); the number of sister chromatid exchanges in bone marrow cells is increased in smoke-exposed mice (8. 9); PAMA is ineteased in smoke-exposed mice; DNA repair capaeity of lung tissue from smoke-exposed mice is approximately 50% inhib- ited (!2); DNA synthesis in lung tissue from smoke- exposed mice is increased up to twentyfold (10): and 16010 Chrortk Clgar.tt. Smoke Inh.latlon Studls squamous cell ardnotna can be produced in these mioe at high incidence with known cltemial csrdnogens (1.6). A further alternative to this latter observation is that polycyclic aromatic hydrocarbons are poor model chemi• nls to use for biologic activity of dgarette smoke. In generat, rodents have not been observed to develop high incidences of lung ancer following exposure to cigarette smoke (see reviews (22. 2J)l. Notable among these many studies is that bronchogenic.aquamous cell nrdnoma has been observed only in I rat (FS44) after exposure to cigarette smoke (21). In this cast, 10 respiratory tract tumors were observed in 7 of 80 animals (9L), with the difference between smoke-exposed and sham<xposed rats reported to be significant at P5.05. The tumots were reported to be I adenonrdrwtna and I squamous cell carcinoma of the nasal cavity and 5 sdenomas, 2 alveologenic cazsinomas, and I squamous cell nrts- noma of the lung (21L Extensive studies by Dontenwill et a). (24. 2f).using randombred Syrian~o lden hamsters have indicated sig- nifiant changes in laryngeal tissues after smoke ezpo• sure, including laryngeal tsrcinoma, with little or no changes reported in the lung. Using inbred Syrian golden hamstezz. Bernfeld et al. (26. 27) have reported neoplastic changes itt the larynx of hamsters exposed to sraoke, with few changes reported in the lung. Bronc(to- genic squamous cell carcinomas have not been reported in either mice or hamsters after exposure to cigarette smoke, although the incidence of AAC has been reported inaased in anice (28, 29). A second alternative condusion to this study is thas the smoke exposure conditions did not reflect the "true" biological potential of this 2R1 cigarette smoke. Ap- proaci.taately the first one-half of the cigarette was used. and relatively low levels of TPM were deposited. #nainly benuse the high nicotine content ot the cigarette limited the dose. Additional studies with other dgarette types will have to be done to test this alternative. A third alternative is that cigarette smoke alone has fairly weak'biolofic activity and that the data in rodents refleu this activtty. The high biological accivity and response observed in human smokers (23, 30) may be a result of a combination of faaors, which so far have failed to be duplicated in the laboratory setting. REFERENCES (1) Htr+av CJ. ltttturs LH. HAti wC_ et al. The et(M of li(atuee eapowee to :+hde eipreue emote in EC1F1/Cyeq ntiee. In: Prot..dinss of the 'Il:ira.e.uh Intemuional Csncer Conpess. Seaulc. WA. Genera: lnternational Union Apinst Canees, t9fz (2) Plu MC. HtwnusoM EE Epidemiolor of Wl.c7clie aydns orbonc Qtaantifyins the oneQ ri.k hern drareue imokina and air pollution st(ecu. tn: Gelboin H, ed Polyeyclic hydrs orbons and eanca. New York: Avdenuc Ptesa. 19a1:317-3lt (3) Dott R. lLto R The ou.n o( tancer. Quaruiuti.e ntinwto o( a.oidakh riaiu et aneR in the Untwd Suto today. J Nt1 1901: f6 1191-130L (4) HtMa. C,). lStts.ua t.H. AvnY MD. e+ aL Luns aneer model eyunn tuin` 3.netl+yldwlandvt- in inded utaint of taKe. Cancer Rss 1911; 41SOZJ-!mi JKp. VOL T!. MO. 1, )ULY 1!L CTR I-IN 04:3'71 S4.

I . 02: :1: 97 09: J7 $212 895 1111 D&P NY 212 Henry and Kour1 (f) HENaY CJ. C1TON JE. STOatLY JR. et al. Depasition and distri- bution o( the toul paruculatc maua of cigarette smokc in mice using a IarReapaoty smoke e:posure system. Tozitol AppI Pharrnacol 1911; 3U99-403. 16) Kol:at RE. B:LLtns LH. Rt•ot TH. et al. Correlation of induci• bility o( aryl hfdrooarbon htdroxylaac with susceptibility o( 3-methylcholanthrenrint4ued lung ancets. Caneec Lnt 1980: 9317-4tJ. (7) KouRl RE. Rt°ot TH. Ct:aatr RD. n a). Biolofieal activiry e( tobacco smoke and tobacco unokrrelated eherntols. En.iron liealth Penpect 197l: l9:S3-69. (8) Bt:ao1CT WF. BANUt)tt A. KA..AUU:+cASr RK. et aL Inaessed sister chromaud ezchsn8e in miee es:posed to whole ogarette smoke. Muut Res 191N: 136:7f-80. (9) Pcr.tAr: DL. D6vtD RM, S/ttstoaN J3t. et aL Sister ehnomadd e.change induction in bone marrow cells of mice ezpoaed nose-only to whole ciprette smoke. Mutat Res 1985: 156; 181-186. I/0) 1COLNACAUxcAat KR. Rtio SM. D.xut DR. et al. Auteradio- snphic analysis of DvA synthesu in pulmonary tiuuea of mice eaposeQ to whole cigarette smoke. The Toaienlo=iet 1912; 2:46. I11) Ht.~aY CJ. HwANG KR, ICAxACwNCAU fJL et al. Reant devel• opmenu in inhalation tozicoio8l: Evaluation of selected ahors- term endpoinu (allowing "noseanly" exposure of rodenu. ln: Homburger F. ed. Saiety evatuauon and retulasion of chemitals. wer York: KarBer, 198l;233-242 (12) AASwtssE.-; RE. BoTO CH. DA.Kue DR. et aL DNA repliation and unscheduled DNA synthesis in IunBs of mice exposed to eigarette smoke. Canea Ra 1981: I l:2bb3-2588. (13) HExar CJ. GArLt T. FLoRaNT L et a1 Chronic inhalation .tudics in miee. 4 Facilities and equipment for no..enly ezpeuure to cigarette smoka Beitr Tabakfotscit Int 1985: 12:37-54. 114) HAtt WC. LutttTRA. HL%*Rv CJ. et aL Sendai vitus-disease processes and rssarels eomplintions: In: Hamm T. wi Com- plieatlons of viral and mycoplaWmsl infections In rrndeat toxicology researeh and testing. Washineson. DC: Hemisphere Publiar9ons. In press. /1S) MONEyNVN JH. STOttLr JR. FLOUSrr L Proceu and Instru• menu Corporation Autonutic Smoke Ezposure W@sine- SEM U. In: GuFrin MR. Slokely J0. Higgins CE, eds. DOE report ORNL-5424, Oak Ridge National LaboratorT. Spring- field. VA: Natl Technital Information Service. L:S. Dept Co m mcrcc. 1979:19-33. JNl]. VOL 77. NO. I. JULY 1la6 (16) Ju+Ems RA. WMrTi S1L GuEST WH. a al. Cherniel charaetensa. tion ol the smoke of selected US commetaal asxena: T nicotine. carbon rooeoside. ozida of nitrogen. hvdco c7anide, and aaolein. DOE repoet ORNLJltihe749. G. Ridge National Laboratory. Springfietd. VA: ratl Techmea: Information Service. U.S. Dept Commerce. 1963. (17) International Agency for RexareD on Cancer. Lont-urm ar.e shorr-tam tasaoiop as.an for ordno8ens: A aitiol appraiusi. IARC blota8t 198ih suppl Y:1-{26. (li) Ms'ttHELL TJ.TuaNa= BW. Long-linear models in the ans:v. sis o( disease pre.akrnce data from survivaVsacriitce eapers- ments. Biometria (47l: l3:221-224. (19) M.nTtL N. HAtxsuL W. Stitisdal aspects o( the analysis ot data (rom retrospective studies o( disease. J Nat1 Caneer lrst 19iY: Y2:719-748. (20) ICaPLA.v EL MuEt P. /Yot>parametric estimation from incom. plete obscrruiona J Am Stat Assoc 1958: S3:4S7-i81. (21) DAUtt WE. Nt7Tisttttsl P. GatuDtu R. es al. Chronic inhaia- tion o( eipeeue smoke by F344 rau. JNCI 19t1? 6i;383-390. (221 Pirttao W. Ezpsrimental respintory oreino8enests in smail animals. inviroa Res 1084t 33:14i-U8. (27) lnternatlonal Aeenclr fo[ Raatch on Cancer. Tobacco smoking. IARC 3rionogr Eval Csreinog Risk Chem Hum. In preas. (2e) DoNTSNWttt W. Ctttvauta HJ, Haatlt AP. et al. Studia on tne effetts of chronic tspreue smoke inhalation in Syrian golden hamsters and the imponanee of vitamin A on morpholof:ea: alterations after smoke ezposure. Z Krebslorsdt 1977; f's. 1i3-18Q (2t) Dor'TEHWUt W. Cnteauta HJ. HAarE HP. et al. ln.estiptiar:s on the eEfeets of chronic ei8srette•smoke inhalation in Srnan golden hamueer. J Nad Canar Inst 1l73: 31:1781-11u2 (26) BEaNSCSa P, Hosutatasa F, RussrrtLo AB. Strain diiterenees in the response of inbred Syrian hamstars to cigarette amo.e iahaladon. J Nat1 Cancer Inat 1i74: 33a 141-1137. (27) Eu:drttn P. Hosiouaeta F, SOTO £, et aL Ciprette smoke inhalation studies in inbred Syrian golden hamsters. J%*C. 1979: 65:673-11e9. . (Yd) HAttltIS RJ. NtGtoKt G. Production of lung aresnomat C378L mice espated to a eiprette smoke and air miature JNed 11967: 4:8l741L (lf) HAarus RJ.:•Jtoaono G. Luoowtt S,ec al. Tlte incidence of lung tumors ia CS7fL mice ezposed to oprette smoke: Air mtattires for prolonged periods. [ns J Cancer 1974; 14:130-136. (J0) Lota L.U, EaxsTta VL. tYAaxtR KE. et al. Smoking and Iung anttr: An oveniew. Caneer Res 1984: 44:3940-S9SL C TR PIN 04 3- r~`.-;00 )