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=~-aff r tfcres; r:c.~raln-Ue ~s:r: cross,~ G.E. Dagle and J.A. Mahafley (Eds.), ° Technical Information Centsr, U.S._ Departlaent af Energy, NTiS, 1980. 0 0 4v The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice: Comparison of a Large Smoke-Exposure Machine. (S€PA) with a Small-Capacity Smoke-Exposure Machine (Yyalton) C. J. )ff.NRY,o C. E. WFi1Tb1IRE.•t A. LOPEZ.° D. R. DAN91E.• y(. Q AVSRY.° 1$ CATON.t J. Fl Sl'OI{MY.t R. W. 1IOtrY®It:AG.t YI. R. GLiEB>1V,t emi L E. gOIJNe °Departmeffi of Bsperlmeatel Oneoiogy ead Dopaetment of 9ioahemieal Onmlogy. 34(atobw1ogicel Aeeociatoa. Bothuede, Maryiaad: tAnolytteal Chemdeay Div®ion. Oa7t Ridge National Lborutory. Oak Ridge. Tennaserie AesMACr The diepwieton and the intensEl distribution of total partieafate mecWr (TPM) of aigatette smoke were esamned under conditione of high TPM deposition in the lunqe of miee. Two differeat. smoka-etposura systema aara urd. The smoka espoaute taeehtae (SEd1 In is a la,gecapaetty (480 miae) dynamic system in wbidi uaoko 4s routed through the aNmad containment vnie as a coutinuouolq lloving maem. bqee ata nseeaithd about the nedt fa stock-type holdets for naroely eapoaure. ':te Walton 1loraontel 9mokinq 1llaehine (Walton) is a tmell-capscetp (12 to 70 mies) smtia systsm ahare the smoke is introduced ioto e:poture cherobers into which mxe (resQained in either atock-eype holders-or sbole-body tnhea) rapir'e directly. Both macftian wene operated under oeandatdimd conditions for puff duradoo (2 see). averap)e putT volume (38 ml), puff froquoncy (owe per minute), and butt length of c4arorte (33 mm). . rndioactive nacer (' `Cdabeled dotsiacontane) ema wed to auaatjtate the depodtion of 'CPM in tiesuea of the mawo after esposure to smoke. Whea the Walton maehine wer uNd, the d.positbn of '1'P!1 for 2A1 Kemucky reference cigarattes iaeraa.ed with inceeaein; smoke coocenemation and ,mokre:poaure time but wae indnpondent of,mt or tttaio of neuaea Detectable Ie.Na of'PPi41 +sme found in the head, laryna» IuoW, and stantaeb. The dittsdretien of 'PP'.11 among tbime tfsum did eot chaage m the total espeeuse time iodaasedd to 300 see or as the amolte con,enttattoa aaeieer.eeed to 14.196 (voLl.ol.) ReSerdlss ot total e:poaun) timu or amoke comautratlon. 73 to 7'. % of the TP94 waa found in cmo IaaQr .nd'88 to 91% of the TPMI vat oontained in tM entire raspieatory tsuct. '1'hua the aooditionf of 10% (aoLlvo!) emoke eoacetutaeion for 300 tee of *Psesent.tddtnse: National Caucer Institute. 8edtesda,.Marpland. 177 -._-Z =-1 H r®o12008' \

ME(YflY EC AL 178 HT®0120082 ., total e:pouue aars chosen for compeefeon of the depoutioe and dietribution of TPM from the two emohev:posure systems. The average depooition of TPM in the lunp of 8C3Fi1Cum mice waa 123 end 134 9C after e:poaure on the Walton and the S0N 11 rmohmet, reaepeetively. CoefIIeienta of variation for a totd of 40 oaieo espaaed on the Walton and a total of 120 mice esposed on the Smw II were 0.20 and 0.00, reepeettvely. The pereent of tonl body dhcribation of TPaf for aaiets4 ettpased on the Walton sea 80% in the luno and 86% in the entire mpiretory -rect; for aaunels e:posed an the S8b1 !Z. 70% was found in the lungs and 88% in the eespiratory tract. Several systems have been developed for the study of the effects of cigarette smoke on the respiratory tissues of laboratory aeimeJs. IY the results obtained from experiments using these systems are to be eatrapolated to humans. the smoking machine must simulate human smoking under reproducible conditions and the particulate phase of the smoke yerosol generared must reach the lunge of the animal. Human smoking can be simulated by a mechine. it rept+esentatave smoking conditions are choeen. These conditions are the averages of several human smoking variables that influence both the total particulate matter (TPWI ) of tobacco smoke and the individual smoke constituents. Four variables have been standardized for machine smolang: puff volume, puff frequency, puff duration, and butt length. Under standardized conditions a cigatette is puffed once per miinute. which generates a puff 35 ml in volume during a 2-sec period. The cigatette is smoked to a 23-mm butt length (Wynder and Hoffman 1967). Dosimetry studies were performed and compared for two types of smoking machines. the Walton Horizontal Smoking Machme (Waltoni and the smoke exposure machine (SEM [I). These cigarette smoke inhalation dosimetry studies are presented to document and quantitate deposition and distribution of smoke 'PPM in cercam inbred and hybrid strains of mice as well as to determine which factors influence deposition and distributior. MATEAIALS AND ARE7'HO OS Smoke Oeneretion Walton Horizontal S-noke Sxpoefere .Nacltine (The WalEon). Figuae 1. parts a and b, shows the Walton, which is manufactured by Process & Instruments Corp.. Brooklyn. N. Y. This mltchine, designated the Walton, was designed to eapose a small number of experimental animals to cigarette smoke under the conditlons of a 2-sec ptlff of 35-rnl volume once every minute. The features of this machine are

DOSIMETRY AND OISTRIBUTION OF CIGARETTE SMOKE IN MICE 179 H T® 012 0 0 8 3 1 Fig. 1(a) Walton Hasizoeml Smolung Machine. Mice aie shown rescnined in the eyiindricai tubes. completely described by Guerin et al. (1979). The Walton provides exposure to standing or static smake from a single puff that is unif'ormly mixed by a rotating fan. The animals breathe the smoke for a preselected time ranging from 5 to 45 sec. The particle size of the smoke aerosol has been measured by methylcyanoacrylate fixatioo. Data indicated that the geometric mean diameter of a 10% 2A1 cigarette sn:oke aerosol (see section on Dosimetry) increased from 0.40 to 0.63 µm during 30 sec in the exposure chamber (Holmberg, 1979). The chamber is flushed with fresh air between puffs. The smoke concentration can be varied by changing either the i

HENpV ET pL i80 HTQ0120084 Fig. 1(b) Walton exposure chamber. One side plate has been removed to sbom the inaide of exposure chamber (C8) with the ¢ooleal-shaped e:posuee ha(ea (1i) visible. Dueing an exposure the cigmetta dome (0') movee forward and automatically iattites the ea8atstte (Cll and 35 ml of pog aa is foeeed through the c(gorette into the chamber. A continuously rotating [en (F) ie required to eenue rapid and uniform mixiag of tbe tmoke. At the end of am exposure intsr.ai. ®olte ia vented through the exhaust port (E). chamber volume (between 384 ard 1150 mll or the number of ci8'are'rum (1, 2. or 3) simuitaneously smoked. For example. in using the 384-mi chamber, smoke concentrations of 10. 20, or 30°5 will be generated by simultaneously burning 1. 2, or 3 cigarettes. Total smoke exposure is represented by total exposure time; e.g.. 30 sec of smoke exposure per minute for 10 consecutive minutes results in 300 sec of total smoke exposure. Automatic Sntqke Expesw+e Machine (The SE.N I!). The SEM II machine (Process & Instruments Corp.. Brooklyn, N. Y.) and animal-containment unit are shown in Fi.gs. 2 to 4. The SEM II (described by Moneyhun. Stokely, and Ftorant, 1979) automatically and sequentially loads, lights, pufis, ejects, and extinguishes a series of 30 cigarettes. The smoking conditions chosen are: puff frequency

OOSIMETRY AND DISTRIBUTION OF CIGARETTE SMOxE oN MICE 181 , HT®0120085 c FGg 2 Aotomatie emolc.-upoeana msehia. (.4SM Q). Ci®atettee (C) ew loaded hom a hopper (HO) into a ea;stlaQ draor hotder (DH). The ItQ(tta (L) automttlq0y ipitei the cioatette. and puff.ir a iaeced thsonah each ciamnte in tuea. Fiaw is eaoW by a aaastontty hed diBeeestiti pieewie ptovMed by the pe.ttiw peasaea io the domo (DO ) b.toaae the i4nftod md.nd tbe buct sud of the aiguelte. Dilution ait (DA) is ioqoduced at the butt end of the cioar.tte. a

CH pig. 3 SEIN 11 espoaure module and atoch•type animal holder. Each doublt-eided module hse the cspacity to e=pose 60 miee at one time. Modules when on the e:posare rack ere held closed by electromagnett; (EM) (Fig. 4). An air seeal is provided by the arins (0) so that either.rnoke or air is delivered through 1he channel (C!1), The animal holders are prtwided with chin reata iCn) k i t i W N . nee spr ng c tS (-i..nd metal Foot grtde IFU). s -i . CO CII\

e l ( f DOSIMETRY AND DISTRIeUTION OF CIGAREITE SMOKE IN MICE 183 Ftg. 4 SEM II expoaure tack with eigbt expo.ure modules. Tho .yetem hs the capacity to expoae 480 mice at one time. Oyee ddt is visible Ya this photograph. (one per minute). puff duration (2 sec). average puff volume (35 ml), and butt length of cigarette (23 mm). Thirty cigarettes are loaded into holders fitted on the surface of a rotating drum (Fig. 2). The drum advances one bolder position every 2 sec, which results in all 30 cigarettes being sampled every 60 sec. Air is forced through cigarettes by a constantly held differentaal pressure between the ignited end and the butt end of the cigarette. Variable amounts of a:r for diluting smoke can be introduced at the butt end of the cigarette. thereby producing a range of smoke concentrations from 5 to 100% HT00120087

194 MENAY ET AL which can be offered to test animals. The diluted smoke flows at a predetermmed race through a progismmable distribution valve tltat sequentially directs the smo1E®. at precise time inteevaia, into one of four channels leading to the animal-containmenc units or to an exhaust system. Air is provided to the channels in the absence of smoke. Each of the four channeis has the capacity to provide smoke or air to 120 mice ac one time. Cigarette stroke is delivered to the test animals within 2 sec after generation. The particle size has been determined by mechylcyanoacrylate fiaation and was found to be log8ormallydistributed with a geometiic mean diameter of 0.34 µm (Holmberg. 1979). In the scudias presented here, two of the four cbannels were used. Smoke was provided to one channel for 30 sec while air was provided to the second channeL Ac the end of 30 sec, the smoke and atr were reversed by the distribution valve. Smoke was vented to an eshaust system for exposures of less than 30 see. Total sffioke- eaposurs time was 300 sec (30 sec of smoke esposune per minute for 10 consecutive mstturxs). Aniraola Male and female BC3F1/Cum. 86C3F11Cum. C5'TBIl6 Cum. and C3H/Anf CUat mice were purchased from Cumberland.Yfew Farma (Clinton. Tenn.) at 4 to 6 weeks of age. Male and female DBAl2J mice, 4 to 6 weeks old, were purchased from The Jackson laboratory (Bar Harbor, Vlaine). They were housed 5 animals/cage (stainlesa- steel cages equipped with plastic fivnts and filter bonnera) on corncob bedding (Bed-O-Cob, Chesapeake Feed Co.. Bedtsville, Md.) and were aL'owed Iee access to Purina Lab Chow and water. B,acks containing the animal esgeP were kept in a room at 70 to•T4°F with a cycle of 12 hr light from fluorescent lights and 12 hr dark. The animals wQre initially exposed to 120 sec of 10% 2A1 Kentucky reference cigaretre smoke (see section on Dosimetry) twice a day with a 10-min rest beta--r 4sp_m*es. Eacn day both sessions were increased by 30 sec each until a total exposure of 600 seclday was achieved. Mice were exposed daily for 600 sec for at least 8 to 14 weeirs befora any dosimetry studies were sciteduled. Anhoel Notder3 or Re»atnts In the Walton machine, mice are held in cylindricsl tubes that aliga the heads of the animals wrth the conical-shaped openirgs on the etpoaure chamber [Fig. 1(b) ). The nose of each animal protrndes into the chamber [FiQ. 'ta)) . Twenty mice. 12 hatnsters, Hr20120erc-8 f

~ - --- -- --- - - •, _... .. . .. _ _-- ~-- -__. --_----------~-- - 00SIM@TKr aNo o1S'rA11BUTION OP CIGI+aeTTB SMOKE IN MICE +e6 Hf19Q 12 0 0 8 9 l or 6 rats can be exposed to smoke simultaneously by using this resteasat system. The animal contamment system for the SEM a is shown in Figs. 3 and 4. Aauaals are held in a stock-type nolder (Fig. 3) with a neck slot and restraining spring. A chin rest ensures that the nose of the animal is aligaed with the conical-shaped opening of the exposure module (Fig. 3). The nose of each animal peotrudes through a dental rubber dam diaphragm (Proc®su & Instrunents Corp.. Brooltlyn. Y. Y. ) which forms a seat to prevent body eaposure. When adequate air and/or smoke is provided to the exposure mcdule, restraint twice daily for up to 3 hr results in no mortality and no obvious ill effects to the animals. 0a9matry Kentucky reference 2A1 cigaretees ( University . of Kentucky, Lesangton, Ky.) were used tlsroughout these studies. Each 2A1 agatette detlvered 40 m8 TP'ar1, 0.5 mg mcotme. and 7.7.3 cros carbon monomde (Guerm et al.. 1979). Cigerettes were selected for use on the basis of weight (L12 - 0.10 g) and resistance to draw (85 s 5 mm H;O). Cigaeett®s arete labeled with [" C]dotriacontane ([' ° C] DTC, elmerican Radioclwmicel Corp., Tanfotd. Fla.L either at Oak Ridge National Iaborarory IIORNL) or at Microbiological ?ssociates (MA) by previously published procedures iCaton, 1979). For the Walton studies the 2A1 cigarettes contained 1 to 2 µCi of [`° C] DTC per ci5,arette, whereas those for the SEM II studies contauied 0.25 to 0.5 mCi of [' + C] DTC per cigarette. For dosimetry studies uaing ;he Walton, three nmdioiabeled cigarettes were used for each exposure condition. The E7rst and third cigarettes were butaed in the smoking machine equipped with a Cambridge filter (Cambridge Filter Corp., Syracuse, v. Y. ) ta collect the generated TPM. The filCers were weighed immediately after exposure. The two Cambridge filters were placed in 20 ml of pyndine, the TPM was eluted, end an aliquot was talsen for deiermmation of radioactivuy and for analysis of nicotine content. The mesaured specific activity for the radiolabeled smoioe generated on the Walton was 50 to 100 dpm (disintagration$ per minute) [" C] DTC per microgiam of TPM. The second cigarette was burned to expose the animels to radiolabeled smoke. As a check of the reproducibdity of the animal exposure from the labeled cigarenes, a sample• of smoke was withdrawn duiing each puff (20 ml total). This was collected anto a Caabndge Blter, the TPM was elutqd with pyr[dine. and the amounts of radioactivity and nicotine aaerR L

:86 MNAr $T AL , determined. In each instance the TPM and nicotine values deter• mined in these p;ab samples were within 10% of the expected values for 241 refererrce cigatettes. For dosimetry studies on the SEbI U. the smoke TPM was determined with an optical scattering sensor 1 H'iggins. Gayle. and Stokely. 1918; Jenkins and Gayle, this volumel placed in the smoke line immediately preceding the animals. Total radioactivity associ- ated with the TP!4[ was determined by withdrawing known volumes of smoke from the sampling port built into the optical sensor. collecting the smoke particulates onto a Cambridge filter pad. eluting the TPM with pyt>diae. and counting the pyridine sample. The countang employed a liquid scintillation spectrometer which used simultaneous internal standatds to correct for quenching and counting efficiency. The measured disintegrations per minute per TPM specittc activity for the radioiabeled smoke generated on the S&lrt II was 15 to 25 dpm of ["C] DTC per micrflgram of TPM. , ?,fter 8 to 14 weeks of adaptation to 2A1 cigarette smoke, the deposition of TPM in the animals was determined. From the.:. [' ° CIDTC•labeled cigarette, mice were exposed to 2A1 cigarette smoke and then were sacrificed by csrbon dioxide asphyxiation. Selected tissues were removed, trimmed, blotted to remove escess blood and fluids. and frozen at -20°C. All tissue samples were coded at MA and shipped to ORNL at =20°C for radioactivity determina- tion. Tissues were thawed and solubilized in an alcoholic potassium hydroxide solution, as described previously by Caton (19791. Tissue samples from mice exposed to nonradiolabeled smoke were used to determine background values. Background values varied among the varfous tissues and averaged 165 dptn for lungs. 155 dpm for larynx. 83 dpm for head, 113 dpm for stomach. 60 dpm for hide, 55 dpnt for liver. and 88 dpm for remaining internal tissues. Because bacKgmunds wera var.able. deposition was considered not signi$cant unless tissues were found to have radioactivity levels three times above bacitgeound and a dose-dependent increase in radioactivity was observed. The only tissues that met these criteria were rhe head. the larqnx and upper tracheaL the luags and lorver trachea. and the stomach and esophagus. The measruements on the larynx and upper ttachea had the highest inberent variation after exposure to either radiolabeled or nonradiolabeled smoke. Tt>js unreproducibuity may be a ftmction of the solubilization ptocedure. The ["C] DTC measurements on the luugs were at least ton times above background. all radioactive samples were corrected for background and • for quenchin;f and counting efficiency, and data are presented as micrograms of TPM deposited per tissue. H IV, 01 200, 50