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! Preprint ~T®012005E December 198 Accepted in Toxicology and Applied Pharmacology C Depositiot and Distributioa oY the Total Particulate tSstaer of Cigarette Saaice in Hice Osing a Large Capacity Smdse Esposare Systeml Rusoi,ag Title: Cigarette Smoka Dosimatry ia Mice C. J. Bearyao5, J. ::. Caton3, J. $. Stokaly3,. 3. R. CaeriXL3, e. LapesZ, •L'. D. &very2, D. 8. Dansie2, G. 11. Sea3ersoa3, T. vayle3, C. E. Wb,itmire2•4, and R. E. b,ouri2 - f 1. f'reseated ia part at the 18th Aaanal aeetiag of the Society of Tosicology, :TSw Orleans, Louisiana, aarch 11.-15, 1979. 2. Department of Fxperimeatal Oncology and Department of 8iochemical Oncology, aicrobialogical Aesociates, 5221 River Road, Hethesda, 1larylaod 20016. 3. The Aaalyticat Chemistry Division, Oak Ridge Nettonal Laboratory, Oak RidBe, Tennessee 37830. ~ 4. Preseat Address: ;Iationsl Cancer Iaetituta/fttiooal Tosacology Program, Sethesda, c;arylaad 20205. 5. To wham correspondenta sbould be aeat. l

HTe0120057 "sTSacT Deposition and Distribution of the Total Particulate Matter of Cigarette Smoke in Mice tJsing a Larga Capacity Smoka ' Eaposure Systea Henry, C. J., Caton, J. E., Stokol.y, J. R., Guerin, M. a., i.4pea, A., Avery, M. D., Dansie, D. R., Benderson, G. M., Gayle, T., Whicmire, C. 8., aad Rouri, 8. E. (1981) Tosicol. Appl. Pbarmaaol. 00, 00-00. I A nevly developed autoaatic Smoke Eaposare Machine (SEM II) ees ased to generate 14C-datriacontane labeled tJniversity of EemtuCky reference 2AL or ZR1 cigarette smoke. The SEM II is a large capacity (480 mice) dyaamic smoke eaposdre systen -ia which smoka is routed throngh the anjagL contaiament system as a contimioualy flowing stream. Mice ure restrained about the neck in stocklike b,olders for °nose-oaly" exposure. Using standard amo[ce .ocposure conditions, the deposit3ou and iatesaal deposition of thm total particulate matter (TPZi) frcm cigarette smoke vas determinad in SC3F1lCum male and fematm mice. Results show: a) smoke exposure conditions can be varied so that deposition from 30 ug to 200 µg TIW]amg caa be obtained, b) 80-90X of the TP!i deposition was found in the re9piiatory tissues, c) the monse-to-mouse variation for TFM depogitioa in pulmoti;ary tisssue was ti20x, d) similar deposition and distribution of T8PH was observed in sale and f®ale mice, and e) depoeition and distribution iF TPM was not altered in mica exposed to smoke oa a daily basis over a 6 month period of time.

a- - HT®0120058 1 UrTAODUCTION There are two major objectives when studying the effects of cigarette mmoke in esperimental animal systems: a) the s+aoking machine ssast stimu- late bs-- making conditions and these conditions must be easily ra- yroduced on a daily basis, and b) the particulate phas® of the seoke aerosol generated must reach the Langs of the an3mald. Every lamma amoicer has aa almost unique puffing pattern, within which there may be consider- able Eluctuat.ioa (Wynder and goffman, 1967). Although no s®oUeing device can duplicate h-a smolci.ng habits, machines can approsimate human making by usiag standard smoking coaditions which are the average values obtained from a large ottatber of saohsrs. Four aariables which influence the yield of total tobacco s®oke, total partiauiate matter (TP:!), and iadividael smoke constituents, have besa staadardized for machine smoiciag: puff voLrme, puff frequency, puff duration, and butt length (Wynder and Hoffman, 1967). ® standardized making regamen'tsas'been established where the cigarette is puffed onee per minute generating a 35 ml puff during a two second period. ihe cigarette is saoked to a 23 = butt length. In the present studies, SC3F1/Cuu (C57B1/Cue x C3S/Aaf CuR) =ls and fe'ma7.m mice eere eaposed to whole cigarette smoke %sing this standard smoking regtaAen wxth a nev Smoke Esposure Machine (SFId II).'The S}2t II is an automatic smoking system, designed to generate and deliver a con- tirnsoue strum of fresh snoke for large scale inbalation studies. Ic is a dyaemic systan in that the aai.4als do not rebreath the s®oke, In contrast to a static systen where animals breaths into a chamber. The SSti II uses revarse making (positive puff) and automatieally loads, lights, puffs, and distributes the smoke from as maay as 30 cigarettes to special holders where "nose on.ly'r exposure is effected. A total of 480 miee can be eapoaed to smake uVing various ®aposnre regimens with the SIIK II anSmal contaicaent system. The distribution r,aa dosimetry of whole cigarette smoke was deter- m3aed using this SIIi II system and the results are presented here. LiSTHODS Smoke Generation The,SF2f 11 (Process and Inetruments Corporation, Brooklyn, .i) shoM.n in Figure i, operates in a maaner simulating bmaa making and automatically and sequentially loeds, lights, puffs, ejects, and eatinguishes a series of -1-

NT®0120059 ( 30 cigarettes. The operating principles of this machine are presented else- where (Moaeybua,,a~t al, 1979). The staadard smoking conditions employad ars: puff fsraqueacy (oaeaminuta), puff duration (2 seconds), average puff volume (35 ml) and cigarette butt Length (23 amt average). These conditions are maintained completely independent of the aaimel contadment systea. Thirty cigarettes are losrled from a hopper into holders fitted oa the srsr- face of a rotating drum. The drum advances one holdes position every Z• seconds resulting in all 30 cigarettes being sampled every 60 secoads. Puff air is forced through the cigarstte being sampled by a constantly held d3fferential pressure betueea r.ha lit end and the butt end of the cigarette. 7ariable amcuats of dilatioa air can be introduced at the butt and of the cigarette. This a]loros a range of smoke concentrations from 5-100Z (v/v) which can be offared to the test aaima2s. The diluted snake flows at a predetermined rate through a programeabla distribution valve which a+eqnen- tially directs the smoke, at precise time iatervals, into oae of four .hsanals leading to the aaimal contaioment.onits, or to an esbiust system. A diagran of the smoke generation system and aaLm,l containment unit is presented in Figure 2. Smoke can be alteraated with.breathing air for various intervals within a one minute cptle. dir is provided to all a!an*+pla ia the absence of s9aoke. Fach chanaeal hsa the capacity to provide smoke or, air to L20 mice at one time. Cigarette smoke is delivered to the test animals within 2 seconds after generation at the cigarette. Preyious studies have shosra that smoke particle siae was (as determiaed by methylcyaaoacrylate fixation) log normally distributed with a geometric mean diameter of 0.34 microns and geometric standard deviation of 1.35 microns (Holmbarg. 1979). In the studies presented here, two of the four chaemels were used. Smoka was provided to aae clauaa®l for a set amoumt of tima (e.g., 30 secomds) while air aas ptovided to the sacosd•chamael. At these predetermined t3me intervals, the distributiov va.lve was aucomatieaLl.y activated so tbac the first channel received air whila the second chaanel received smoke. ftoks exposure is presented in terma of total smoke eaposure tlme, e.g., 30 setoads smoke eapoeare per minute, alte=aated with 30 seconds of air par miauta, for 10 consecutive minutes resulted in 300 seconds of total smoke ( euposura.

H r®o120060 I ( Smoke moIIitoriIIg A smoke monitoring systam was developed for the SFM iI. Tha monitor provided• coatiauous measurement of smoka particulate phase ('rpri) and smoke gas phase carbon moaozjde (CO) i;mmadiatel.p up stresm from the aa3male (see Figure :). Smoke particulates were measured with an optical-type scattering detector (Siggias st a1., 1978), while a.noa-dispersive infrared aoalyzar. (Beci®an Model 864-Source Infrared Aealyzer, Fullerton, CA) was eatployed for CO determiaaticns (Si,ggias at al., 1978, Gayle ae al., 1979). Each exposure was doewaeated on a•Speedamaag M 2lark II Recorder (Leeds and Northrap, ttorth hiales, FA) which provided a permaaent recor.d of the puff by puff profile and fita]l ictegrsted values for both 1'F!i and CO. The detector signals vere e processed to interface with 'approprixte saf ety appara- tus to protect the aaima.ts from accidental over exposure. - The integrated sigaa.t was coaverted to mg TPM by using a correlation factor derived by simultaaeously. cpmparing this sigual to lmoan weights of ~ '=PM which were col.lected oa Cambry.dge filters (Caabridge Filter Corporation, Syracuse, NY, see Gayle at al., 1979, for details). For smoke concentrations between 5% and 20g, the sensor readout oas linear with respect to TPM. The concentration of a 10Z smoba' aerosol was approximatelp 9-10 {ig I3Wml (v/v). Male and female BC:3F1/Cum mice (C5781 x C3H/Aaf - Cumber].and Vi®w Farma, Clinton, TiQ) were purchased at 4-6 weak9 of ag€, housed S mice per cage (str.ialass steel cages equipped with plastic fronts and filter bonnets) oa Bed-0-Cob coYa cob bedding (Chesapeake Feed Company, Beltsvil7.e, !ID) and allowed free access to Pnscina Lab Chow and water from an autamatic watering systea. 8aclc9 containing the animal cages v®re kept in a room at 70-740r with a light cycle of 12 • hours darimess and 12 lsours light from fluoreseent lights. dica were initially esposed •to 80 seconds of 10Z 281 or 120 seconds of l0x 2A1. Xwtwcky reference cigarette smoke, twice per day with a taa mimata rest between exposures (see Doaimatry). Eaposnre was inr.zeased rnily by 20 or 30 seconds of amnke•per exposure until a total of 360 or 600 seconds per day was achieved for ZR1 ar 7A]I smoke, respectively. Nice were exposed on a daily basis (5 days/yeek) to this Level of emoke natil dosiaecry studies were performed. .

H T®0120061 ( i ansmal Hoiders The an.ima1 conta3ament system for smoise esposure Ss showa in Figure 3. Groups of f ive aaimals are held Ln a "stock like" holder using a co®biaation of a neck slot and a restra~A~ spring. The chin rest insures that the nose of the anima3l is aligaed with the cone shaped openin8 on the eaposure modules (Fig,use 2). The mesa of each saimal passes through a deatal rubber dam d3aphragm (Process and Instrsmtents Corporation, Brooklyn, NY), Foraang a seal that preroats eaposure of the body to the smoke aerosoL. Bubber dsm diaphragms must be changed frequently (at least monthly) to ma+n*9tn the seal around the nosa. Daily restraint for up to three hours (twice par day) resulted in mortality and mo obvious ill effects to the anIma].s. Cigarettes Kentucky refereoca 2A1 or 281 cigarettes (University of Santneky, Leu3agton, SY) wore used in these studies. These cigarettes have beea characterized at Oak Ridge N,atioaal.Laboratory using fhe Phipps and Bird Analy- tical Smotcing Machine. tTnder these analytical conditions, 2A1 cigarettes delivered 38.9 t 1.9 mWTI+M, 0.54 t 0.04 mg nicotia, 19.0 - 0.6 mI. C0, (yoneytum et al., 1979) and 2I4 cigarettes delivered 45.+ = 1.9 mg T!!i, 2.10 - 0.13 mg nicotine and 21.6 s 2.0 ml Co. Cigarettes were preselected on the basis of weight (1. 7.Z t 0.10 g) and resistance to drav (80 = 5= IL20), measured with a Filtroua Pressure-i?rop Tester (CSgarette Components L.imitmd, ttembley, England). Gigarattes were labeled with 0.Z5 - 0.5 uCi 14C-dozria- coatane (14C-DTC, 3merican 8adiochemical Corporation, Teaford, FL) as described ,previonsly (Cacon, 1979). Dosimetry 8ine esperimental measurements 4ra required for each eigarette smake doshoatr3+ study. These are siaaoarized in Table 1. The smoks TPti aae - datecm3aed using the opt3cal seasor (see Smoke Monitoring) place directly in the stooke Line lmmediataiy prsceeding the +`aioals, iass than 0420 csn fram the eaposure module. Total radioactivity was determined.by withdrawing tmofan volumes of smoice from the sampa.ing port (see Figure 2), col.'.ecting this sample oan Camyridge filters, elatir.g these,seoke particn]ates with pytidiae, and caunt3ag the pyridine solution by liquid scintillation speacro+s.etry simo.l.taneously with interna]l standards to correct for quenching and counting effienGy. The specif ic acxivity for the 14C-DTC radiolabeled

snoke was 15-25 disiasegrations per minute (dpm) /ug TPM. Ia addit+i0dq %!!e~ 00 62 smoke delivered to che aalmals was further moo.itored by withdrawiag samples of smoke from the first and Iast an3mal, eaposnre port in the aniesl costaia- mear, unit. These mpnsuremeat9 were asad to qua].itatively check the r.adio- labeled snoice bafore and after esposure•of the anima]s. Differences of lose than 5% of L4C-DTC content saera observed between those two points. Tha amouat of TP".i res,oved by the animels in Chese eaposuras was less than C 4X of the total generated. Mice were exposed co smoke for various tiae periods ranging from 3 weeks to 6 months to allow for adaptioa to esposure to cigarette smoke. For the dosimatry studies the fo]lowing protocol was used. Mice were espoaed to either 180 seconds of lOT 2IY1 onlabeled cigaretta smoke using an eaposara cycle which alteraated 20 seconds of smoke with 40 seconds of air for l0 consecutive minutes, or 300 seconds of 1OZ 2A1 unlabeled cigaritte smoise using an exposure cycle which alteraated 30 seconds of smoke with 30 seconds of air for 10 consecutive miautes.' After a 10 minute.rest, mice were then exposed to selected esposure conditions utilizing 14C--DTC:-= of 14C-=-2Al cigarette smoke. Imeediately after ssak,e exposure, mica were killed by C02 asphyaiation and selected tissues were removed, triaed, blotted to remova excess blood and fluids and stored at -60oC until aaalyred for total radioac,ivity. The following tissues were selected: head, larynx and upper trachea, lungs and lover trachea, esophagus and stomach, liver, hides and a eomaosite sample of all remaining tissues. Lung and lower trachea ci.!sue samples were weighed before storage. All tissue samples were coded at Microbiological Associates, packed in dry ice and shipped to Oak Atdge Nat.ional Laboratory for detera.iaatioa . of radioactivitq. Tissues rere qutcklT thawed and solubiliaed in an alcoholic potassiom hydroside solutioa as described previously (Ceton, 1979). Tissue samples fro® mice exposed. to noa-radiolabeled smoke were used to determine background values. Background values varied among the different t.Asaues and averaged 165 dpa for lung and lower trachea, 155 dpe larynx end upper rrachee, 82 dpm for head, 13.2 dpm for stomach and esophagus, 60 dpa for hides, 55 dpm for lAver, and. 88 dpm for the remain.tag tissues. Because of background variabi].ity, deposition was not considered sigaifi- cant imless test tissucs ware found to have values three times above 'i3ack- ground. The only tissue sample which met these criterala were the head, 3aryoa and upper trachea, lung and lower trachea, and stomach and esophagus. -5-

H T10120()63 . l All radioactive samples were corrected for background, quenching, and counting efficiency, and final data are preaented in terms of ug TPM deposited per total tissue. The affect of total s®oke exposure time oa the deposition of TPl9 in BCiFl/aam mice is shown in Tables 2 and 3: Mice were exposed to 10Z (q/v) 2R1 cigarette smoka for a. totaL of 180, 360, or 540 seconds• (Table 2) or to 20Z (v/v) 2A1 cigarette smoke for a to.tal of 50, 100, 150 or 200 seconds (Table 3). Depositioa increased in a].L tissues arLh increasing; exposure times for both smoke concentrations. L tjme dependent linear increase is evident from Tablee 2 and 3 for deposition ia the long and iower trachea, the total respiratory tract and the total body. Correla- tion coefficients (r) of 0.99 were calculated for the deposition in thaa Lung and lower trachea as a function of total smoke exposure time for both 281 and 2A1 cigarette smoke. the amount'o! radioactivity detected is the lung sample was always 5 to 10 times above background. No other tissue samples reflected this amovat of radioactivity, however, the head, laryna and upper trachea, and esophagus and stomach were generally 2 to 3 times above thetr respective backgrounds. The liver and composite remains semples were not above background levels and eere not considered in furtber eaperiments. TPM deposition on the hides of the animals was found to be a functiaa of the seal around the nose provided by the rubber dam diaphragms. Changing these diapbrages routinely resulted in TPM deposition of less tbatt 25X of the total internal tissue deposition (data not shown). The intesaaimal variation in TPM deposition in the lung and looser trachea vss found to be dependent upon exposure time. The coeff icieat of veriation (cv) for deposition in the lung and iover trachea from 13 nice per group decreased from 0.30 to 0.14 for 10Z smoka when exposure time vas increased froq 180 to 540 seconds. For 20S amoke, the cv's for '0 mice par group decreased from 0.67 tc, 0.21 when uposure '.ime vas in- creased from 50 to 200. The deposition in eatrapulmanary tissues was more variable and these variations were not dependent upon exposure tjae. Oeposi.tion in the laryns and upper trachea was the most variable, as reflected by cv's ranging from 0.16 to 0.80 (see Tables 2 amd 3). At a 10$ smolse concentration, equivalent deposition of ZPX from 2Aa1 or 2Rl cigarette moke was observed. For a range of exposure tiaea, -6-

I ( HTQ0120064 the rate of deposition of TPH in lung and Lower trachea was 0.45 ug/sec for ZLL cigarette srake and 0.43 ug TFlVsec from 2RL cigarette smotca. As can be dsearmined fram data in Table 3, the rate of depositim for 20% U1 smoke, i.e., 0.9 u6 TP"_s/ssc, is double that for I0X 2Al smoke. Compsrisoe of 10% and 20% 231 cigarette amolce could not be made because of eoaieity to the anioals of 20X 2R1 saoke. The SEi tI is a dyoamic smoke exposure machiae which generates smolse in a flowing stresm to the animals. The snoke flow rate to the aaimels is 5.0 Iiters/miautPs far = saoks concentration and 2.5 Litprslminute for 20X smoLce concentration. Dosimetry studies were p®rgormed to determine whether TPH deposition was altered by a change In flow rate. 8C3F1/(am fema7.e mica mace esposed to 10Z 2A.L cigarette smols.a for 300 seconds total eaposnrs time, with smoke flow rates of 1.0, 2.5 or 5.0 liters/minuts. The smoks scream was mechsnically altered to provide the 1.0 and 2.5 liter/ aiiaate flow rates. Palmouary TPH deposition was 123, 142 and 123 ug TPH for smoke flow rates of 1.0, 2.5 x* 5.0 liters/minutd, rsspectively. No change in deposition was observed when the flow rates were changed. The distribation of TPH within the,mouse was analyzed in all o: these esperiments. As prqsented in Table 2 far 10X 2R1 cigarette smake, the percent of total deposition of TPH in lung tissue was over 802 for the three exposure timea evaluated, with an average of 882 found in the total respiratory tract. The esophagus and stomach contained the rema-detectable radioactivity. Data from Table 3 for 20X TAL cigarette staake, demonstrated that the percent TPTI foand Sn lung tissue was 60, 78, 80 and 81Z for 50, 100, 150 and 200 seconds aaposure tiee, respectively. For thess same erposare times, the percent TPH found in the total respiratory tract was 83, 89, 81 and 944, respectively. As seen in dal:a from Table 2,' the rema ins radioactivity was in the stomach and esopbagtss. The effect of previous ®aposure to smoke on '!PM deposition and dis- tribution was determined in three groups of SC3F1/Cum female mics and compsred to one group of BC31!'l/Ciaa male mice. One group of female mice and the male group were esposed to tan-T0 minute ~asposnr®s per day (5 days/ week) for 6 manths. A second group of fe9ma].e mice was exposed to ten-10 minute suposusrea per day for 3 moncbs, and the third group of female mice ums espoacd to ewo-10'miasite exposures per day for 3 veeks. The female mica from these three groups were exposed simultaaeously during % doaSmetry eaperiment utilizing 102 ?A]l cigarette smoke for 300 seconds total eaposare

i / time. The ®a].e nice were exposed to che same eaposur.e conditions "MS~' 2 a u 6 5 a different day. As shown in Table 4, TPM depo9itioa vas observed tobUe equivalent for ell four gsoups, with no sigaifi.caat differences observed between any of the groups for deposiLSoa in th® luag and lover trachea. Thus the average daposit ian in luog and Lower trachea f or 40 fema].a mico was 145 pg TPH (average cv • 0.15), 167 pg TpK for the total respiratory tract, and 183 Ng for the total body, compared to deposition ia 30 male mics of 1&6 ug TPM (a+v ° 0.26) in lung and lower traehea, 170 {ag VN in the total respiratory tract, and 198 µg TPM for the total body. No sigaS.ficant differewe in TPM distributiaa was found between m®le and female asice. The percent distritwtioet vas detsrstiaed from aI]. TO nice. Bn average of 78% of the TPM was found in the luog and loseer trachea and 93Z was fosmd in the total respiratory tract. These distributioes vere qaita reproducible with cv's of 0.05 and 0.03 for lung and low®r trachea and tota]l respiratory tract, respectively.

r i ( DISCQSSION HT00120066 The SFH II described here was designed for iahalation esperiments of long duration and large-scale, both in tarms of numbers of exposures and numbers of mica exposed to smoka. The SEK II (Figure 1) is completely auto®atic and is equipped with monitoriag devices to document exposure levels and aLso provide safety far aecidental toxic ezposwres or for aachine malfunctions. The amount of smoke and the exposure times can be prer.isely varied, while maintaining standard smoke eaposure conditions. Ovar the last two years, the SEK II has proven to be a re®artRably trouble- free precision instrument, used 6 to 8 hours per day, 5 days peti week. While reliability and quantitation in saoke generation are prerequisites for chronic inhalation studies, an equally import4at prerequisite is the capacity to expose large msmbers of animals at one time. Animal holding trays which caa be rapidly Loaded and unloaded because the mics are re- strained only around the neck (Figure 2), allow the full capacity of the SEK II to be atilised. In this m~ner, 480 mice esa be "nose only" exposed to s®oke under the same esposure.conditions (Figure 3). The key elements which were measured in these experiments and are necessary for effectively quantitating the Level of smoke exposure using the SEK II are presented in Table 1. The most important paramaters are the continuous monitoring of smoke during exposure and the continuous sampling of the TPti onto a Cambridge filter. The simultaneous measurement of both parameters greatly decreases sampling error and assures the repro- ducible conversion of the optical migaal to TFM. The only problam that should be kept in mind is that th® sampling time must be sufficiently long so that adequate levels of radioactivity are counted. The qnantitation of TFM in various tissues of the exposed mice is probably Influenced by the method of.solubiliaation. Tissues such as the larynx and loover trachea were observ«ad to have relatively high background radioactivity (ti155 dpm) and the large variabilities observed among the differeet mice (intra-asaay cv's from 0.16 to 0.80) seem to reflect these high background couats. tu lung tissues, however, deposition of TPi was quite reproducible. For similar exposure conditions repeated savea times, an average ev of 0.20 was found (intra-assay variation). Over an 18 month period, the cv of the means from seven experiments was 0.08 (intza- assay variation). Over 2500 tissues wore collected from 500 mice in these stud,Ses and the subsequent manipulations of these data required

i l H TE0120067 computerized data handling (+Catoa, 1979). ZPPt deposition was quite dependent upon total exposure tfine and smoU.e concentration (Tables 2 and 3) and TPM dose was additive for multiple exposures (Table 2). The rate of deposition in pulTnonary tissues Lncreased two fold as the amoke concentration was doubled. Deposition was simil,iar in male and fema].e mice (Table 4) and over a 6 monch tims period, mice did not Ieara to avoid the smoke (xable 4). Predictioe.s of dose for smoke iubSJ.ation studies based upon aerosol concentration, exposure times and frequency of exposure can be made from these dosimetry experiments. Variabilit-7 in deposition in earapulmoaary tissues (e.g., b,ead, laryax, s+tomach) maay result from the low Lc+rvels of radioactivity in these tissues relative to background. • Eaposure conditions described here demoastrated that ti70-80x of the P.PIi was found in the Lung and lower trachea, 't-80-90X of the TPM in the total respiratory tract and ti10X in the esophagus and stomach. Shorter ezposure times (i.e., 5 seconds af .smolce/minute for 10 minutes, Table 3) resulted in lower percentage deposition in pulmonary tissue, suggesting that the mice could possibly influence deposition at these short esposare tim$s, perhaps by altering their breathing patterns (Keadrich, et a~,l 1978). It should be pointed out, however, that short exposure times result in less total deposition and, therefore, the sampling error for detecting radio- activity is greater. Depositi.on data ean also be presented in terms of TPM depositioa pes given amount of tissue. Using data in Table 4 for BC3F7./Cum female mice exposed for 3, months, ].ung deposition can be calculated to be 0.63 Itg 'IPFUmg Lung weight and 6.2 µg TPWg body veight, with deposition ia the total res- piratory tract of 7.1 {tg TPM/g body weight. For BC3Fi/Cam male nice exposed for 3 months, the values are 0.65 Ng• •IPi/mg lung weight, 6.0 1ig TPWg body weight for lung deposition and 6.2 ug TAM/g body weight :or deposition in the total respiratory tract. These data for deposition and distribution of TPli from smoke generated and delivered 3n the SIIi YI system agree siell with dosimetrp rmsults obtaimed with mice exposed to smoke using the Walton 8orizontal Smoking :lach.iLne, a Maller capacity, static exposure machiae, (g®szrq, at al, 1980). Mice asposed to moke aerosol concuatrations of lOx or 202 for exposure times of 100 to 300 seconds on the Walton or the SEM 11 de$onstrated similar emount of TP'9 deposition, distribution aad rate of deposition of TPM in pulmoa.ary -10-

® , ! tissue. other reports for dosimetry studies in mice indicate TPlfl &§Q4:QPQ 0 6 8 of 30Z (Leax.a, er a1P 1973) to 60x (Page, at a_,,1 1973; Biaas, et, AL, 1976) ia the Lower respiratory tract usiag 14C-hexadaeaae or decacblurobipbenyl labeled smoke, respectively. Dapositi.on data for mice expoaed to cigarette smoke caa be compared to data for other species, including b.amaas. Data for humans are frequently given in texms of _ummilative '!T"M deposition after expoeure to 20 cigarettes. Hakiag such a comparison from the avai,iable astimatas (Binas, 1977), the as=t of 'CPi"t deposition in mice under exposure conditions of 10% smoke concentration and 300 seconds total smok® esposure was found to be approai- ms,tely eqa:Lvalent to TPM deposition in a huesn vho has smoked 20 cigarettes (1 paclslday smoksr). This estlms,ta Qas based oa the following ass®ptions (after Binns, 1977): a) body weight of the subject is 70 kg; b) subject smokes 20 cigarettes, each arith a delivery of 37.6 ag/cigaratta; c) 70Z of the inhaled TPM is retained, and of this portion, 10Z ia retained ia the mouth of the smoker. That is, dejosition of '%,6.8 ag YPlS/kg body weight or ~%09e ug ZPH/g lvag weight has been estlmated for hueans smoking 20 cigarettes, while the studies presented here ia mica abom that deposition of '%6-7 mg '1TM/kg body weight or 'v640 ug TPrS/g luag veight is foand in mica following esposure to seoke from the equivalent a.f one cigaratte (Table 4). These studies and others carried out in our laboratory using the Waltos 8ori.zontal Smoise Exposure Machiae (Heary, a al, 1980) have demonstrated the suitability of the mouse as an aaima1 modal for inha]ation studies. Results wil] be comm^n{^=ted elsevhere regarding carboxyhamoglobia levels in miee after exposure to smo&e (Panay, It al, 1981), as wall as the deposition and clearance of other smolce constituents (Renry, et al, L981). Results presented b.ere detonstrate that using the Sffi II, matsei eapoaw:-e reg3 mans can be daqeloped so the tosic effects of cigarette smoke can,be reduced and survival kept high (no mico died during any of the doeimetry aaperiments). Ia fact, recent studies have shown that at least ten eaposures per day of 300 seconda eace to 2A]. cigarette amoks (10T, v/v) can be given.to BC3P1/CLn mica for at least one year with less tban lOx death oheerved coBOpared to sbsm egpoaed aajma].a. -Li-

f - - . _ .- - - ~ .- - - - - , ~ -T - - - -- - I ac~o~c~rrs HT90120069 Y'ho authors grat®fv1ly acnowiedge the 9apport of 'tm Coaacil Eor Tobaceo bteseare3s-FJ.S.A., Iac. We alsa wisti to GbSak Mrs. Patricia 8whia, Mrs. ?racia argaeblo, and Ms. tsary Zacic for preparatioa ol the mWusrsipt. 11 l

.~ TABLE 1 SU!@IARY OF NINE EXPERIMENTAL MEASUREMENTS RF.QUIRSD BOa CICARBTTE SHDKB QOSIMBTRY STUDIES Qeantitation of the Smoke Generated *lumber Description_ Source of Measurement 1. 2. 3. 4. TP11/cigarette . . . . . . . . . . . . . . Dpw,/cigarette . . . . . . . . . . . . . . TPK/dps ratio . . . . . . . . . . . . . Total amount of TPl1 generated ....... during an exposure . Optical monitor during exposure . Cambridge filter sampled continuously during an exposure . Calculated from optical monitor data end the Cambridge filter data Optical monitor 4uring exposure Quantitation of Deposition in Animal Source or Method 5. Dpm/tissue . : . . . . . . . . . . . . . . Each tissue from smoke exposed animals aolubiiized and radioactive content determined 6. Background dpm/tienue . . . . . . . . . . .,Tissues from control animals solubilized and radioactive content determined 7. Tplf/timaua . . ... . . . . . . . . . . . . Calculated from TPN/dpm ratio, , dpm/tieaue and background dpm/t}asue a. Percent of total deposition ........ Calculated from TPM/tiesue s ln each tiesue ~ ® 9. Percent of total amount of TPN ...... Calculated from the total amount of TPH 0 ta:en up in each tissue and in generated during an e:posure ~ each animal and the TPH/tiaeue N3 C7 U V O

0 t H 11012007' EFFECT OF TO'tAL SZlORE EXPOSURE TIlM ON DF.POSITION OF TOTAL PAILTICLILATE MA1TE8 IN 8C3F11 CDM FMIALH MIC6 AFM EKPOSIIBE TO 10$ 2RL CIGABEZTE SMOIE Deaosition of TPM (uR) Larynx Esophagus Total Smolsa osure (sec) Lang and Lover Trachea sad Oooer Trachea • Bead TR1& and Stomach Total 180b 83(.30)e L(.16) 7(.21) 92 11(.a7) 102 360c 143(.25) 4(.75) 10(.26) 157 22(.26) 180 54Od 239(.14) 2(.k0a 13(.l8) Z54 3S(.21) 289 a. Total respiratory tract. b. Esposstre condi.tsoas 2oasisted of aa exposure to 20 seconds of 1Gi smolc®~ followed by 40 seconds of air each a:innte for 9 consecutive miautes, resulting in 180 seconds total smoka e$posure. Fifteen mice per gr.oap, 25-27 weeks of age9 were used. c. As in foataota b, after which mice were givea air for 10 minutes and the smoke eupoeure described in foutaote b vas repeated, resultiag in 360 .secondn total smoke esposure. d. As in footnote c, after which mice were ginea'air for 10 minutes and the smoke ezposure described in footaote b was repeated, resultiag in 540 seconds total smoke eaposnre. e., Coefficients of variation are given in parenthesis.

t N T ®01 2007'e" ( raaL$ 3 EFFECT OF TOTAL SMOKC PXPOSiJRE TINE ON D8P0SITION OF TOT®b PA@1°YCi1L6TE MATrBB (TPAI) 0 SC3F1/C1ai FF.MALE lICF A= f2F0SQRE TO 20T 291 CIGABEm 5wxE Dagsition of 2! (usr) IAa9oa Faoptra8us Total Smoice B=osure(sec)a Lung and Lower Trachea and U2per Trachea Head TRTb and Stomuch Total 50 24(.67)c 5(.80) 4(.50) 33 7(.25) 40 100 83(.32) 3(.bQ u(.a0) 97 L2(.58) 109 1S0 149(.31) 7(.75) 14(.29) 170 16(.63) 186 200 184(.21) 12(.66) 18(.22) 214 13(.57) 227 a. Total smoke eaposures of 50, 100, 150, and 200 seconds resulted from eapoaures to 5, 10, 15 and 20 seconds of Z0Z smoke followed by 55, 50, 45 and 40 seconds of air, respactively, each miauta ior 10 consecutive minutes. 'rhirty mise Per group, 28-30 weeks of age, were used. b. Total respiratory tract. c. Coeffieients of variation are givea in parenthesis. t

- ~ r ~ l A®l.6 4 I f F h DSpOSI.TION AdD DIST[RIBUTI01! OF TOTAI. PARTICULATE HATTF.N IN gC3F1/Cu10 111CE AFTER PREVIOUS EXPOSURE TO CICAFlB'fT8 SNOXE FOR 3 HSBtC9, 3 i0N19iS, /l,Nt! 6 2!0l8TIiSe Hicrograms TPH im Exposure Perlod Number of Nice Hean HOan Lung Larynx Mouse Lung and and Neight Weight l.ower Upper (g) (n8) Trachea Trachea liead Total 6teepira- tory Tract Stomach Contentsb Total Body } I F 3 Weeksc 10 28.3 236 137(.16)d 14(.42) l0(.38) 161 23(.SS) 184 F 3 lionthae 15 24.4 24'~~• 152(.14) 14(.6S') 7(.20) 173 12(.65) 186 Y 6 Honthsf 15 24.1. 288 145(.16) iS(:48) 8(.80) 168 12(.23) 179 h 3 Honthug 30 27.5 225 146(.26) • 11(.39) 13(.28) 170 28(.38)h 198 Percent Total 78(.05) 8(.1) a8L 93(.10(~40) 8xpusure conditions consisteb of an exposure to 30 seconds of Ox 2A cigarette smoke folloued by 30 secon$a I of sft-each minute for 10 consecutive minutes, resulting in 300 seconds total swotke exposure. bStomech contents were separated frpm the stomach for these itrellf waa lees than background value. experiments. • The amount found in the stomach cHice were 32 weeks of age when ezposed. dCoefficient of variation is given In parenthesie. eHice were 20 weeks of age when exposed. fHice were 32 waeks of aNe when exposed. BHice were 35 weeks of age when e=posed. pmi 4rnmaeh cnn[ente.

HiC0120074 RHFEBENCEs BL13S, R. (1977). Inhalation tosicity studies on cigarette smoka IV. Espression of the dose of smoEce particulate material applied to the Ismgs of expe;:imental anima.ls. Toxfc=l. 7, 189-195. B1NNS, 8. , BEyF.DT, J.L. IiIL10N, • L. . 9. AND LUGTDrT, W. G. D. (L976). Inhalation tosieity studies on cigarette smoka II. Tobacco moks iabalation doeimetry studies on small Laboratory animals. Tosi . 6, 197-206. CATON, J. E. (1979). A metbod for the deteamination of tobacco smoke iahalation dosimatry using carbon-L4 labeled dotriacontane. In Tobacco Smoke Inhalation Bioassay C!s<mr_ (H. B. Guerin, J. B. Stoks.ty, and C. E. Higgins, eds.) DOE Report OBNL-5424, Oak Ridge Natiooal Laboratory, NTIS. _ GAYLE, T. X., SIGGANS, C. E., STORELY, J. 1. (1979). e& cigarette amoha monitoring device for contiauo.us, aaimal exposure systems. In Tobacco Smoke Inhalation Sioassag ChemisM (M. R. Gueria, J. H. Stokaly, and C. E. Higgins, ads.) DOE Report OBNL-5424, Oak Ridge National Labora- tory, NTIS. - BII48Y, C. J., WMM=, C. E., LOPEZ, A., DANSIE, D. 8., AVEBY, X. D., CATON, J. E., STOEffi.°, J. 8., HOLNBEBG, E. W., GUERIN, N. Bo, AND KOURI, R. E. (1980). 'Ch® dosiaecry and distribution of WhoLe cigarette smo}:~ particulates i~a inbred strair.s of mice. Ccmperisoa of a large capacity smok,e-ecposure machiae (SEM) with a smsll-capacit7 smoka- esposiare machina (Walton). In PalmonarY Toxicoloeg of Besoirabla P~art (C. L. Saaders, F. T. Cross, G. E. Dagie, J. A. Mahafley, eds.) CM-791002 Tecbnical iafozmation Center, 0. S. Department of Ener.gy. HENRY, C. J., BRETH, L. A., GEBHABT,• J. M., 1i8Iffi1tS, C. E., AND Ep08I, B. E. Carbozyhemaglobin Levels as a measure of cigarette amoiae esposure in mica. (In preparation) 1980. HCW, C. J., LOPEZ. A., DANSIE, D. &., AVFRY, Id. D., 1d$1'II=, C. E., CATON, J. E., STORELY, J. B. , CUE833, K. E. , CDRREM, S. -D. , AND 1~ ROMZ, R. E. Distribution and clearance of three cigarette amotse con- stituents, Docriacontane (DTC), Nicotina (NiC), and Beozo(a)pyr®ae (BP), after szvpoaure of mice to whole cigatett amoke. Tai=losX and Agp7.ied Phazmacoloes+. Abstracts of Twentieth lleeting, Society of Toxi- cology, ia prass, 19@1.

H iCG 12'U"J 7 5 Figure l.. ihe SM IZ with front aad aide panel$ opem to show eZMRronic componeats. Sae teact for fu]1 operati.ooa]l deeails. Ci.garattes (C) are loaded from a hopper (RG) into a roratiag dzum holaler (DB). The ( Lighter (L) aatomaticalLy i.gaiteo the cigarette and pnff air As forced through each eigarette in turn. F'loor is caused by a coaatantly hald 3:.ffareatSal pressure ptavided by the poeitive pressure ia the doma (D0) betrreen the ignited ead ead the butt end of the cigarette. Dilution air (AA) is intsodsacad at thq butt end of the cigaratte. Side stream smoke is restoved through the ven (Q). Cigarettes ase removed by the astomatic ejector (8) and d3scsrded ia the butt ahutte (S). (

d 1 1 Figure 1 H TE01 ?007(; '

I Fsgura 2. sch®ati.c diagram of sM! ZI, smoke g1ot+ moaituriag 9ysRT® 0 7 20077 aad aalml contaiameat aast. ( l 1 C"

.-. Flgure 2 Aa4md CoaeohwncnQ ®eed SmoEco floaro Syaemm Cloa-up View oi Aaimal Rastreiat In Ralceion o® Folyea"n.u aludule NEAQ RESIRAINS NOSE SEAL V 00

y----- - - - Fi.guse 3. sL•1K u aasmei coa*a{*zAt system. one ssae of r.lsa **10120 079 is sbom, vith the capacity for Psposia6 240 mice pes side (480 ~ mica total.). Bnbber stappers are use.d ia the asposare holes wtfea less thaa the full capacity of mice are to be exposed. ~-

: Mlgu~ 3