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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). (