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259/12 TECHNI~ES FOR TOBACCO ~IOKE Z NHALATION TOXICITY STUDIES AUthOR Dr. Richard ~inns DATE 7. IO. 1974 British--Amerlcan Tobacco Co. Ltd. Group R. & D. Centre Southm-pton. C~ C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-2- ~NTRODU CTZON ~ODOLOCY Smoke Expos ure Sys tern Inhalation Dos isetry Phys£olos£cal mm£tor£n£ S~ STUDI~S ON A~I2~A3~ Z32OSZD TO ~40KE Deposition o£ Smoke F~t£culate Hatter Selection of F~posure CondiC£ons £o= Shore-Term Scudies wich Rats F~GURES I.-4 TABLES 1-4 CO OD BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-3- Ci) (ii) J I/~rRODU CTION The £nfocmac£on conca£ned in ch£s document .is a brief revim,r of low," of the cechufques being used a~ B-A.T. Co. Lcd., .G.It. & D.C., South•upton, £or .studies on ~lacion CoWrie7 of cLzareCte smoke. Of p~ccicul~ i~porcance are the deecrlpcion of the smoke exposure system which has recently been deveioped for this type of works and • auzm~ry of the technique used for demon•craCkS peneCrac£on of smoke particulate mccec ~nco the lungs of sell laboratory animals. Work has bern carried our on a number o£ laboratory species, but in this report the information ~vmn relates ~ainly co me-dies o~ •he 18boracory zac. This an£mal will be used for shore-term inhalation studles ~equix~d as parr o£ • "So•Be l" submission to the HLuzCer Cous~ccee on ohm chmrmccer£stlc~ of • substitute smoking mterlels ~o~d '~ATFLA~Z". $~aclsed results ~e ~ven of two £mporcanc up•cos of chic work: prelin~nary dace .on dos~mEry ecud4es ~Ch the race selection of the exposure cc~d£c£ona Zor au£mals subjected co smoke £rom one of the ripped ~lue-cured e£sarmCtes Co be used ms the lOOZ tobacco control sample £or 6-~eek comparative toxLcity studies. NETHODOLOCY Smoke E~posure System A Ken•tel v~ew o£ the smoke e~poeu~e system is given in ¥iSu~e 1. Eseencia~lys the |yst•m is based on • mod£g£md Maso~ emokin& •chines as umed fo~ smoke condensate producClon, ~rLch 8 sequence controller which m~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-4- operates various valves to 81Zow movement of smoke and diluClnS air co the exposure chamber around vhich animal holding cubes are attached. Some advantages of the machine are: (1) Cigarettes are smoked under closely controlled standard conditions of pu~£ volume, frequency, etc. (2) Smoke exposure conditions can eu£ly be varied to give either conclnuous or ince~uitcenc exposure to smoke. Xntermlttent exposure can be varied from 5. seconds co 55 seconds in 5 second intervals. (3) For continuous exposure of animals, which ~ the preferred method o£ exposure based on respiratory monicorin& studies, the smoke KeneraCion system ensures ~ atmosphere of fresh smoke. (4) Smoke mi.X~ £S rapid, with no visible evidence of layerinK or uneven distribution o£ smoke in the chamber. (5) Particulate loss in the anClre system, be~veen the buCC end of the c£saretca and the exhaust port of the chmmber, is approxi~ately IOZ o£ the TPM zeneraced. There is virtually no loss due to deposiClon ~rLChin the exposure chamber itself. (6) The system is readily adaptable for exposure of a wide range of animal species, and Ir~Ch this machine york has already been caz~iad ouC ou mice, hamsters, rats and Su/~e8 pigs. (7) The smoke dilutlon zange can be readily set aC any level ~r~th~ the likely useful =arise 1:5 to 1"20 smoke:a~r. Zf required, dilutions outside Ch/~ range can be achieved. (8) KonicorinK o£ chamber atmospheres can be carried out durinB exposure of animals co smoke. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-5- (9) Animal holding tubes can be used to monitor breathing patterns of animals durinK exposure to smoke. DeCa~ls of operatinK characteristics of the mach£ne are 8vailablej buC aze not S£~nn in this brief :epo=t. Inhalation Dosime tr7 A method has been developed* to determlne ,'he penetration of smoke pazticulate phase into the various :egious of the re•pit•tory system of animals exposed to smoke. The method also a/low some use•amen, of the "dose" of particulate me,trial which animal• receive during an exposure to smokeo C£garettes are ~'spiked" v£th unlabelled decachlorobiphenyl (DCBP), • d~ch dur£ng burning of the cisarette transfer• unchanKed to the particulate phase of smoke. The tmlabelled particulate phase mLrker i• tetaLned ~th~n the respiratory system of animals exposed to DCKP-teKsed smoke. After recovery £rom tissues the DCB~. hence TI~4, load can be determined to K~ve an indication of smoke "dose" retained by test animals. In the e~periments described in this report, iuntdi•tely after exposur• to DCBP-taBad smoke, animals were removed ~d • 8a:qpla of blood taken for cArboxyhaemoKlobLn (COHb) maanu.t"emant. The an~mAls vere then killed, rapidly dissected and the luns8, l~rynx and trachea removed. Heads were skinned and separated into upper head and lover jaw (with tongue) sections. These smnples vere veiKhed and stored under deep freeze condltions until requ£red for analysis. j " j IIIIII I II * ~ased on Levis, C.I., et el., (1973) Am. J. Kesp. Dis., ~08, 367-370. BAT Co LTD - MINNESOTA TOBACCO LITIGATION F '[-'1

--6-- LunK aalpIes vera homDsenised in the presence of cyclohexana and sodium sulphate and the homogenata yes extracted rich cycIohexana in a Soxhlet un£t £or 3 hours. All other tissues ware extracted vichout pretre4~Camnt. The crude extracts were concentrated by evaporation, s then applied to colunms of alumina (Activity 1). A first £ract£on yes collected in cyclohexane and d£scarded; r.he DCKP fraction was eluced with 15Z ether in cyclohexane. eluace vss evaporated to small volmm then made up I:o 8 stamdard volume (5, 10 o: 25 ml) vith added DDD* as £nte:L-nal standard. The solutions we=e m~alysed £or I)CBP by £njecclng 1 ,I samples onto a glass column (2 m x 30 san i.d.) packed with 3.8Z OV-I on 80-100 mesh Chromosorb W (I~P) us£ng a Perkin ELmer Hodel F17 GC ££tCed with a Hi63 electron capture 4erector at 300°. The amount of DCBP present was determined by comparing the DCBP:DDD peak height ratio £or the samples wlth chose obcait~ed for a series of standeds o£ knovn concentration. PhYs £olosical..H0nitorinS Systems have been established £or the measurement o£ :eaplraCory function Lu animals under normal cond£clons and for monitoring changes in respiratory function during exposLure to smoke. l~Lonitor£ng of the breathing patterns o£ amimals will be carried out routinely durins exposure periods. For sho:t-term stud£es terminal measurement o£ basic pulmonary characteristics, such as C£dal volume and respiratory rate, ~r~ll also be made on test and control animals. In addlclon, rapid routine measurements of blood COHb levels can be tattled out. This parameter KLves an Lndlcmt£on of penecrst£on of me , , * DDD- p',p- l,l-dichloro 2,2-b£s (parachlorophenyl)ethane C~ C~ C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION r-,O

-7-. at least one saseous phase component of smoke into the respiratory system of exposed Rnimsls, and to some extent this may complement the particulate phase dosimecry measurements made using DC~P labelled smoke. SOME STUDZES O~ ANY~ALS EXPOSED TO S~IOK£ Deposlcion of Smoke Particulate Hatter Work has ken clr~ied out involv~nK exposure of animls to DCBP- taKKed smDkeo The objectives of these studies were: (£) to assess the effectiveness of the B-A.T. smoking nuach~e for exposure o£ test animals to smoke, (ii) to determine whether the use og ma unlabelled marker material in smoke could be used got quantitative dos~netry studies. (iii) to beBin the collection of comparative doslmetry data £rom several species of laboratory animal, which may in the £uture help in the selection o£ animals £or inhalation studies, Experiments on rats ~d hamsters, the two species which are currently being used most extonsively for J~haletlon studies with tobacco smoke. ~e ~eported briefly here. S~ test ~imals o£ each species were exposed in p~rs for 10 minutes to a continuous stream of emeke (l:l& dilution with ~r) from unripped c~Karette8 (Code BO) u~ich were loaded ~th DCKP (1.95 u~/~K). Control m~als vt~e exposed to smoke from untreated BO ~Karettes° All c~Karettes were smoked under stanKard conditions Co a 23 nm butt ma:k. Blood samples for COHb determ£natLon and tlsst~ samples £or 8nalysl8 £o~ the particulate phase mrker were taken as described previously. BAT Co LTD - MINNESOTA TOBACCO LITIGATION c_)"1 c~

-8- A sun~ry of some of the rat data from test animls is ~ven in Table i. The experiment need not be discussed in detail here. The principal points of. interest to be noted ac this stage are: (i) (ii) (iii) smoke particulate phase marker was detected ac all levels of the respiratory system examined. the B-A.T. system was therefore an ef£eetive "system for exposure o£ tats to dilute smoke ~m~et t/~ condit£on.s described. ~mde= the conditions used, althouKh there was some evidence of depos£tion of particulate materla2 in the head res£on of tats, a large proportion o£ DCBP recovered was £ound in the lower respiratory 8y8 ~em. (iv) cor~iderinK the technical di£Zlculcies in cmrrying out quantitative inhalation dosimetry studies, the dose o£ particulate material zeachlnK the lower respiratory system, ezp~essed as ~g DCKP/K tissue, az~d blood COHb levels veto reasonably consistent in this study. A simila= study has been carried out us~nK Syrian Kolden hamsters, and this allows some interestin8 inter-speciflc comparisons. Essential detalXs have been condensed into Table 2. F~fective exposure of animals can reasonably be inferred from the data presented. In the hamster, deposition of particulate materiel in the lover respiratory system was hiKh, with mote than 90% of deposited e DCBF beinK detested there. There.was therafore less evidence of nasal filtration in the hamster compared v~th the rat. Particulate load, expressed as ~K DCBP/E tissue appeared to be 2OZ hisher in the lover respiratory tract o£ the |~unster compared with the rat, when both species mmmedb C~ c7% c~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-9- were exposed under similar conditions. The deposition is further sub-divided Co compare "dose" of DCBP to the various regions of the lower respiratory tract as sho~n in Table 3. This interesting comparison suggests chat although deposition of particulate matter per unit weight of tissue was similar in the funks of the two species, deposition in the trachea end in particular the larynx" of the hamster appeared Co be greater than in fihe rat. For both the rat and hamster, blood COHb level tended co increase as particulate deposition increased in the respiratory system. This ~a noc unexpectedp but to our knowledKe this has not previously been investigated experimentally in this way. For 8 E/vet lower respiratory systmn load of particulates, the tendency was for blood COHb to be hiEher in rats than in hamsters. The details of dos~metry work are not ~ven here in full, and the fiKures have not been subjected to detailed analysis. Several important points of fundamental interest arise from the results obtalneds but at this point the information 18 given only to demonstrate the potential of the techniques u~ed for doeimtry work. Hopefully, this has been made clear in Chls b~iof presentation of dace. Selection of Exposure Conditions for Short-Term Studies in Rats Work has been carried out to investiKate the tolerance of rats to various dilutions of make from a flue-cured tipped cigarette. This ciearette~ coded G29__~5, is one of the lOOt tobacco cigarettes which will be used as the standard aeainst which other c£Karette8 containinE added foamed BATFLAEZ in various proportions will be cqmpared. The experiments mmmmb Cr~ C7~ CXD BAT Co LTD - MINN~TA TOBACCO LITIGATION ¢,.rl

-10- described briefly here related to the selection of a maxLmum tolerable exposure level (~frEL) for exposure of rats to smoke from G29_~5 cigarettes. Tt iS expected that exposure reKimes for studLes with BATFL.q/~-containlng ciKarettes will be related to the N~KL for G29__~5. I= planning the work for determination of smoke tolerance of rats, various conditions were pre-defined : (1) Continuous cachet than inCermitte=t expoeure to smoke would be used dur£nS the burn,s8 o£ el••reties Co a standard butt lensth. Konitor£n$ of breatKin$ patterns dur£ns exposure of animals %ruder both continuous and .intermittent ¢ond£tions indicated to us that the former •de would be preferable for this work. (2) Since episodic exposure to smoke throughout r-he period of the ~erLment is preferable, it was decided to expose animals twice da~ly, vlth approximately 4 hours between exposures. (3) E~osures would be carried out on 7 days per week for the short~ tet~n study. In addition to the smoke tolerance schedule, come race were also subjected to daily restraint in holding tubes to determine the effect of th~s stress on the animals. This aspect of the work was done as part of • study oa the back&round paCbolosy o£ various batches o£ race kept for several weeks ~n the facilltles which will be used £or the ma~.n r-omparatlve inhalation experimance. m O Cr~ Cr~ co BAT Co LTD - MINNF_.SOTA TOBACCO LITIGATION

-11- Because characteristics of atmospheres ~ exposure systems tend to be characte~ist£c o£ the machines used to generate the smoke, chamber atmospheres should, if possible, be defined more precisely than by the initial smoke d£1u~ion, since this may thanks. Xt is expected that some chmnber smnpl£ng for determination o£. for example, smoke particulate phase concentration or carbon monoxide lev~l will be" ca~ed out du~£nS Iong-te~m experLmantSo For the purposes o£ chLs report, re£e~snce to smoke dilut£on ratios will be adequate. For the work summar£sed here, smoke d£1ucion £8 expressed as the ratio o£ volume o£ smoke to volume og dilutinB a£r. Preliminary studies were carried out to determine the extremes o£ the ran~e o£ tolerance o£ rats to smoke £rom G295 cigarettes. Under the conditions no~ed above, usinK the B-A.T. exposure system, e smoke dilution o£ 1:3 was not tolerated, whereas a d£1utlon o~ 1:19 was eas£1y tolerated by the animals. Work van c~ried out to wssess H~ZL using concentratlon8 from the less dilute end o£ the smoke tolerance ranks. Three dilutions of smoke were used: 1:5, 1:8 and 1:12. Groups of ~ an~nals were acclimatised over 5 days to exposure at these dilutions, and subsequently exposed t~rice da41y for approximately t~o weeks. DecaLls o£ body vaight changes and survival o£ ~nimals under these cond4tion8 are Ki~en in ¥£Ku~e 2 and 3 raspacti~tLy. Tube control anLmals ax~ includLexi £o3 conwarLson. Compared w~ch case control rats, tube control rats showed scene reducclon £n growth race. However~ th£s was slight and much less marked than the reduced srowch rate seen in 811 smoked animals compared trLch Cr% BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-12- controls. This effect of smoke exposure on K~owth =•Ca has been veil documenc~ for several species. 7~ this study the reducc£on ~n grov~h race was noC ~ela~ed to the smoke dilut£on (FiEure 2). The effect o£ smoke d£1utlon on survlval of animals was clearly related to dilution, and this information is most useful in the esti~uation o£ HT.KL £or a study o£ up Co 6 weeks. On the basis of the survival data sho~n ~ Figure 3, a smoke dilution in the r~gion 1:12 vc~id probably be used for such a study w£th G295 cigarettes over • period o£ •ix reeks. &t the end of this ranSe-£indini study blood samples vere oaken ~lnediately £ollov£nK the final exposure at the various dilutions and COI~ levels detained (Table 4). For the Do ~coups v£ch several surv£v£nK animls, blood COHb levels were quite s~lac at • p•rt£cula,c smoke dilution, £nd~c•tlng consistent exposure o£ anlmals within • frcoup. Blood COHb level was extremely high after exposure to 1"5 smoke dilution, Xt is interesting to compare blood COMb levels in those animals accl~nacised to various dilutions of smoke, with COMb levels £ound in rats a£ter one un•cclimac;aed exposu:e under identical conditions (Figure 4). For all rats, over m vide range o£ dilutions, CO~ levels increased as smoke dilution decreased. At co.non dilutions, blood COlib levels in non-accl£umtised and •ccllmatised rats vere very siaL~laz. Th£s Indic•ass, i£ one •ccepts COHb levels as at least • rough index, Chat smoke dos•Ks £or particular conditions may not change markedly as annals become acclimatised. However, there is no question that the tolerance o£ an;male to the exposure, there£ore ~he smoke 'dose'. is much improved i£ • short period o£ ac¢l~tisation precedes the proposed max~um exposure zeg£me. m 0 C~ c~ c~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION Co

-~3- SUMMARY A brief description has been ~ven of the B-A.T. an~l exposure system developed for iuhe~&tion to~ci~y studies ~r~th smoke. Su~ies are gLven of phys£ologlcal m~nitorin$ methods and the dosimecry cechr~que, using a~ ~labelled p&rticulaCe phase marker, which wilI be used for inhalation work. These special methods will be uses in addition to sCand&rd pathological techniques for assessins tissue response to smoke ~osu~e. Some details are 8£ven of experiments Co assess tolerable smoke exposure condicionJ £or ciKaretce G295, one standard cigarette to be u~ed £or comp&rst£ve s cud£es on smoke. Th£s ~eport should be taken principally as an introduction to some /xLhalation ~thods to be used in investisa~ions of the potential biological activity o£ foamed BATFLAK~. BAT Co LTD - MINNESOTA TOBACCO LITIGATION l_,r-i Lrl

-14- TABLE 1 DEPOSZTION OF DCBP IN RAT RESFZRATORY SYSTEH AND ~- -- BLOOD -COHb' -I.EVELSt- FOLLOWI~i(;- ~.X-POSt[RE~ O'F- - - - 3 4 o 7 8 J TOTAL MEAN Total DCBP Recovered (ps) L2,096 7,911 4.463 9.676 6.853 6..501 Z Distribution of DCB3P DCBP ~n Lover .............. Kmsp~raCo%~/ SysCe~ Head Lo~r Jaw mL L 10.69 3.07 10.52 4.39 13.44 10.51 • 6.63 1.70 9,15 4o28 0.84 2.33 21.43 8.44 15.33 6.98 ~.34 14.55 I_ - _" I m - l ..... I' _ l 9.368 8.95Z 6.62Z Trachaa L.40 3.60 4,20 Z,44 6.61 10.94 IL _ 4.87Z Blood Lung (Lat-y~x, Trachea, Lunl) COHb (~S/S T£ssue) (Z) ,L ........... J~ .......... m _ L u 83.14 8.965 48.2 68.28 71.79 76.31 75.81 46.45 4. 838 2.409 7.013 4.071 2.312 28.6 24.0 24.4 23.0 17.6 70.30Z & .935 11A8/8 27.63Z 18.3ZZ 81.79Z BAT Co LTD - MINNESOTA TOBACCO LITIGATION ,,..3-1 -O", "O"-

-15- TABLE 2 DZFOSI'rloN OF DCEP ~ tL~HSTER RESPXKATORY SYSTEH AND J BLOOD CO!Lb LEVELSF ¥OLLOWZNG EXPOSURE OF- - ,~z~s To S~,ozZ, FR~~, DCpp-~^aC~D +~+FxGu~rzs IL, 17 18 ToTal DCBP Recovered CPs) Larynx 4. 440 Z DLscrlbuCion of I)CI~ Trachea 0.97 8.504 1.339 0.798 6. 790 4. 388 "A'0~AL HE/d~ _~=- 0.63 6.31 7.64 1.56 7.17 4.33 4.26 3.63 5.01 2.12 6.72 1.21 J | IN, 5.56Z 2.25Z 7.81X | it _ 15.99 .~. g6 8.zs 6.63 3.19 i i i 7.37Z 3.35 1.64 1.25 0.46 8.55 1 2.56Z L II 92.05Z * Animals subjected ¢o 1.4m4Ced exposure only. Lung Ill 89.53 71.44 82. gO 82.71 85.79 8O .33 82.12Z DCBP in Lover Respirmcoz7 System (~I~rynx, Trachea, Lung) (IPK/I Tilmue) II J |1 6.77g 9.882 2.211 1.. 182 IO.O57 5.343 5.9O9 US/S Blood COBb (=) 1.5.9 25.4 8.4 10.0 27.4 13.8 16.82g TA~E 3 Co.r~so. OF .c~ D ZmS=m. O. ~GZONS- oF z.z LO~R RESPLRATORY TItAC~ OF AJ, i,I.HJ~S EY~L*OSI~D TO TAGGED SMOKE o..,,zxo,S .... ....... UNDER SIHILAR C 8pee£es 4.243 2.977 Hamster 11.421 5.340 ptiX:llP/8 iislue |,. Larynx Trlrhil Linsi 5.175 Jt 15.648 i ,::ND J, BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-16- TAmE msmummmmmm~ BLOOD C~BO~GLOBr.S LEVELS IN RATS EXI"OSZD TO A -RA-NGE OF-DZLUTIONS OF' SNOKE FROH czGARETTES Saz~les taken £~mediet:ely eft:er she second 10 minute exposure on day 17 (121:h full smoking day) of the exposure period (Cigareel:e G295). Smoke Dilu~iou Anlmal Blood (SmokezA£r) No. COHb (Z) 1:5 14 59.6 92 35.8 1:8 9~ 29.0 96 26.7 21 24 97 101 102 _ Jm ...... * Blood sample clo~ted. 22.7 18.4 15.5 18.1 Mean COHb 59.6Z 18.7Z emmmmmumm m 0"-, BAT Co LTD - MINNESOTA TOBACCO LITIGATION

• _T0 S)~KE EXPOSURE_" SYSTEM, SHOWING SMOKE GENERATION UNIT AND E~pOSURE CHAMBER ~¢I~H A'FTACHED~ AN '~[AL HOLDING TUBES BAT Co LTD - MINNESOTA TOBACCO LITIGATION CO

J:'JG. e THE P, ELATIONSHIP .P,~TWEEN FO~ RATS SUBJECT'ED TO A GROUP MEAN BODY WEt~HT ~ND T!~,~r, RANGE OF DILUTIONS OF SMOKE Ir~.OM CIGARETTES rIVE DAY ACt'.,L, rMAT|ZATI~N PCRIOD, I,'DL.LOWED. BY ~ X IO mJn IEXI~OSUR£S/DA" I~OR UP TO I~- DAYS. CAGE (:ON'rROL. L 1:8 1:18 I: S ! [I~,~I.IMATIZ, A?'ION ! P.X I0 iii MINUI"C r.:~DOSUIRI=S I=l -I~R DAY T,M= C=AYs) BAT Co LTD - MINNESOTA TOBACCO LITIGATION I cr,,, c~

THE REL ATION~.HIP BETY~/EEN rio. 3 ~;URVIVAL OF ANIMALS AND TIME, FOR RATS $UDJECTED TO A RANGr OF DILUTIONS OF SMQKE FROM 62 95 DAY ACrwI.IMATIZATION ~OERIOOt UP TO I1"~ DAYS. CIGARE TTES ~0660WED BY E X I0 mm EXPOSuRES/DAY, ANIMAl.S/OROUP v w w v . SMOKE: AIR = = ,,:~-- i - : : - = : ~1 "12 ' J ' I:| | X '0 MINUTr I[|XPO,,.','II,,II=:IES m~R DAY I • r,,,,,= CoA','=) L..rl L-P,, CO (_rl f.jrl BAT Co LTD - MINNESOTA TOBACCO LITIGATION

BLOOD RANGE VALUES ACCI.Ih4 A'I'IZED RATS. ~'01~ P..,RACK c'r's INDICAT¢ NUMBI~R FIG. 4 CARBOXYHA~MOGLOBIN LEVE:i.,S IN RATS EXPOSED TO A O1=' DILU.TION,S OIr 5MOKC FROM G295 CIGARETT.E5 ARE ,SHOWN FOR ACCLIMATIZED ('I'ABLE 4.) AND NON- THE LATTER, Ir'IGURES IN OF" ANIMAl. l SAMPI.E~. SO (4) ACCLIMAT/Z,~D NON.ACCI.JMA'riz~ ID O I :aO 1 : 15 I : .SMO|tC DILUTION I0 c~ c~ -:::x:) 0-, BAT Co LTD - MINNESOTA TOBACCO LITIGATION