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:ri})ution: `.''6 . 'Johnson (Library r. A.-~W. Spears r. r'.. `d'. 5chultz , k n , 'd. I G c- a a f 3's- :-2 0, P a SUBCHROAIC EXPOSURE OF RATS AND HAMSTERS TO CIGARETTE SMOKE - A PROBE FOR COMPOUND lll TREATED CIGARETTE SMOKE INHALATION STUDIES ACGe551oFf' {1Umher -.:185 ~~vb~mitted by: Dr. H. S. Tong ~" R epor~ nurr~bnr: . , . Oa~e: '3/9/73 ~Surnmary or Abstract: -s . . , ~ .: .., . . , t -W!, Young Male Sprague-Dawley rats (225-235qm) and male Golden- , Syrian hamsters (75-85gm) were -subjected to 5 weeks of 85mm nonfilter cigarette smoke exposure at a daily schedule ,`of 3 cycles of 8 puffs of 16-second puff, with 44 seconds .;. of air after each puff, every 20 ininutes for 6 rounds. The rest.period between cycles was 2 hours. Body weight gains in ~the "smoked rats and hamsters were completely suppressed in the 5 weeks. .The suppression in body weight gain was due partly to decrease in food consumption. This condition was.. reversable after termination of smoking. At the end of , .5 weeks, the rats showed exhaustion and debility, while .although the hamsters showed stimulation, they seemed in p. .good laal;:h. CCBP smoke tracer studies su7gested that: the 1,4 mean respiratary minute volume of the smoked rats was half ~ of that of fresh rats, and that of the smoked hamsters was tn- slightly higher than fresh hamsters. Because of spontane- C)h ous chronic,respiratory diseases, histopatholgic examina- N tion off lung-trachea can not he u:.-ed as an indicator of the degree of cigarette sr.ione toxicity. Based on'clinical . impression, it is predicted th~~.t the ha~,sters can be subjected to this s,~vke regimen for an ac; I'iti~~aal 8 months and rats .for 3 months before mortaZitv s'ets in.

SUBCHRG.lIC: E;ZPOSUP.D OF RATS AND HNP•"STrRS TO 1?r('1T?F: PnP C(1','"T(lrT`]1) 1.11 T"I'[s`i1: D CTr'~«°^,ET'_'E ST:^I:;, .T.?::i":T_•:'.TIn':" S'1'UDIUS . The anti-ciliostatic and anti-inflamrll"dtory activities of Compound 111 (3 -pher.yl-5-r•iethyl-1, 2, 4-o;:adiazole; P2I0) against .cigarette smoke in the respiratory tract of laboratory anir.:als have been documented by Dalhamn and Rylander (1), Jones and associates (2) and Dahigren and Dalhamn (3). We also have demonstrated in our laboratories tnat Co::.pound .lll possesses anti-inflammatory activity which is equipotent to that of phenylbutazone on a weight basis in the carrageenan-induced . edema of the hind paw of the rat test (4, 5). More recently, : we observed that it also e::hibits anti-inflarn-iator.y activity in the Freund adjuvant-induced.polyarthritis of the rat test (6 Acute and chronic oral toxicity studies in rats (7, 8) and dogs (9, 10) and acute dermal toxicity studies in rabbits (11) indicate that this conpound is relatively.nontoxic. Ho%,ever, since Compound 111 is a potential useful tobacco.additive (12), its potential toxicity should be evaluated, when possible, in the form in which it is intended for use. . ~. - . . ~ . . ' _.~.'. . . M~:. . ' . ~ .. ~. . . - :. .. : .' - . . . - ~ .. ~'_ ' . The present experiment, in which nonfilter production cigarettes were used on rats and hamsters, was undertaken to determine ntaximum tolerable smoke dosages and suitable animal species for.use in future smoking experiments in which Compound 111 treated cigarettes will be employed. By using the "_.30-Port". reverse automatic smoking machine, which was developed at our laboratories (13, 14),• smoking conditions with respect to the volume, duration and frequence of each puff of smoke generated can be closely approximated to those in human. •The concentra- tion of the smoke can also be regulated. Since it:is.~known: ; that rodents.are obligatory nose breathers, it is expected, and has been found (15),.that nasal absorption of smoke will oc.cur. ''Accordingly, decachlorobiphenyl (DCBP) was used as a cigarette smoke tracer (16) to estimate the extent of pul- monary exposure to the smoke in smoke-acclimated animals. Body weights and food consumption data were recorded. Histolo- gical sections of lungs and tracheas of the animals were taken ,immediately before and at the end, and 28 days after•the end of smoke exposure. Materials 'and riethods O IJ Male albino Spragu^-Da~~:ley strain rats (151-175gm) and .%I. male Golden Sa-rian hamsters (50-60gm) were purchased fromL Cn 0 ARS/Sorague-DIn:-1ey, i-.adison, [Viscons,in. These animals were received . i n filter protected 'crat'Ls via air freight. They were housed 5 to a pol, . Li~S~lc ne czge (15" x 13" x 7") containing t co~_ncoh p ~~r`icl: s bedding (San-I-Cel). These ca'.TPs c.Ie::e pl3ced on a•acl~: in a laminar air flow tent (l3ioClean-100, C':S In:iu:jtries, Conshohoc'_en, Pa.) in an

t air conditioned. room (21°C, . 55% R. FI. ). Food (Wayne Lab- }sloz, ialied v!ills, Chicago, Illinois) and watcr were nrovidcd ad lihitu~ : The cage's, bedding and food were st^~ili- :. - 'J , ~:.~ tcsla-rin - an d the : r.irl:i:~c ~~atcr by hoilincJ, The efficiency of the laminar air flow tent was periodically monitored by bacteriological,studies using-nutrient agar plates (6 hours day time exposurel`48 hours culture at 37°C) . X g w~~ eA a Typical American type;~blended 85mmpon-filter cigarettes. .(approxir:.ately 65i rim water pressure drop) were used. °The smoking machine was set to generate a 35-m1 puff of smoke. at 1:14 dilution (smoke: Cambridge filter filtered compressed air) in two seconds, every 58 seconds, for a.total of eight puffs (5Yi each cigarette. The rats were placed individually'in plastic cone-cylirider holders with their heads in the cone portiqn (center'hole. .on apex of cone through.which smoke enters was 5/8" dia.). A plastic foam plug was inserted into the cylinder portion of the holder (2.3/4" diameter) after a rat was placed in it to prevent'the animal from escaping. A glass cup : (1 3/4" dia. x 1 3/4".h) fitted with a`rubber membrane .gasket was placed over the cone end of the plastic animal .holder. -There are two glass tubes (1/4" dia. x 3/4"l) located on the opposite side of the dome of.the qlass .cup. One of them is for the smoke from the machine to enter through a teflon tubing (1/16° dia. x 33" 1) and the other is for the smoke to escape via a Tygon tubing (1/4" dia. x 31" 1) , The hamsters were placed individually in a glass, instead of plastic, cone-cylinder holders fitted with glass cups. as described above for the rats. Except for day 1, 2 and 3, during which the animals received 1"cycle and 2 cycles of smoke exposure, respectively, otherwise, the animals were subjected to a smoke inhalation -schedule of 3 cycles of 8 puffs of 16-second puff, with 44 seconds of air after each puff, every 20 minutes for 6 rounds. The rest period between cycles was 2 hours. The animals remained in the holders between rounds in each .cycle. Iiowever, between cycles, they c•rere removed from the holders and had their eyes and faces wiped clean with gauze sponges moistened with 0.9% saline (for rats) or 2% boric acid (for hamsters) solutions. They were then placed in their cages in the laminar air flow tent. All animals were provided with water between cycles, but only t::e rats given food during that time. The hamsters were fed -i,,--t.veen 7:30 P. ;I. and 8:30 A. M. over- night. Except for t,.e first and t::e last week during which the rats and t he h~:.sters, respecLive1y, were exposed sE'F'a:'a t:1.y to th° SIi:o'.:c, ~ttl: r?:1SE'r they were exposed tor,et;:er t.".r~et:g::out t:zis study.

. At the end of 5 weeks ot smoke exposure (4 weeks for the hamsters),`eight DCBP cigarettes (JLT-8-14-72, pressure drop 77-87mm water, 0.247 percent wj:•z, ~jielding 250mcg of DCf3P 4n the mainstream of undiluted smoke per cigarette or •:.0598.mcct/cc of diluted s;:7ke), instead of regularr cigarettes, were used in the third round of the second cycle-ot smoxing. After eight 16-second puffs, 3 rats and 3 hamsters were sacrificed by spinal cordotomy after cerebral concussion. The trachea was clamped with a hemostat and then the lung and trachea were removed in one piece and then placed in a flask containing chilled (acetone-solid carbond dioxide) n-hexane for DCBP analysis. ".fter 5 weeks of smoking, the animals, save 2 each from the -~ontrol and smoked groups of each species, were sacrificed - by exsanguination via the abdominal aorta and vena.cava following sodium hexobarbital anesthesia (200mg/kg, i.p.). . The trachea <<,as exposed and 'tied with thread. The chest was opened and the trachea, heart and lung were removed together. The organs were weighed with a lead shot and then placed in .formalin (10%)-sodium bicarbonate (0.8%)solution. After .5 days, the lungs and tracheas were embedded in paraffin - (Bioloid) for microtome sectioning. The sections were .``"stained with hematoxylin and eosin-Y. Twenty-eight da:ys following the last day of smoking,.the remaining animals were sacrificed and histological sections of lungs and tracheas were obtained as described above. ,the hamsters apreared quite normal. Eence, they appeared to tolerate the.sn:oking regimen reasonably well. of smoking. On each Rionday morning after c=rnencing smoking, This same condition has not been observed in the rats. hamsters were progressively more excited after each cycle ,hamsters.reversed to normal within a week after its appearance. 1the direct effect of cigarette smoke. This eye condition in.these ~ rubbed-against the wall of the cone shaped holder.rather than condition could have been the-results of the animals' eyes being, second week of smoking, respectively. In.a11 probability this" One and two hamsters developed puffy eyes during the first and =j of th'J jolnts or the' r ~ ys. condition most likZly :i til(2 r C.:..._.i: Cf t:: C.'iC:.~:;~:.. i:0::.~.1i 2 L O: I =, rats to the holc;c_r's rat:::?r tha~n' any Cii:"c.'.ct E?tlecr of the This LC"`CI; ~' ^.- r 1J .,ot.: :_C`pf_'Z_' in }:JT,:S`.C'?" This bFttao-en t}:~~se trro s:)ecies : rcoabl•.• vas clue t,-• anatomic::.t rat+ic:r were caused by hancll ing in placi.:-- the ar.,~Taals in .:.d out of the holder-s. Bv the fi f`.'., ;.reek, t'-ie smo'r>_-d rats seeMed to have The rats did not tolerate the smoking schedule too well. By the ninth day after s::o:ci::g, was heaun, all of the sno},ed rats were sr_ora,..Ln,;. By the foiurt--enth day, the_v seemed debilitated and did not prccn themselves well. Four of• the ten smoked rats had hematomas on the r.iargirn of their ears. T:-...:se traumata tr.an physioloc ic:.l cliffex-unci2s.

Ttre present study. coriiirias the body weiqtit gain inhibitory effect of cigarette smoke in hamst6rs (17). Fic; ures 1 and 2 illustrate the average body weights of rats and it is; .: c,n i.:.: ~t the coatroll rats and period from smoking, *the average body weight of the smoked to that w~hich,has been reported (17). After a similar r.est -that of the control. This weirht loss reversibility is similar average body weight of the smoked hamsters was similar to and that •dur~~iig ~the r.o:i--s...o::irlg ~~~:ekeni~s, they were elevated. After 21 days of res; following t,ic last day of smoking, the smoking weekdays, " the smoked animals body weights were lowered, failed to gain any'weiaht. ' It is also seen that during the - five we^ks; whereas, :` i:Z this sams period the smoked animals hamsters gained 48% and 24 -10 in body weight, respectively, in It is also seen*that during the weekdays the smoked rats and hamsters -v:ere sinilar to those of their respective controls. The food consu.::ption data are shown in Figures 3. and 4 for .. the rats and hamsters, respectively. It is seen that during the weekend the food consur„^tion rates of the.smoked rats and rats was. still 19 $_ lower than their control. consumed slightly more food than their controls during this ing to note that the smoked rats and hamsters which were not sacrificed when. sr",oking was terminated, were found to have was partly due to decrease in food consumption. It is interest- the weight loss seen in the smoked animals during the weekdays controls failed to gain weight. These observations suggest that rats 'sho%,.ed loss of weight. Also, during "paired" feeding, the smoked ha:::sters, as expected, showed loss of weight, and their was instituted, both the control rats a.s well as the smoked their controls. During the weekdays in which "paired" feeding ha.msters cons=ed 35% and 37%, respectively, less food than rest period. This increased food consumption during the rest gain.of.the smoked animals as shown in Figures 1 and 2, period undoubtedly also contributed to the rapid body weight .:The histological sections taken from the lungs and tracheas are listed in Table 1 and 2 for rats and hamsters, respectively. five harsters (Fi:g. 5-b) had what an~cars to have been mild acute broncho7neumonia in the apical lobe of the right lung. In spite of that finding, one can be reasonably confident that Q the remaining animals were "clean" as.defined by Reid (18). ~ ~ C!1 ~ O - • ~ of pulmonary lymphoid tissue infiltration. However, one of the lungs to determine the suitability of the remaining animals for use in the experiment. As depicted in Fig. 5-a and 8-a, 4/4 of the rats and 4/5 of the hamsters were completely free Prior to the start of the smoking experiment, 4 rats and 5 hamsters were sacrificed for histological examination of their

'~,t the end ~at.,5 weeks of smoking, 8/11) and 4/7 of the control .:cre sacri^ .ficec: for histolo^ical `ea:a.~ination.. As depicted in Fig. 5-c, 5/8 of t:e control hl,risters shc;.? no rcrr,i:rkahle histological chan.es in their lungs. Of the remaining 3/8 in this group, one shoc•:s. grede ~I .(<:4$ .of . dorsal section of whole -lung, ) one : shows. grade' I=T (50 Z j•. arad 'one shows grade III ( s30 0, k'igure '5-d) interstitial pneur.io:.itis.' = 1lmona the 55 smoked hamsters, 3/5 show Grz::te I and 2/5 show grade II .•(I'ig. . G-a) chronic interstitial pne»^cnitis." ' in the 8 control.r.ats, ?-show grade I(Fig: 8::-b), 3 sho:.r grade..,ZI(Fig. 8-c) and 3 show grade III : (Fig. 8-d) chronic intpr~.titial pneumonitis. In the 5 smoked rats, 2' sho,i:*. grzde II (Fig. 9-a) and 3 show grade III (Fig. 9-b) chronic •interstitial pneumonitis. ~-;No inflammatory changes or other` :-abnormalities-were seen in the rat and hamster tracheas. Fig. 7-a and 7-b show the longitudinal tracheal sections of the control and smoked har.:sters. The tracheal epithelium shows normal ciliated cells. The multilayered appearance of the epithelial layer is due to the thickness of the slide (10 nicrons). jlynder and Fairchild (19) observed no tracheal epithelial changes in the mouse after chronic cigarette smoke expogure. - Twenty-eiaht days after the end of smoking,.the remaining 2 animals of each group of each species were sacrificed: Two control and two smoked hamsters show grade II (Fig. 6-b) and grade III (Fig. 6-c) lung pathology, respectively. The two control and,two smoked rats both show grade II changes (Fig. and Fig. 9-d). Although the hanster appears to be more resistant than the rat to chronic pulmonary inflammatory diseases, nevertheless the `present study shows that 50% of the control hamsters did develop chronic interstitial pneumonitis ranging from grade I to grade III. As expected, the rats were less resistant and 100% of the control rats develop3d chronic pul:ronary inflammatory changes. Although the laminar air flow tent was used to lower the air born microbial burden to tha animal respiratory systern in hope that the incidence of respiratory diseases would be lowered in these animals, it did not seem to help the rats much. It is entirely plausible to speculate that the observed pulmonary histopathological conditions in•these animals were due mainly, if not entirely to endogenous causative agents. it was stated that epidemic of acute bacteria .pnetz.TMonia in.laboratory rats has not been observed (22) or rarely observed (23). Presently, the etiology of chronic respiratory a disease in the rat remains unclear, although Nelson believes that ~,.i infectious catarrh and endemic pneumonia probably are caused by ~ Myccplas::a pulmonis and an unidn-:ntified virus, respectively (24).~ ,~p,A Since we dici not observe ar_,~ s igns of infectious catarrh, the observed pulmonary pathology could have been caused by virus. O Even if virus-free rats and han:stcrs c~:a~r.e obta~ n,~ble, to I~zin' :in `~ thon free of the viral agents. in this type of cftp_-rim:nt is icalt. Table 3 sho',~s thi' air aualit'y ins1C'e the tent and outside of it. The colonies ceu::~:ed were consistant to those which werc. 9-c

reaorted for another laminar air flow system (20). In the inste4d of 6 hours as in our work. The 30-minute data when .:-rC c: ;cccd ~or 3 ~ 0 mir.•a~c8 support (which would be equivalent to the front of the tent of Lab. No. 19 in our stu&%) and 18 colonies on the internal 62 colonies on the wire cage 3.2 tir^a-s (I.e., 62/19 = 3.2). Dnrinb the tir!n when our This approxinately produced a reduction in microbial burden lar„inar flo-7 shelf (er.uivalent to the tent in our study) . present data, it is unlikely that histological evaluation of level of cacte cleaning activity in Lab. No. 19. Based on the level of traffic in and out of the tent was well as to the and the floor in this area were cleaned with Lysoliv disinfectant daily. The plate golonies in Lab. No. 20 during period B and C were lower than that during period A. It seems that the level of plate colonies in the tent was...directly related to the authorized personnel were allowed in Lab..No.- 20 and the furniture were not specifically controlled. During period B and C, only Lab. LTo. 20, where the "30-Port" smoking machine is located, This also occuYre.d during period C after most of the experi- mental animals were sacrificed and traffic in and out of the tent and animal cages cleaning activity was at a minimum in Lab. No. 19. During period A, house keeping and traffic in even though the plate colonies outside of the tent were high. of it was at a minimum, the tent plate colonies were near zero, respectively. It is interesting,to~note.,that during period A, when there were no animals in the tent'and traffic in and out , experiment, during thn- =qa . xme,nt and after -the experiment, 3.7) . Periods A, B, and C~were- the times before the smoking -_ tent to those in the tent i•ras-of the same order (i.e., 82/22 - ratio of the nu*-,d:)er of agar plate colonies in front of the..; siroking experir.!ent was in progress (Table 3, period A) , the. After 5 weeks of smoking,.DCBP smoke tracer cigarettes were assay for cigarette smoke toxicities. the tracheas and lungs of smoked animals would be a useful used in place of the regular cigafettes for smoking•in order to sssay any changes in smoke.exposure pattern between fresh animals and smoke acclir.^.ated animals. Table 4 presents data ~:42o increase of deposition of the tracer in these organs over trachea-lung deposit of half of that which was obtained earlier (16) in fresh rats; whereas the acclinated hamsters showed a which indicate that the acclimated rats showed a mean DCBP the fresh hamsters (16). It is to be pointed out that the esimated respiratory minute volume used-in the calculation was that for fresh normal rats. Since the :_;:?oked rats showed exhaustion and debility, it was possible the respiratory rate and : tidal z'olu.me were ~rasticallv reduced. As pointed out earlier, the'har,2sters hecaMe more excited- pror,•ressively from I-A the first to the last cycle of snokin:r during the day. Except ~ for t'ie cle~~ression in iaou,1 i•rezght c-ai::' a nc~ stimulation, the .~ hams ~.:.__., `.--?ec:=- =C.1 nori;::'i . .SJ_T' '"` t:i~'~ tracor work :-:a s ~7or'_orIT:c:C~ ~ . in the third rG:_ n•:: of the second c,- cl(-- oz-sm oriincr c:) th::;_ day;--. :.~ the hicher derosj ':i c-~ ~y r:C:3P cou.Ld 'r.ave been the result of (b elevlated res pirator.• -:i.nut~ voliw:~~a c<:used by an increase in r~~ ~r:,taY r :llc.

Young male Sprague-Dawley rats (225-235gm) and male Golden Syrian hamsters (75-85gn) were subjected to 5 weeks of 85mm nonfilter cigarette smoke exposure at a daily schedule of 3 cycles of 8 puffs of 16-second puff, with 44 seconds of air after each puff, every 20 r.tinutes for 6 rounds. The rest period bet~•reen cycles was 2 hours. Body weight gains in the sr.toked rats and hamsters were completely suppressed in the 5 c:,eeks. The suppression in body weight gain was due partly to decrease in food consum-ption. This condition was reversable after termination of smoking. At the end of 5 weeks, the rats showed exhaustion and debility, while although the hamsters d showed stimulation, 'they seem2d in good health. DCBP smoke tracer studies suggested that the mean respiratory minute volume of the smoked rats was half of that of fresh rats, and that of the smoked hamsters was slightly higher than fresh - har:sters.- Because of spontaneous chronic respiratory diseases, histopathologic examination of lung-trachea can not be used as an indicator of the degree of cigarette smoke toxicity. Based on clinical impression, it is predicted that the hamsters can be subjected to this smoke regimen for an additional 8 months and the rats for 3 months before mortality sets in.

:'ubc2:ronic Cigarette Smoke Inhalation---Rats 'and Iiamsters. Lung and Trachea Histological Sections. Pre-In!i`i l.at i.on f10tCLJoo'f. n 6, i1 P o s t-T:inaon 72-26 11 u . . bi 11 Lung 72-27 72-:% 8 72-29 72-30 72-31 72-32 72-33' 72-3•1 -i. 33 -1 ; i2-1`6 -) 3 72-37 -i:) 72-33 Post:- ~ion ;%1us Rest 13-4`7:-6=1 11 11 1 -2 72- :3 ' n u n -3 72-54 OIVS49T0 - f 72-26-1 72-27-1 72-26-1. 72-29-1 72-30-1 72-31-1 72-32-1 72-33-1 72-34-1 72-35-1 72-36-1 72-37-1 72-33-1 72-52-1 72-53-1 72-54-1 72-55-1 1/ I,#5 n 0 II,#1 II,#2 0 II,#3 n n III,#1 III,#2 III,#3 I1"I #4 11 , III,#5 n a n IV,#1 IV,#2 IV,#3 .! Cage'II,#4 ° .' II,#5 m n IV.,#4 IV,#5 Smoke,exposed n of Norinal Control u n grade II. grade III grade III grade I,II grade II grade III ,: grade III grade I grade I grade II ' grade II grade III grade II .

Subcronic Cigarette•-Smoke Inhalation---Rats and Hamsters.. Lung and Trachea Histological Sections Prc-InhalE_tion Hamsters ~_... ',':'t.eboo}_ ` Lung Trachea Animal# Remarks RIO-4-97-5 72-21 n m m n 72-22 72-23 72-24 72-25 Po;t T-..':.;;,ation. B~fJ-.:- L1~^1 72-39 " -2 " -3 1~ U 114-6 ' 1/ 11 " -7 O t/ il -8 11 Is 1t -9 -10 ,1 11 -1 p /1 ` II 11 u 11 -3 '11 . . it . 1, -4 -5 72-40 72-41 72-42 72=43 72-44 72-45 72-46 72-47 72-48 72-49 72-50 72-51 72-39-1 ;:.: Cage I II 72-40-1 " III 72041-1 " III 72-42-1 " III 72-43-1 " III 72-44-1 11 IV 72-45-1 " IV 72-46:-1 1/ IV 72-47-1 72-48-1 72-49-1 " II 72-50-1 . n II 72-51-1 Post-Inhal.ation Plus Rest B10- :=11G--5 7'2=56--72-56-1 " " " -6 72-57 72-57-1 " -•7 72-58 72-58-1 m -8; 72-59 72-59-1 tIVS49I0 Cage.I1 11 I.L : it IV /, I V 11 11 .u Smoke Exposed n n n it Not remarkable Not remarkable Not remarkable Not remarkable Not remarkable Chronic interstitial m m pneumonitis, grade I " d , gra e I: , grade I: , grade I , grade I,