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CTR HN 042270

, c•r•R. Gehtra,1- #sA 1.'olnrn, ttee; Sornn" a rt, .}ndeevenT, (.ardnsr anl l.osl i .Tun 21, I ;>i- 'W0yr;/fp ProRress Report For tbe Current Contract Period and Renewal AvI)lication f.. # S' S (with Budget) of the Project Entitled The Determination of the Usefulness of the Golden Syrian Hamster as Model Animal for Inhalation Studies SuDrnitted to The Council for Tobacco Research - U.S.A. By Bio-Research Concnltanta, Inc. (BRC Project No. C-191) April, 197t --~i n'1 Peter Bern(eld, Ph. D. _ Vice President and G11 Director of Research r r CTR CCINTRACYS 005B93 11225634 i.J' r/ i"r I 1 1 1 7..h ) i ~.. •1 J.

Abstract Syrian golden ham,ters from two inbred line are being exposed to chronic inhalation of cigarette smoke. The two inbred lines had been selected on grounds of ttie following consideratione: (1) higher susceptibtlity to chem- ieal carcinogenesia in one of thern;(2) greater resistance to nico. tine toxicity and (3) absence of kno.vn genetically conditioned dic- sases or disorders. Smoke inhalation is administered in Walton.Mor. rissey reverse smoking machines. Every ania}al, i. e. a total bi 102 hamsters, initially, from each of thesc twolnbred lines, in- halas unfiltered smokes from IRI Kentucky reference cigarattes at a 1;5, 2 overall dilution from eight consecutive puffs, twice a day, five days a week. Each puff of smoke has a duration of 60 seconds and consists of three separ+Lta perioda two seconds of smoke generation, 15 seconds of actual exposure to smoke and 43 seconds of exposure to fresh laboratory air. Two control groups of 60 hamsters are being main- tained for each of the two inbred lines; one group of animals is ex- posed to all of the conditions of smoking, exeept for the omission of cigarettes in the smnking machinec (11sham-smoked" controls), aAd the other group consists of cage-held co,-.trel anirnals. The length of treatnsent now* ranges from 32 to 69 weeks. Mortality of thc animals during this time has been ex. tremely low, in spite of the high doses oi smoke adminiatered, i. e. 6. 396 and 15. 6% in the smoke-exposed groups, 16. 7% and 1. 9% in the aham-smok.d control groups, snd 14. 8% and 5.6% in t,he cage-bald control groups. Moderate toxicity of smoke inhalation is apparent, especially in the 87. 20 line of hamsters, since there is a con- siderably smaller weight increase in these animals than In the cage.held or sham.smoked control hamsters of the same inbrad line. * Status ae of April 3, 1972. LY CTR CONTRACTS 005894 11225635 CTR ~""~ ~""~ 042272

After 45 weeks of exposure to smoke or to control conditione, six animals of each of the six groups of hamcters were sacriticed s.nd autopsied. Among all the parameters studied at that time, including gross autopsy observations, organ weights, carbon monoxide and triglyceride levels in blood and histopathology of tLeir lungs, trachea ssid nasopharynx, the most important ob. servations were made in the lungs. There was an extensive, granuioma-like elumping of macrophages in the lungs of all six 87.20 hatnsters exposed to smoke, whUe none of the sham.smoked or cage-held controls of the same line exhibited this phenomenon. This manifestation is obviously genetically determ'.ned, since it oceuired in only one of the six smoke-exposed 15. 16 hamsters, although isolated msero. phages were present in the lungs of these animals. r (.n r r N ' _2. CTR CnNTRACT9 005895 11225636 V rI I! 1 I I ! 042273

1. Experimental a, Selection of inb:ed lines of hamsters - The tolerance of intraperitoneally injected nicotine tartrate in five lines of inbred bamsters was from ten to 23 timeo as high par unit of body weight than in mice, and from 1, 6 to 3. 6 times as high'in rate. Aaimals from these same hamster lines stiirvived from 10. 5 to 30 times as long as mice, and from 4. 1 to 11. 5 as long as rats, when exposed to inhalation of cigarette smoke under standard conditions. By far the highest toleraace to both i. p. injection of nieotine tartrate and to smoke inhalation airiong the inbred ham.' sters studied was exhibited by the 15. 16 line. Coincidentally, this same line of himsters bad also been found previously to display the greatest susceptibility to chemical carcinogens. Hence, this line was selected to be one of the two to be employed in the present study. Tbe second highest rankiag line of hamsters by the criteris of i. p. nicotine tartrate and smoke inhalation was'the 82. 62 strain. Since a large proportion of animals from that line were known to be affected, however, by myopathy and cardiopathy and since, consequently, the mortality In this line is markedly higher than that of other inbred strains of hamsters, it was decided not to use the 92. 62 line for the present study. Accordingly, the next most resistant strain to smoke; i. e. the 87. 20 was chosen as the second line. It should be noted that a high incidence of tumors upon e. c. injection of DMBA was described with the 87. 20 line of ham. sters, approximately equaling the response in the 15. 16 line. b. Design of experiment - Three groups of hamsters from each of the above-named two inbred linec were established, i. e. an experimental or smoke.exposed group, second a sham-smoksd control group in which the anirnals are exposed to all of the condi. tions of the first group, except that no cigarettes are inserted into the smoking machines and third, a cage-hsld control group. Male hamsters are being used because, in contrast to other rodents, male bamsters are more docile and less prone to fighting than the females. All harn,:ters, even tnose in the cage-held control -3- . . . r ... . . . _ CTR CChtTRACTS 005896 11225637 W, C""R HN 0~~~- 74

groups are permanently vearing snugly fitting felt collars around their necks, which are reinforced on both sides by thin aluminum rings. T1use collars allow to insert the heads.of the hamsters into the smoking chambers and to immobilize the animals in that position (see below). Since six harasters may be exposed simultaneously In each exposure chamber, the anisnils are also housed In lots of six and the tnitial numbers of animals In each of the six groups were, therefore, multiples of six. In anticipation of a higher mortality In the experimeatal than in the coctrol groups (not borne out until now), the experiment was started wit}t larger numbers of animals in the former (17 lots of six, or 102 hamsters) than in the latter (ten lots of six or 60 animals). The design of the experi- msnt is summarized in Table I. It was not possible to obtain the required numbers of hamsters from each line (222 animsis) at the same time so that they would all be of comparable age or weight. For this reason. exposure to smoke, to shatn-smoking conditions and maintaining of cage.held controls were rtarted, as the animals became avail. able, buc always in lots of six animale, and the beginning of the experiment was, thus, staggered over a period of about 30 weeks. Inasmuch as this was possible, for every two lots of six hamsters stsrted In the exoerimental groups, one lot of six animals of each of the two control groups was set up for the sarae Inbred line at the same time. In all instances and in the case of both inbred strains and all six groups, smoke exposure or admission to the control conditions was only started when the hamsters had at- tained a body weight of 107 = 3 grams. Several lots of six hamsters In the smoke-exposed groups, especially in Group A, had been lost in the early stages of the experiment, because of mechanical malfunctions in the ex. baust or purge-air system of the exposure chambers. These mal. functions were subsequently corrected, and the chance of their recurrence was reduced to a minimum. The animals lost were then replaced (seven lots of six in Group A and two lots of six in Group D). The replacement of tbese animals at a later time ex- plains the relatively large margins between the earliest• and latest beginning times In Groups A and D. The number of animals presently (April 3, 197Z) .4- CTR COMTRRCTS 005897 11225638 C°~R H~~ 0~~~~'-9 5

included in me experiment and the duration of their exposure to smoke or control conditions are lndicated !n Table II. c. Smoke exQosure - Five Walton.Morrissey re- verse smoking machines are being used for this study, i. e, three machines for the smoke exposure in Groups A aad D (204 animals) and two sriachines (always the same ones) for the eham-smoke treatusent in Groups B and E (120 animals). The heads of six hashsters are inserted into an exposure chamber of 725 tnl volume, and the animals are immobilized in this position by felt collars they are wearing permanently around their necks; the felt collars are reinforced by thin alumi.uum rings of appioximately the came size, one on each side of the felt collar. For cmuke exposure, four eigerettes are being smoked simultaneously: 35 ml of air, corresponding to the con- veintioaal average pufY volume of a oirarette smoker, are passed simultaneously through each of them, while smoke is generated. Hance, a total of four times 35 ml or 140 ml of smoke are intra. duced into the exposure cbamber of 725 sal volume, and the over. all dilution of cigarette smoke in the exposure chamber thus amounts to 140:725, or 1:6. 2. Tbis is the concentration of ciga sette smoke actually inhaled by, and rapidly reached in, the lungs of the hamsters. In comparison, the average dilution of cigarette cmoke in the lung of a human smoker ranges from 1:10 to 1:14, as arrived at from the average values for the puff volume (35 ml) and of the human tidal volume (350 to 500 ml). if the hamsters were forced to inhale cigarette amoke of. that concentration uninterruptedly, they would die from carbon monoxide poisoning within 60 seconds. To avoid this, they receive alternately smoke and fresh air in 60-second smoking cycles, as does the human smoker. Each of these smoking cycles consiats of a smoke-gonerating period of two seconds, followed by an actual smoke exposure period of 15 seconds, and finally by a period of exposure to fresh air of 43 seconds. Each hamster in Groups A and D is treated in ses- sions of eight coneecutive smoking cycles (eight consecutive puffs smoked from four simultaneously lighted cigarettes during eight minutes) twice each day, at least four houre apart, on five days each week. r _5. ~. ~.. ..C~ cTR coNTRRCTS 005898 11225639 CTR I~I~~ 0~-~~`~`~

Kentueky iRI reference cigarettes are being used for this purpose. They are smoked in the eight puffs to a butt length of approximately 30 mm. d. Special care of the animals . Because the ham- sters are wearing the felt collars permaneatly, they are unable to grootn their Laces, and eye Infections would develop: In order to prevent such eye infections, the eyes of each hamster are bcine washed with a penicillin solution once every day, five days a week, by means of cotton swabs. t Individual body weights of each•hamster are being determined once a week throughout the expericient. 2. Re sulta a. Mortalof the animals - Nine lota or 34 animals of the smoke-exposed groups died~ accidentally during the first 26 weeks of smoke inhalation because of malfunctioning of the exhaust pump wh.ch replaces the smoke in the exposure chamber by fresh air during the 60-second smoking cycle. These animals were re- placed subsequently. In add:aon, six hamsters of each group (one lot) were sacrificed after 45 weeks on experimental or control con- ditions. From among the remaining animals, i. e. a total of 408 bamsters, only 4Z *or 10. 3% died for other undetermined reasons. According to the mortality figures for each group in Table III, the death rate of the smoke_exposed 15. 16 hamsters in Group A is Lower than those in the two control groups (Groups B and G). On the other hand, there is a somewhat higher mortality in the smoke-exposed 87.20 hamsters (Group D). than in the corresponding control groups (Groups E and F). These differences are not statistically eignificant, howeve r. It does not appear, therefore, that smoke exposure or sham-emokfng conditions cause mortality in these two inbred lines of hamsters. In an overail appraisal of the mortality, there has been a very small death rate of the aairnals for unspecified causee in this experiment. b. Average body weiahts of the aaimala - The figures tsi the last three columns of Table IIi clearly indicate that chronic smoke inhalation markedly af#ects the aor.aal growth rate of both -6_ cTR coNTRACTS 005899 11225640 „ „ 9 . l. n . C~"R MN +~~#~ ~~ ~ 7

inbred ltnes of hamsters, while exposure to sham-smoking condt- tions does not, This phenomenon is considerably more pronounced in the animals of the 87.20 line (Group D versus Groups E and F) than in the 15. 16 hamsters (Group A versus Groups B and C), and the differences are highly significant by statistical considerations in both inbred lines. There is a considerable disparity between the adult body weights of the hamsters of these two inbred lines. Since ex- posu.re to smoke in the 15. 16 line hamsters (Group A) was started at a time when this adult.body weight was almost reached (see Group C). smoke ezposurc had, of course, much less effect on their body weight increase than in animals of the 87. 20 1ine, where the•body weights in the control group (Group F) 'continued to In- crease by about 36 grams. When, the changes in average body weights for all six groups are followed as a function of time, a second, much more subtle effect may be perceived, as seen from the data in Figures 1 and Z. Tbere was a slight but significant loss in body weight in all six groups of hamsters during the Initial week, and during the second week ar• well in Group A. Thereafter, body weights started to increase, more or less rapidly, in all groups. This initial body weight ioss was probably caused by the stress inflicted to the hamsters in all six groupe, including in the cage.held control animals, by making them wear the felt- aluminum collars. Additional stress may have been produced in the animals of Groups A, B, D and E by the conditions of rnachine smoking and, finally, even rnnrs stress is expected to have been produced in hamsters of Grosos A and D by the actual smoke in. halation. The time-weight curves in Figures 1 and 2 suggest that wearing of the felt colla.rs by the animals may have been the rttain caase of the iaitial body weight losees which wers very similar in all six groups. e. Changes in organ Weights atter 45 weeks on eY rimental or control conditions - Atter a duration of 4S weeks on the ezperimenta or control conditions, six animals of each of the six gronps, as well as six untreated hamsters from each of the two inbred lines having the age and body weights of the animals at the beginning of the study (107 * 3 g), were sacrificed and an- topsied. The results on their organ weighte are shown in Tables ZY and V. -7- CTR CoNTRACTS 005900 11225641 G11 .,, r CI I! I I/ I W' I2bv NI -

The statistical significance of the dtfferencss be- tareen the various groups are shovn in the last four columns of Tables IV and V. In many instaaces, the organ weights of the young animals of 107 * 3 g(status at the beginning of the experi- ment) are eignittcantly lighter than those of the cage-held control animals, 45 weelu later (Groups C versus CC and F versus FF). In view of the body weight increases of the animals during the ex. perlrnent, discussed above, these findings were to be expected. :iutilarly, the differences in organ weights between the young ani. mals of 107 * 3 g and the animals of t}he samc inbred lines after 45 weeks of smoke exposure or of sham.smdking are without im. portance, (See Group CC versus Groups A and 8, or Group FF versus Groups D and E.) ~ In addition to thcee di.tferences in organ weights between young and old animals, there are only a small number of instances, wbere emoke exposure and/or sham smoktng con. dltions may actual]y have caused a change in certain organ weights. These ar^. (1) increased adrenal weights in sham-smoked animals of both inored lines, but not in the smoke-exposed hamsters_• (2) increased weights of lungs plus trachea in sham-smoked hamsters of the 15. 16 strain; (3) decrer sed liver and kidney weights in the srnoke-exposed 87.20 hamsters, but not in the sham. smoked ani. male of the same strain, and (4) decreassd spleen weights in smoke-exposed 87.20 hamsters and, to a much lesser degree, in the sham smoked control anirmals. d. Changes in serum triglyceride levels after 45 weeks on ex erimental or control conditions - There appear to occur no changes in serum triglyceri e evels of the hamsters as a consequence of exposure to cigarette smoke or to the con. ditions of sham.senoking, as seen from the data in Table V2. The only tignlfieant changes in this parameter observed are increases in serum triglyceride levels during aging in the 87. 20 line (Groups E and F versus FF). There are no corresponding increases In the 15. 16 line of hamsters. However, there was a uiuch smaller increase in body weights during the same period of time in this latter strain, and the absence of increases in se- rum trillyceride levels may be connected to the lack of changes in body weights. E. Blood catbon monoxide le+ele after 45 weeks .... C!1 of exposure to smoke or to experimental con itions - Blood was r r .B. CTR CONTRRCTS 005901 11225642 ~-rRHN 0'~'~2f~9-