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CO ~i~?~ im,E ~IT! 5,L Not to be taken: from t, his room PROJECT 6900 PHYSIOLOGICAL STUDIES (Semi Annual) PERIOD COVERED BY REPORT: October, 1966 - Ap.ril 30, 1967 DATE OF REPORT: May 9~ 19'67 PROJECT LEADER: R'. D. Ca,rpen,ter I. Introduction The objects o.f this project are to develop and apply to cigarettes biological tests which have meaning in the area of smoking and health. Mice, guinea pigs, cats, and monkeys have been used~ with in vivo procedures. These tests were performed by researchers in contract laboratories,: where qualified staffs and a,dequate facilities for animal care are available. There a~re three health: areas in wh~ich methods are bein,g sought. Lung cancer has been widely acclaimed as being caused by cigarette smokin,g. More recently, cardliova~scular disea,se and the respira.tory diseases of chronic bronchitis and emphysema have been a.dldedl to the list of strong, indictments against smoking. We are actively seeking tests which, will be meaningful in the areas of can,cer andl the. oth,er respiratory diseases, but we have no tests under study for cardiovascular disease. II. Summary of Results A. Prima=te Inhibition Study Cyanamologous monkeys given, cigarette smoke for a period: of ii months showed n.o observably microscopic ch,anges. Three of the six monkeys died during the period, presumably because of extremely high, carbon monoxide levels and trauma. The smoking monkeys did exhibit shortness of breath when they attempted to avoid being caught following their exercise periods. A fourth monkey was sacrificed! before the en,d of the stu,dy becau~se of problems unrelated to. smoke exposure. B.. Long-Term, Mouse Skin Paint~-~g The tw0-year skin painting program begun in April, 1965, was terminate~ at 21 months. The histo-pathology and the final~ report are expected; about June i, 1967. The gross pathology

-2- indicated that filtered cigarette smoke was no less tumorigenic than non,filtered smoke.. Smoke from, an all-burley cigarette was less tumorigen,ic than smoke from the blended, cigarettes. Smoke from a cigarette incorporating most of Wynder's suggestions for lowerin,g the tumorigenicity (reconstituted tobacco from, burley who,~e leaf, addition of sodium nitrate, and a filter) resu~ited in. the highest incidence of tumors found for any of the smoke samples. Tobacco p,yrolyzate produced by. pyrolysis a~t 4100°C and processed like smoke was more tumorigenic than any of the smoke samples tested. The above results are based on total tumors, in,cludling those which~ may have spontaneou~sly regressed du~ring the study. A judgment of the carcinogenicity of the samples must await the histo-pathology report. C. Mouse-Skin Hyperplasia Test Th.is test is used by some investigators to screen chemicals for ca~rcinogenic potential in 3 days. We have abandoned~ this test because we found it to be in,sen,sitive and inaccurate with pure compou,nds. D. Mouse. Irritant Test This test is. based on the respiratory response of mice exposed to cigarette smoke. While differences between cigarettes seem to result,~ the test is inconclusive at this.time~ It is planned to determine the effects of several different cigarettes by a protocol design~ed by Mr. Tindlall. These ciga:rettes will vary wid!ely in their p~ara~meters and will be thoroughly analyzed chemically. E. Carbon-Monoxide Uptake IOOO~4~O~ Inha~led substan.ces which are irritatin,g to lung tissue frequently cause changes in the permeability of the alveolar walls~ resulting in a lower diffusivity for gases which are i- exchangedl between the a,lveolar air and the blood. One method of determining if such changes have occurred is the mea:surement of th,e rate at which ca~rbon~ mon,oxid;e is taken up. during breathing. A study to test this effect with ciga=rette smoke has faile~ to date because of the high level of carbon monoxide in the smoke. It was not possible to expose the animals to. a h,igh enough smoke

-3- concentration to~ cause changes which lasted longer than the time necessa~ry for the CO level resulting, from the smoke to be lowered sufficiently to allow the uptake rate determination. An attempt will be made with acetaldehyde at the concentration foundl in smoke. If this fails also, this method will be abandoned. F. Neonatal Mouse Testin..~. Recently, efforts have been made to shorten the time required for testing ca~rcinogen,s by the use of new-born mice. We have conducted a~ pilot study to determine the tolerance of new-born mice to whole cigarette smoke con,densate. Th,e conditions for a full scale study have been established. G. ~cus Flow in Cats - Acu~te Extensive stu,dies of the mu,cus flow changes, cau,sed by the acu~te exposure of cats to cigarette smoke have shown that smokes from different brands of cigarettes are different if the tramsit times differ by 5%. in n~ine animals. This method, which, uses three consecutive puffs of smoke after the lightin,g puff, can also show a diifference between whole smoke and! gas phase smoke (Cambridge filter). This is not possib~if only one puff of smoke is given to the cat. H. Mucu~s Flow in Cats - Repeated Exposure A six-month study is in progress with cats which are equipped with tracheal windows. The study is at the mid-point with 90% of the cats in good condition. III. Results and Discussion 10003420(;6 A. Primate Inhalation Sturdy This: study was originally designed to be a pilot study for a long-term test of the effects of different cigarettes on the respiratory system of monkeys. The estima=ted duration of 3 months was exten,ded several times,~ until the surviving monkeys were sacrificed at ii mon,ths. All of the 6 control animals survived, but only two of the smoking animals su~rvived. Three of the four that diedl are presumed to have succumbed to ca:rbon monoxide poisoning,, since carboxyhemoglobin levels as high as 72% of -- saturation were found in the surviving, animals.

No conclusive evidence of bronchitis or emphysema was found, although there were indications of emphysema in the gross appearance of the lungs of the two smoking animals sacrificed at the end; of the study. Th,e microscopic appearance in the his.to- pathologic examination failed to show. conclusive changes, because of over-distention of the lungs during fixation. The greatest effect noticed wa:s the shortness of breath, of the smoking monkeys following vigorous exercise. These monkeys had smokedl 35: cigarettes da:ily for 9' of the ii month,s. This is equivalent to about 30 packs per day for a man. The final report on, this stu~. is expected later this month. Bi Lon~-Term, Mouse Skin Painting. The moulse skin painting program which was started in April of 1965 was terminated a~t 21 months. The completion report is due June I, 1967. The results from gross pathological data, shown in Table i~ confirmed other investigators findiings that cigarette filters do not reduce the tumorigen,icity of the smoke on a per weight basis. An all-hurley cigarette was less tumorigenic than blended cigarettes. A cigarette made from reconstituted hurley tobacco with added sodium nitrate andl a carbon filter was the most tumorigenic sample tested. It is of interest that this sample h,ad a benzo(a)pyrene content below that which Wynd!er considers carcinogenic to mice. (No.te that tumors were counted. The number of carcinomas has not been reported.) The most active sample was a pyro.lyzate from tobacco. It was intended that this: material wouldl be free of b.enzo.(a)pyren,e~, but it contained about 1 ppm benzo(a)pyrene. This result invites the speculation that the most active portion of the smoke is d;istilled from the tobacco, or that it is produced by a relatively low tempera=ture pyrolysis. If it is distilled, it may be possible to reduce the effects of smoke by solven,t extraction, of the tobacco during cigarette manufacture. 1000342067 C.~ Mouse-Skin Hyperplasia This test is bas.edon the increasein the thickness of the dermis following application of a ca,rcinogen.. A correlation between. thickness and carcinogenicity exists if certain known carcinogens are applied in, sufficient con,centrations. At lower concentrations the result is not significant. No effect was found for smoke conden,sate other than, topical irritation.

-5'- Table I Mou,se Skin Pain,tin:g-Gross Patho,logy Percent With Mean Time SamDle Skin Lesions + S.E. To Skin Lesion, Days Acetone 0: .. - - B (a)P i00; 148 Nonfilter 44.5 +8.4: 494 CA Filter 66.9 +9'.4 326 CA + C Filter 66.0 +9'.0 5.64~ All-Burley (60 mg.) 23.5 +7.0 607 All-Burley (45 mg.) 30.4 *7.7 567 TFP + NaNO3, 75.8 +13.6 393 Pyrolyzate 89.9 +3.6 465 Pyrolyzate + B(a):P 98.3 +1.4 278 F. Neonatal Mouse Testing The length of time necessary to observe a tumor is dependent on the rate. of tumor growth. It is presumed that the rate of tumor growth will be a function of norma=l growth rates, and that new-born mnima:is will produce a visible tumor in a sh,orter time than. ma,ture animals because their ra=te of growth is infinitely greater. This ha,s been demonstrated for known, qa~cinogens. xqoo342o6 We have beg~n, a pilot study with new-born m~ce to determine the dosage that can be used for a much larger test. Whole smoke from nonfilter cigarettes was used. The experiment has been valuable, because the solvent is as critical as the nicotine in, the new-born mouse. Dosage levels have been, established for a program with 5 experimental grou,ps, a negative control group, and: a positive (BaP) control group. It is anticipated that the period of observation, will be 6 months, but this will depend on the outcome of the pilot study. The expected end;-point is t~e nu.mber of lung adenomas produ,ced insteadi of skin tumors, although skin painting will be the method! of application after the first month. Subcutaneous injection of smoke condensa:te will be performed at 2, 7, ands28 days after birth~ andi prior to the skin painting.

-6'- H. ~cus Flow in. Cats - Repeated Exposure This experiment utilizes the window cat technique to compare the effects of repea~ted exposure of cats to cigarette smoke. Six groups of 5 cats each are given the smoke from 4 cigarettes d~ily (3 puffs/cigarette)5, daTs a~ week for 3 weeks. The order of exposure is designed so that each group is exposed to each sample and to a sham period. The entire study will require 24~ weeks, since a 1 week rest period is given between dlifferent samples. Figure i shows a typica=l resuit for the. first half of the study. When the sturdy is completed; the advisaSility of publishing the. results will be considered. IV. Plans A. Primate Inh,ala~tion~ Studies No dlefinite plans for further chronic studies have been formulated. We plan. to suSmit the results of the present stu,dy to management a~s a desirable publication in the fieldl of smoking and health. B. Lon,~-Term Mousse Skin Pain,tin~ No further plans. Mouse Skin, Hy~erp.lasia Abandon, this work. D. Mouse Irrita~n,t Test Test a group of carefully contro,lled samples, includiing high filtration/low delivery cigarettes prepared by Mr. Osmalov. E. Carbon Monoxide Uptake Determine the effect of acetaldiehyde at smoke concentration on CO uptake. If meaningful,., continue for other compounds in, smoke having, suspected a:ctivity. F. N!eonatal Mouse Testing I000~4~06~ Begin a 6-month study with 5 cigarettes on June 5, 1967. Continue the pilo= study (30 mice), to the 6-month point.

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-8- G. M~cus Flow~ in Ca,ts - Acu~te No further studies except as necessary in conjunction with other projects. H. Mucus Flow in Ca,ts - Rep,eated Exposure Complete the present study and consider publica~tion.

Physiological! Studies Carpenter DATE LOANED I

E}~TE II ISSUED TO