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159 Report on In'vestigation anent IRRITAN'T EFFEC'T'SI OF CIGARETTE SMOKE AS INiF'LUENCED BY THE HYGROSCOPIC AGENTS DIET'HYLENE'GLYCOL AND GLYCERINE by SAMUEI, J' .: KpPET. zKY, M D.:, F'. A. C S. Professor and Director of'the Department of'Otolaryngol- ogy, New York Polyclinic Medical School and Hospital•• Int,roduction' A perusal of the literature on the irritant proper- ties of cigarette smoke as influenced by hygroscopic agents shows that s!ome work has been done from clinical and pharmacological angleso Mulinos & Osborne (1)' (2) and Wa11'ace, Reinhardt & Osborne (3), using, the conjunctival sac of the rabbit as a test membrane and the resultant edema produced as a.criterion, reported that cigarettes made with dietlaylene glycol were less irritating,than those made with glycerine. Hlaag (4) using the same method, re- ported no d'ifference. Flinn (5) (6) reported a series of clinical investi'gations and concluded that on smokers there was a distinct difference in irritant effect of cigarettes treated with dicthylene g].ycol and glycerine -- the diethy- lene glycol cigarettes bieing definitely less irritating (1) Pro:c. Soc. Exp., Bioll.& Med'., 1934, 32, 241-245 (2) N.Y. State Jour. Med., 1935',11,590 (3) Arch. Otolaryngolog3', 193Fi, 23, 3', 306-309' (4) Jour. Lab. & Clin. Med. Jian. 19'37,22',4, 341-346' (5) Laryngo'scope, 1935,45 149-154! (6) Laryngoscopey 1937 , 58!-60 47 1 , , , N GJ Q. ~. G~. ~ C1i N

160 Ballenger & Johnson (7) and Ballenger (8), using clinical r observations, failed to find any di~ff erence. Carlson (9),using salivation by smokers a~s a measure, came to the same: conclusion as Ballenger & Johnson and Ballenger. Without discussing in detail these papers, it is very evident that the results these authors present leaves the question open, because the results are controversial,, In an endeavour to clarify the questions involved I make a study ofthe subject from an entirely different angle and one which had not heretofore been undertaken, Chemical Considerations It is known that among the combustion products of glyeerine are present glyceric, tartronic, oxalic, tartaric, acetic and formic acids, together with acrolein, Fromidiethylene glycol, among,the combustion products, are foundiglycolic, oxalic and formic acids, but no acrolein. It can be readily seen from the structural formulae of glycerine and diethylene glycol that the former, being a three-carbon, straight chain, trihydroxy alcohol, could produce aicrolein, whereas from diethylene glycoly having a chain made up of twoica~rbonss oxygen, and two carbons, acrolein would not be expected. Acrolein itself is exceedingly irritating and its presence could (7) Arch. Otolaryngology, Jan. 193 7, 25, 1, 75-80 (8) Archo 01tolaryn'golo'gy, J'an:: 192 9, 219, 1, 115-123 (9) Proc o Soc. Exp. Biol e& Med,, 1937, 36, 302--307' -2-

161 ` ` be responsible for a differen.ce inithe irritant pro- perties of cigarettes in whi.ch these two hygroscopic materials were used, respectivelyo Before any comparative study of'the irritant effects of cigarette smoke as influenced by the use of glycerine and diethylene glycol as hygroscopic agents can be made, the various problems involvedimust be: studied. First it is necessary, to_cancel out variables other than the hygroscopic agents. This is accomplish_ed..by using the same blend of tobaccos made up into identical cigarettes., the only difference being that in.one group the tobacco wasd treated withiglycerine and the other with d3ethylene glycol -- both in the amounts regularly used on!pppular ciigare ttes . Observation of the habits of smokers shows that some take a short puff and others a, long one, that some puff'slowly while others take~ twice as many puffs pex~ unit time. The type of smoking also has an influence: on th~e combustioniproducts and consequently upon!their effects. To obviate these variables, a definite smoking rate must be used whenitesting effects inr ordier to make a; fair comparison withibut one variable. Most important, however, is the choice of thee membrane or tissue, the reaction of which is to serve as a criterion for estimation of the irritant effects ofthe s,moke.- In the study herein detailed', the effects observed were the results of smoking the cigarette as a whole, and noting determinable comparative histopathological effects on the living tissue of the respiratory tract. The two types of cigarette tobacco contents were tested, -3-

and the results compared, the tests being conductedd under exactly alike conditions and undler controls, established by exactly alike normal tissueso Fizrthermore,, it was deemed necessary for the accuracy of'the findi~ngs to make a fundamental study ----------- of the effects of th~e two types of processed cigarette tobacco smoke upon living, tissuie of the respiratory traict,, using a virginal tissue. A word rega~rding my use~of the termi"virginal tissue'~ is necessary. By this term is meant tissue never before subjected to tobacco smoke irritation, This type was found in the experimental animals selected, Their tissues furnished material for autopsy findings, not only of gross pathology, but permitted microscopic histopathological studies to be undertaken. The specimens are preserved for future study and demonstra- tion. The sections were photographed as well as were the microscopic~slides. The copies are part of this report. Thus th~ere is at hand concrete evidence of definite paithologicail reactions in the tissues examined. This concrete evidence presented by the tissue themselves of the body reactions to the two types, of processed cigar- ette smoke are immeasurably more conclusive in making determinations than are observations upon the human nose, throat, pharnyx and larynx by a clinician. The latter sees; and tells what he sees, but he cannot make whalt he sees a matter of permanent record. In the study detailed herein, the record is permanent and any compet-- ent microscopist can study the cuit slides and see thee findings for himself. Furthermore, one could assume~after careful exam- iniation that the selected animal's respiratory tract was -4-

163 C normal. Such an animal when killed furnished both gross and microscopic evidence that it was indeed a,normal animail.. Onlthe part of' humanibeings living in a civilized community, a normal respiratory tract would be difficult to find. A clinician examining a person can only state that no pathology is found at his examinationio Almost all human adults before they reach adult life halve:had'numerous "inciidents" happen to them which leave marks upon the respiratory tract.~ In addition it must be remembered that most adult humans, in this time and age, smoke tobacco in some formo Therefore, observations made upon th~eir respiratory tissues a~s to the effects produced by cigarette smoke must take into: accoun~t, those reactions of a protective character which the bo~dy builds to protect itself from irritating substanceso Th~e effects of'smoking cigarettes must therefore, in humans, be pro-- duced over and beyond the protective barriers which nature already has established in the individual- Greater effects observed in one individual human a~s compared to those observed in another, may depend on the extent to which the given individual has established'his own tissue defense mechanismsa This statement must however not be construed as a criticism of comparative clinical observations made upon humansto record' their perceivediphyaical reactions while under clinical study and control to test the effects upon them of'cigarette tobacco smoking: My study was concentrated upon the respiratorg tract, in general, and upon the epith.elial lining of this tract - ---- ----- - ---- in particular., This epithelium, and its ciliary covering especially, is probably the most vitally important tissue of the body> _5_

164 In smoking,, especially when smokers inhal'e the tobacco smoke, this vital tissue comes into direct contact with the epithelium of the upper respiratory tract, andd often with that of the lower parts of this tract alsol. Thie rabbit was selected as the test animal~ This wais. done because of its convenient size for handling, its availability, and particularly because the epithelial lining of the rabbit°s respiratory tract possesses exactly similar features and histological cell characteristics to those found in humanse The rabbit°s epitheli!an lining of its respiratory tract, its ciilia:; its cell character- istics, subepitheliial stroma are identical with those found in Mumans~ Obviously the anatomical structure of the rabbit differs from humans, but its mucous membrane and component parts nevertheless are identical tissue.,. As far as ascertainable, rabbits of'the same sex (miales) equal size and weight, and even coTor or fur were used. The animals were tracheotomizeda That is, a small cannula was introduced into the wind-pipe (trachea) and the tobacco to b~e tested was s!moked', by the animal using, the automatic smoking machine devised by Dr.Arthur Walter Proetz of St. Louris, The tracheotomy tube in itself caused some local irritation~ These!melchan,ical irritation factors being, present in both series of cigarette tobaccos tested can-- celled each other out. This mechanical irritation factor could easily be visually recognized. It was totally dis- regarded in making determinations fromiboth th~e gross and' the microscopic findings of the tissue reactions from both series of cigarette tobacco contents which~were studied. Another factor needs notice, In the test animals

165 C the insertion of the tracheotomy tube doles not entirely block,the upper respiratory tract as it does to humans upon whom a tracheotomy tube is used to permit respira- tion because of some tumor or other obstructionof res- piration through the larynx and the nose or mouthe Because the tracheotomy tube in these test animals did not entirely block off the upper respiraltory area it gave our t.est ain additional a~dvantage, The upper respir=- atory tract is open andfree, in the test animals, and the tested smoke can be exhaled through the nasal pass- ages. This made possible the study of the effects of the tested smoke, on, the tissues of the nose, the nasopharynx and the larynx. The automatic smoking ma~chine walst set to smoke "puffs"' at the rate of'two puffs per minute. By "puff" is meant an inhalation, an exhalation followed by a period of rest from smoking~ The animals were permitte4to smoke for only one hour eachiday in those tested for more thanone hour of smoking ti'me o Following each smoking period the animals were re- leased in their cages, free to roam abouit, take their natural nourishment by moutha They reacted well, andd acted in no way different from other rabbits in the laib-- oratory animal quarters. A few animals died of pneumonia.contracted inter- currently during,the experimental work~ An autopsy was performed on these. The resultan~t findings are includedi in my report. Curiously all those that died were those who had inhaled the gllycerine treated tobacco. While this finding has no effect upon the conclusions drawn in thle problem upon which I am here reporting, it -7-

16'6 opens a new avenue of research into ancillary problems which have other connotations and which therefore I do not report herein'e In reporting on the experimental study I necessarily must make record of them. REPORT ON EXPERIMEN'TAL WORK Comparative Study between th'e irritative quality of the by-products of identical Tobaccos when treated with (a) Diethylene g1y¢ol (b) Glycerine. TOBACC© USED' One blend of tobacco made up into two ------- ---- groups of cigairettesa ('`) One group treated with Diethylene glycol (r'D) 275% () The othex group treated with Gllycerine (" G) 3'65% (r) Cigarettes furnished'thru courtesy of Plailip' Morris & Co. Ltd. () Amounts commonly used in commercially produced cigarettes ANIMALS USED': Rabbits PROCEDURE: Two no_rmal rabbits used as controlis. Other rabbits trach,eotomized Cigarettes smoked by automatic smoking machine, into a reservoir from which rabbit inhaled from different periods of' timeRabbit never smoked for more than' one hour at a time- Tota1 numiber of hours smoked never more than si'x- - 8'-

167 RABBITS STUDIED: During experiment, after ) Gross autopsy) ) Microscopic GROSS: Nlasal chambers ----- Pharynx and Hypopharynx Larynx Trachea Carina Right and Left main bronchus and' surrounding,Iung tissuee MICROSC'QPIC': Sections taken from Hypopharynx Larynx Traichea, C'arina Right and Left main: bronchus and surrounding tissuea C PH~O'TUGRAPHIC: )Entire respiratory system, Gross )Nasal Chamber )Trachea, Carina and Bronchi Mi.croscopic: STUDY OF NORMAL TO PROCURE NORMAL SPECIMENS Two Rabbits Used. One rabbit killed' and entire.respiratory system including Lung Trachea. Larynx Hypopharynx Pharynx Nasopharynx Nc„sa1 Structures was preserved in formailin~ N C Q C.?. ~ --9- €D (b (n0'

168 Second Rabbit killed - respiratory system removed and ------ ------ .sections made from the following,diesign- ated areas for microscopic study: Hypopharynx Larynx Trachea Carina Right main Bronchus, Bronchioles, Lungo FTNDI NG'S': HyPaPh,arynx: Covered with stratified squamous epithelium Sub-epithelial layer containing blood vessels, connective tissue an~d, cells, occasional plasma cell, mucous gland ducts. Numerous mucous glands~ Muscleso Larynx: S'quamious epithelium except in ventriculus area, where ciliated mucous membrane with numerous ga~blet cells are presento- Very thin sub-epithelial layer - cartilage - muscle fibre striatedi, Trachea• Pseudostratified ciliated column epithelium with several goblet cells. Benealth this layer is a thinicompact connective tissue layer, Sub-.epithelium consists of loose areolar type connective tissue rich in blood vessels, Connective tissue cellscartilage. Carina- Simple ciliated columnar eplitheliumlin folds_ Some goblet cells. -10- ( C

169 C Sub-epithelial layer (loose areolar) contains blood vessels, connective tissue cells, somee smooth mus.cle fibres, lymphoid tissue, plasma cells,- Right Main Bronchus: Bronchiolies - Lung_ Silmplc Low columnar ciliated epithelium on a balsemenit mlembraneo Some goblet cell - submucosa is a loose areoliar connective tissue containing thin blood vessels, con- nective tissue cellso (Some plasma cells) mucous membrhine in folds posteriorly where some smooth muscle fibres are present - C'artilage, . LUNG: C Very low simple non--eiliatedi epithelium (Simple Siquamous) alround air spaces< Some spaces contain geletinous material with aa few polymorphs, and red bloodi cells in it.• In some areas around bronchioles may be seen collections of'lymph cells making up lymphoid patches:, In these areas maly bee seen occasio al giant cells probably for fighting off infectiono Left Main Bronchus - Lung - B'ronchioles: . Description same as for right siide, EXPER'IMENTAL ANIMAL,J1 In which animal was smoked with Diethylene_gjycoj , treated cigarettes for one hour through a tracheotomy tube, killed,, and studied. Experiment_performed_September_26,_1942,. 6& Cigarettes u edicontained Diethylene-glycol Q Respirations 34-36 per minute O Smoke inspired through *racheotcrmy tube and expilred 0 ~ ~

170 partially through nostrils and partiailly through! smoke reservoiro No moisture was observed from nostrils. Autopsy revealeri no gross changes Mi.c,roscopic Findings: Hypopharynx: Somie hyperactivity of mucous glands Larynx: Epithelium normal - very little infili,,ra~tion of' sub-epithelial layer Blood vessels slightly dilated Trachea: No changes Carina: Some secretion in lumen Mucous glands Right Main,Bronchuis: Narmail epithelium Slight oedema of submulcosa Slight perivascular infiltral- tion EXPERIMENTAL ANIMAL #2' Rabbit "2G" September 28, 1942. Animal was smoked for one hour with cigarettes in whichi tobacco contained glycerinic-. R'espiration 34-36 per minuteo Vapor was observed in inhalation tube (tube connecting smoke reservoir to Trachea of animal) . AL'topsy: Showed slight discoloration of the trachea with dilation of blood vessels of the trachea and carina. Microscopic Fin~dings: -------- ------ Hypopharynx: Hyperactivity of'the mucous glands, Larynx: Sopne oedema of the sub-epith- lelial layer. Dilated blood vessels- C -12-

171 C ` Perivascular infiltration with mononuclear cells. Trach'ea: Increase activity of goblet cells. Some oedema of sub- mucosal layer. R'emorrhagic area'.. Cariina': Inerease in the goblet cell activity. Some engorgemen't of the blood - vessels. Periva'scular infiltration. Mostly mononuclear ee11s. Right Main Blronchus: There is an increase in gobliet cell activity. Some polymoxphonuclear cells in the epithelial layer. Engorgement of'the blood vessels with perivascu!lar infiltrations Left Main B'ron'chus ~ There is some interference with the ciliary activity. There are some golymorphonuclear cells infilt~ating the mucous membrane. Marked perivasula'r infiltration>. Some polymorphonuclear cells as well as connective tissue cells in the sub- mucosal layer. EXPERIMEN'TAL ANIMAL #'3' Rabbit °3D" Animal was smoked for three hours~ One hour a time for three consecutive days. use~d contain~ed ID'iethylene glycol.'. Cigarettes. Respiration. 5u' per minute (increased respir- ation) Vapor was observed in the inhaling tube after the fifth cigarette..

172 No vapor was observed during the second and third hours of smoking. Autopsy: Revealed some dilated blood vessels in the lower portion of the trachea. There wass some small pinpoint hemorrhage in the lung~so Microscopig Findingsc Hypopharynx: No changes in the epithelium. Hyperactivity of the mucous - gl andis . Some engorgement of the blood. 'vessels.. in the sub-epithelial layer, Larynx: Mucous membrane appears normal. There is some secretion in the lumen of the larynx. Blood vessels dilated. Very little perivasculair infiltrationo Trachea: Ciliary activity diminished. There are some polymorphonu- clealr cells in the epitheliall layera The sub-epitheliall layer shows an occasional polymorphonuclear cell close to epith~eliu,m. There is nolengorgement of the blood vessels. There is some oedema of the sub-mucosa layer; most of th~ee cells are plasma and connect- ive tissue cells. Cairinai: There is very little inter= ference in the cilia of'the mucous membrane.. The submucosal shows very little cell infiltration. There is some engorgement o~f the blood vessels. -14-

173 c Right Main Bronchus: There is some secretion in the lumen. There are a few polymorphonu- clear cells in the epithelium. The submucosal reaction is very mild, showing very little cell infiltration. Left Main Blronchus': There is some interference withicilia of the mucous membrane. There is some engorgement of' the bloodi vessels. _ Very little connective tissue reaction and perivascular infiltration. EXPERIMEN'TAL,ANIMAL #4 Rabbit "'4G" Animal was smoked for three hours. One hour at a time for three consecutive days. Cigarettes contained gl!ycerine. Respiration: 34-36 per minute. Autopsy: Revealed some grayish areas over the mucous membranie of the trachea. Lungs appearedinormal. Mi¢roscopicFi,ndings:Hy,popharynx: No change in thie epithelium. Hyporac tivi ty o f the mucous glands. Some engorgement of'the blood', vessels in the sub-epithelial layers. Larynx: Interference with ciliary activity. There is an increase in perivascular ).ainfiltration; more marked than in Q, ,, 38,; o 01 W Trachea: There is some uliceration of the Q ~ ~ -15-

174 mucous membrane, . Submucosa shows increased oedema, numerous polymorphonuclear cells and' periva~seular infiltration. Carina: Loss of cilliary activity, Mucous membrane denuded in areas. Sub- mucosa infiltrated many pol'yso Right gain Bronchus: The mucous membrane and submucosal layer are invaded with polymor-- phonuclear cells~ Increased connective tissue react- tion> ' Left Main Bronchus• Interference with cilia~ry a~c.tivi~ty~ . Epithelium infiltrated with poly- morphonuclear ce11s~ Perivascu2lar and submucosal infil tratiione (this shows evidence of infectioni,) EXPERIMENTAL ANIIiMAL #5 Rabbit "5'D'r Animal was smoked for six hours. One hour at a time for six consecutive d'ays. Cigarettes contained diethyrlene g,lycol'- Respiration: 3!6-•40' per minute. Small amount of vapor was.observed inithe inhalation tube (tube con- necting smoke reservoir to Trachea of animal,). No vapor or moisture after the first hour of smoking> During the fifth and sixth hours of smoking respiration increased to 55' to 60,per minute., C -16-

.175 Autopsy: Showed a little mucous in the Trachea. Some dilated blood vessels in the larynx. A few hemorrhagic spots in! the lungs. Microscopic Findings: Hypophalrynx: Epithelium appears normal. Some increased mu~eous gland activityo Blood vessels show engorgement. Very little sub-epithelial infiltration. Larynx: Pus in th~e luimen. Very little interference with ciliary activity. Slight sub-epithelial tissue reaction. Trachea: Increased activity of the goblet cellse Some loss of'ciliary activity. Some (jedema of the submucosa. Carina: Diminished ciliary activity. Increased activity of the goblet cells. Very little infiltration of the submucosa~l layer. Right Main Bronchus: Interference with ciliary activity.A few polymorphonuclear cells infiltrating the mucosa. Perivas.cular infiltration. Connective tissue reaction is slight., Left Main Bronchus: Some secretion in the lumen. No infiltration of the mucosa. Connective tissue and peri- vasculair reaction slight~ ` -17-

176 EXPERIMEN!TAL ANIMAL #6 Rabbit "'6!6" Animal wa's smoked'for six hours. One hour at a time for six consecutive daiys~ Oigarettes contained 1 rine g_Yce_ R'espiration: 45 - per minute, During the third hour of smoking,,tobacco stain was observed around' the nostrilsA millky secretion fro,m -•the lieft eye wa's also observeth This same observation' was noted during, the fourth',, fif'th and sixth hours of smoking, Respirs.tions increased to between 50 and 688 times per minute dhiring, the fif'th and sixth hours of smoki~ngo Autopsy: Showed a large amount of mucous iin the trachea and lungs The blood'vessels of the trachea were markedly dilated, The sku11 was dissected so as to bring into view the interior of the nasal chamberso These cavities were lined with a thick mucous film (pyogenic membrane, ) This membrane was attached to the mucosa of the nose on th'e l e f t s lide . Mlicroscopic Finding~° Hypopharynx: Pus collection in the lumen0 Epithelium inta'ct_, Mucous gliands hyp'eracttive: D'ilated blood vessels in the subepithelial 1a'yer, Larynx: Phs in the lumen: Perivascula'r infiltration C E'

177 C Traichea: Sub-epi thelial layer shows infiltration of all kinds of cells including polymorphonuclearo Loss of ciliao Mucous membrane ulcerated in areas. Blood vessels of the submucosa dilated. Perivascuilar i'nfiltrationi. Carina,: Pus in the lumen. Loss of'ci'~liary activity. Engorgement of vessels in submucosa. Increased perivascular infiltration. Right Main B'ronchus: Muchpus in the lumen. Loss of cilia. Mucosa ulcerated iniareas. Mucosa and submucosa inifiltratedd withipolymorphonuclear ce11s. Left Main Bronchus: Polymorphonuclear cells infiltrating the mucosa. Submucosa shows muchiless reaction than th~~eright main bro~nchus:o, EXPERIMENTAL ANIMAL #7 Rabbit "7D" Animal was smoked for six--hours~ One hour at a time for six consecutive days. Cigarettes contained diethyleno glycol. Respiration: 40 per minute,. Particular care was taken to notice any tobacco stain around the nostrils~ Only a slight brownish discoloration of th~e mucous was found- Autopsy: Only a slight amount of the congestion of the mucous membrane! of'the trachea was observed. The skull was dissected so as to bring into view, the interior of'the nasal chambers. A little mucous was.present but no pyogenic membrane could be found. This specimen was preserved in formalin -19-

178 contained glycerineo Respiration~ 32-36 pier minute EXPERIMENTAL ANIMAL #8 Rabb i t "86"' Animal was smokedifor one hour, withitobacco EXPERIMENTAL ANIMAL #9 contained to become part of the gross exhibito Rabbit died - before second hour of smoking could bie startede Autopsy: Reveailed oledema of both lungs and a large amount of muco-pus in the tracheae This specimen was fixed in formalino Rabbit "9G'" , Animal was smoked for one hour,, with tobacco glycerineo Respiration: 32-36 per minutea Rabbit died the following morning.,- Autopsy: Revealed acute pneumonia. EXPERIMENTAL ANIMAII, #10 -------- ------ --- Rabbit "101G!' Animal was smoked for four hours, one hour at a time for four consecutive days withi tobacco: contained glycerine. Respiration: 22-40 per minutea During the fourth hour of smoking - after

179 the third hour of smoking, tobacco stains were noticed around both nostrils. This rabbit died before the fifth hour of smoking could be started. Autopsy: Nasal passages contained a fair amount of muco-pus. The trachea showed marked in- flammat,io.n and a large quantity of muco- pus.- Both lungs were conge ted. Muco- pus was present in the bronchi. Consolida- ted areas in the lung gave evidence of pneumonia. Microscopic: Sect,io.n from th~e right main bronchus and surrounding lung tissue revealed much purule,nt secretion in the lumen of the bronchus. The mucosa is denuded. Sub- mucosa markedly infiltrated with poly- morphonuiclear cells. Lung,tissue shows numerous consolidated areas. Evidence of acute infectious pneumonia.e EXPERIMEN'TAL ANIMAL #11 Rabbi t 1'111G"' Animal was smokedifor two hours - one hovi!r at atime for two consecutive days with,tobacco contained' g1 -ycerine. --- -- , Respiration,: 44 pier minute. Moisture from bothinostrils were noted,, andian increase in salivation. Tobacco stains over the nostrils during thesecond hour of smoking. This rabbit diedd before the third hour of smoking could take place. -21-

180. Autopsy: Revealed large amount of'muco-pus in trachea and bronchIe- Lungs showed evidence of'pneumonia. EXPERIMENTAL ANIMAL #12 R'abbi t "'12G" Animal was smoked for three hours, one hour at a,time for three consecutive days withicigarette tobacco contained glycerine. Respiration: 34-50 pe-r•minute. This rabbit diedibefore the fourth hour of smoking could take placeo. Auitopsy: Revealed trachea and bronchi filled with muco-pus. Lungs showed evidence of acute pneumonia. (#8 - 9 - 10 - 11 and 12 were placed in formalin and preserved for gross exh~ibit.) EXPERIMENTAL ANIMiAL,#13 Rabbi t "'13D I" This animal was smoked for three hours, one houi!r at a time for three consecutive days using, cigarettee tobacco contained diethylene glyc,ol, Respiration: 20 -40 per minute,Rabbit showed no evidence of discomfort. No moisture formed in smoking tube. No increased saiIiivaition: Rabbit wais kililed a~fter the third hour of' smorking,. Autopsy: Revealed nolgross changes in the respiratory tract. Specimen preserved for exhibit ~ -22- (

181 EXPERIMENTAL ANIMALi#14 ---- -- ----- ------ --- Rabbit "14G"' Animal was smoked for six hours, one hour at a time for six consecuitive days using cigarette tobacco contained! giycerine. Respiration: 20-28 pier minute. During second hour of smoking, left eyee showed a, miIky secretion. Increase mucous, secretion from nostrils. During third smoking hour, mucous accumulated in thie trachea, and was removed'with a suction tube,- Sub-maxillary region a little swollen. Milky-secretion from the 1ef;t eye, and small amount of tobacco stain around the nostrils.- During fouirthy fifthiand six,th hours of smoking, the same findings as above were observed. Tobacco stains around the nostrils formed after the thirdicigarettee Rabbit was killed after the sixth hour of' smoking. Autopsy: Revealed muco-pus in the tracheai and bronchi and nasal passages. This specimen was used for photog,raphy. EXPERIMENTAL ANIMAL #15 Rabbit "15G" Animal was smoked for three hours. One hour at.a time for three consecutive days. Cigarettes contained glycerine, Respiration: 35-50 per minute. Autopsy: Revealed some mucous secretionlin ` -23-

182 nasal meatio, Numerous punctate areas over lower part of the trachea coaIescing into a pyogenic membrane. Some punctate areas over mucous membrane of right and left main bronchuso Mucous membrane of trachea and bronchi appear- ed hemorrhagic. Rough Test of the Cigarettes on the Public Markets The two series of'tobaccos tested and upon which I reported above were delivered to the laboratory by the Philip Morris Company. To test the cigarette tobacco in the cigarettess as they are sold upon.the open market,.I bought six different brands of cigarettes on the op~en market in September, 1942. I emptied their tobacco contents and assigned a code number to each, as follows: - #1~ Old Gold #2. C'amels Philip Morris. Chesterfields. Virginia Rounds Lucky Strikes I sent the tobaccos thus marked to, Dr. Samuel Ro~senzweig, of the Synochem Corporation, 80 Fifth Avenue:,, New York C'ity. I happen to know this research chemist very well, andhave full confidence inihis intellectual integrity- He was unaware of thie names of brandso His report is as follows: - . C - 24-

1'83' 'S T A T E M EI N T^" C concerning the resu!lts of the qualitative analysis of six samples (#1-#6) of tobacco provided by Profo Kopetzky. "After a study of pertinent literature, ten gram samples were extracted with acetone following the pro- cedure of chapman, AnaSyst 51, 382 (1926). In quanti- tative work, the final extract obtained upon evaporation of'the acetone and remova'l of resins should be subjected to a Zeisel determination," "However, at this poinit, it was decided to estab- Iiish rather the chemical nature of the moistening agent tha.n its quantitative amount. Therefore, the residues were used' to prepare - naph'thyl urethanes following the procedure of B'ickel and French, Journ. Am Ghemo Soc, 48,. 747' (1926) . " "'In each case,, 05 g',., samples of the extract were mixed in th'e cold with' 0, 5 g of - naphthylisocyanate, and the crude urethanes were carefully recrystallized from ligroin." "'From the observed melting points, it is concluded' that the samples #1 and #3'contained diethylene-glycol, whilie the samples #2, 4, 5, &6contained' g~ycerol. If' the observed'' amounts of urethane should permit to d'raw additional conclusions, it would app'ear that sample #3 contained more diethylene glycol than sample #1, and that samples #5 and #6 contained'more glycerol than samples #'2 and #4 Note:--Samplie #3 was the tobacco co tents which I removed from a carton' of Philip Morris cigarettes, -25-

185' connective tissue cells, (c) blood vessels, and (d) perivascular area~so Five animals diied'before the experiments could be completed. In all these cases cigarettes moistened withiglyeerine were used. In all these cases acute tracheai- bronchiolitis and pneumonia were the cause of death. FINAL OBSERVATION AND CbNCLUiSIO'N From, this study o-f"the irritation reactions from exactly alike cigarette tobacco manufactured, on the one hand using,glycerine, and on the other using diethylene glycol, and noting, the effects produced upon the living tissue of the respiraltory tract of rabbits, it is very evident that more marked evidence of irrita- tion effects are present in these tissues when subjeeted to the smoke of cigarettes containing glycerine than from those containing diethyl,ene glycol, Finally, the data presented in this paper should tend to end any controversy in regard to the effects of hygroscopic agents in cigarettes on the..irritant pro- perties of' smoke. The method of manufacture has a, distinct and measurable effect on the irritant properties of' smoke I C'iiga~rettes in which diethylene glycol is used as the hygroscopic agent in the above study have beenn shown to be less irritating, than those in which glycerinee is employed. ACKNOWLED!GEMENTS ~ ~ Grateful acknowledgement is made to the Laboratory Department of the New york Polyclinic Medical Slchool ~ ~ ~ ` ~' -27=

186 and Hospital for extending~, to ~ my staff and to me~~ its full facilities. Likewise I aim indebted to Dr. Julius Bell, Instructor in Embryology and Histology at the above named institution for making the sections' and controll- ing,their preparation for photography, and for his active cooperation and interest in checking,upon both, the gross and the microscopic findingsa To Mr, Joseph D. Ha~ckel of the New York Medical Film Guide I am indebted for his time consuming`Work inisetting the microscopic specimens in permanent visual records. His phoitography has made easier the preparationiof this report-by producing a faithful record, by untouiched-up photography, of both the histoliogy and the pathology exhibited.in the specimens. Respectfully suubmitted,, SAMUEL J. KOPETZKY, M.D., F'.A.C,S, April 2, 1943 71 East 80 Street New York City C - 2'8 -

187 C ` APPENDIX - MICROPHQT©GRAPDIS Experimental Findings and Normal Controls

1W N 1 Upper Respiratory tract. 4X - Normal showing anterior (Anferior) turbinate, middle tur- binate, tongue, nasopharynx, oropharynx.. 'N! 2' Respiratory Tract. (lower) 4X. - Normal, Epi'gDottis, Larynx, Trachea, Carina, Right NainiBronchus and Left Main Bronchus and Lung,.

A /q N ' 3 Trachea. (18!0X),JMIncosa1, ciliatory columnar epithelium with goblet ce11s. - Submucosa and cartilage and a blood vessel. 18S IL 06~ Ni 4 Trachea - higher magnification 8010 X.

100 N 5 Bronchus and Lung, 18'0 X- C'iliated columnar epithel'ium with goblet cells, with thin sub- mucosa'li layer, containing some smooth muscle fibres, cartilage and lung tissue and blood' v essel (branch of pulmonary artery) wi'th contained blood!. 1D 1 Tlrachea' - ti8OX - After one hour of smokin!g Di'ethylene Olycol treated tobacco. Nlucosa normal Submucosa slig'ht oedema 6light amount of ti'ssue cel'1 reaction. E

191 C 1D 2 Bronchus& Lung - 180 X - After one hour of smoking IDilethylene Glycol treated tobacco. No pathological changes. 3D 6 Some interference withicil!iary activity of mucosa, few polymorphonuclear cells in mucosa. Slight submucosal and lung tissue reaction.

192 ., `.,. . _ Aw 5D 1! Trachea - 180 X - shows some interference with ciliary actYvity and some infiltration of polymorphonuiclear cells. Suibmucosaa slightly oedemaltous - increasedi cel!llular activity andivascular engorgement. Slight amount of perivascuilar infiltration. ~ 5D 2 Higher Magnif'ication. BOO X - finding the same as 5D 1.

193 5D 3 Bronchus and Lunig~ 180' X'. Diminished ciliary activity and infiltration of'mucosa with a few polynuclear celils,. submucosa shows sli~ght connective tissue reactions with increasedl perilvascular infiltrations. Slight lung reaction. 7D 1 Respiratory tract.

194 7D 2 No. gross pathology. r 703 3 Trachea and Lunig - No gross pathology, a slight congestion appears in the mucous membrane of the tracheal.

195 1i3D 2 Nio gross pathology 4X. 130 D 3 No gross paithollogy 4'X. .

196: 13D 4 Trachea 180-X .. Very slight goblet cell activity. Slight increasen in oedema and connective cell-activity. 13D 5 Trachea 800 X - ANagn!irien same as 13D 1.

197 C 2G 1 Tra'chea 180 X- After one hour of 6lycerine trested Tobacco. Increased'gotilet ce11 activity in mucosa Oedema of suhmucosa. Marked increase in cellular activity. Increased' cel!lular activity.. ,' ~ . .~. c c 1;/ .. i '~ •,,? i ~" i " . g l • .~ . . - 2G 2 Trachea - Hig,h Power of' Same as 2G 1 slame area, same find3ngs.

198 2G 3 8'ronchus & Lung - 18OX - Increased goblet ce1i1 activity an(l' mucous secretiloni. Sub- mucosa shows increased lymphoid cells and connective tissue cells. No change in 1ung. IG 1' Trachea. Lonw Power. 7'2X - 3 hours Glycerine treated Tobacco - Purulent secretion in luimen of trachea, - mulcosa shows interference with celiac erosion aind ulceration. Submucosa shows marked oedema infiltrati'oni with connective tissue andipolymorphio.nuclear cells - Di1'ated,blood vessels andiperi- vasculair iinfiltratilon. C E

199: C C 4G 2 TrachW18D X - Three hours - Glycerinee treated Tobacco - finding same as 4G' 1. 4G 3 Lung - 1&0 X Three: hours - Glycerine Purulent secretion in"bronchial lumen. Interf erence with ciliary activity mucosa ulcerated in areas marked infiltration of', submucosa with"P. N. M. celiLs, marked perivastular and lung tissue infil!tration.

200 6G 1' Upper Respiratory Tract. 8 X - After 6 hoursg smoking withiGlycerine treated Tobacco, showing Pyogenic membrane over inferior turbinate extending into inferior and middle nasal meati. 6iG 2 Trachea - 180 X'- After 6 hours.smoking with Glycerine treatediTobacco showing Pyogenic membrane attached to mucous membrane, loss of ciliary activity. Ulcerated in, areas markedd iinf i ltrat ion of submucosa vascular diilatation and marked infiltration with numerous GcuDy- mbsphonuelear celils.

6GI 3 Blronchus and Lung - 180, X. After smoking with Glycerine treated Tobacco, showing loss of ci~Liary activity, mucousme,mbrane ulcerated in areas,, pus in bronchial lumen, increased perivascular and lung tissue reactions. Engorgement of pulmonary bloodl vessels.. 6G 4 Trachea - 8!010 X - After six hours of smoking with Glycerine treated Tobacco, showing pyogenic membrane attached to the mucous membrane, loss of ciliary activity, ulcerated in aireas„ marked infiltration of the sutr- mucosa, vascular dilitation, and markedisub- mucosali infiltrate with numerous poly- morphonuclear cells. 201

202 15 GI 2 lDpper Respiratory Tract 4 X - After 3 hiours G1'ycer.ine treated Tobacco - several smai11 punctate areas on mucous membrane of milddle meatus and inferior turbi,nate. 15G 3Trachea and' Lung 4 X - After 3 hours Glycerine treated Tobacco - numerous small ulceraltilons ever lower part of trachea coalescing to and covered with a, pyogenic: membrane.

203'. C 14a The gross specimen (14GI) after six hours of smoking-°with Glycerine treated Tobacco showed! large areas of uilcerati'on of' the treachea covered'with a thick py ogenic_ membrane. 14 G Lung and Trachea 4 X - Large ulcerated area ini trachea.

204 14'G Head - After 6 hours with Gllycerine treated 'Tobaccos showed: Purulent secretions inn the anterior (inferior) meatus and over the inferior turbinate.

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