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-5- thickness of the epithelium on the vencro-laceral surface and Ohm outer dorsslsurface of the vocal cords. In extreme cases, Che entire lmn 4,, secclon was invested with fully d£fferenclaced s~s cells. Zn sev~al rats, macaplasia was incomplete ~rich foc£ of col~sm~r cells soil1 present in the ventral region of the larynx. Examples of t:his ~ncomplece meCaplasia occurred in most groups of smoke-exposed anlmals and did nov appear Co be related Co exposure or Co dilution of cigarette smoke (Table 2). The ventral depression showed no sips of squmaous mecaplasia but there was some ~ndicaCion of coltwmar cel~ hyperplasla. TABLE 2 S~UANOUS NZTAPLASIA OF THE EPITtELZUM LZNZNG THE LARYNGEAL LUMEN FOLLOWING EXPOSURE TO 2 DILUTIONS OF SMOKE Exposure Period in DaTa 1 7 14 28 42 Smoke d, 1:8 Dilutiou TreaCsmnc t ill Smoked, Sham- Case 1 : 12 Smoked Control Dilution Number of ani:nals showinS metaplasia* &2 vapour phase only 919 717 (2) 8/8 SIS (1) 1o/zo (1) o/zo 717 (2) 9/9 7/7 (2) 919 (2) 919 (1) 016 013 016 013 016 013 016 013 016 013 qigues are numbers of animals s~riq mCaplasla/uumbar of sn~nals in group. Fibres in parenthesis - £ucompleCe moCaplas£a. BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,..,m r',,,a ",,o

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-6- In mnoke-exposed aninmls, chere yes invariably hypsrplasla often with kerat£ui~at£on on the medlal ventral surfaces o£ the vocal cord, where squamous cells are normally found. The degree of hype~plasla was measured by 1£ght microscopy and between-group cotpar£sons made. In both smok~-exposed groups there va~8 a rap£d increase in epithelial th£ckness (Table 3 and ?£gure 1). A~cer the i~ttial ~ap£d Increase in ep£thel£al thickness, no clear pattern of thanks was seen. The degree of hyperplasLa re~ained £airly well fixed, eve~ as exposures continued. There was no clear d£f£e~euce in response in those au~nals exposed to smoke aC 1.8 dilutlon compared vlCh Chose 8ubjeceed eo L:12 d~luCion. TA31~ 3 HYPER.PLA~ZA OF TIlE ,E..PZTHELZUM LINING THE I~D%A/.. ASPECT OF THE VOCAL CORDS~ FOLLOWING EXPOSURE TO 2 DILUTIONS O~ SMOKE Exposure Pe~£od in Days 1 7 14 28 42 Smoked, 1:8 D:/.lut:£on Treatment Smoked, Sham- Cage 1:12 DiluC~ Smoked Con~ol EpiChel£al thickness* 42 (vmpour phase only) .... i 28 (I0) 39 C9) 39 C7) 44 CZ3) 42 (9) 30 (ZL) 42 (6) 36 (9) 44 (8) 39 (14) 3e (9) t9 (8) :,3 (9) 19 (7) 20 (4) 23 (7) 14 (4) 23 (5) 26 (4) Is (2) 23 (9) *Yilures are mean epithelial thi~Jo~tlJ (~/l[) and (S.D.) BAT Co LTD - MINNESOTA TOBACCO LITIGATION cz~ r~,

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-7- ~n many rats there was a fully dlfferentlaced stratum corneum associated with the hyperplaeia (Table 4). Its frequency of occurrence clmarIy fucraasad as smoke exposure continued and was always Ereater in Chose animals subjected to the higher concentration of smoke. In the animals exposed to smoke diluted 1:8 the proportion of rats with kecatinizatlon of the larynx continued to increase up Co 42 days but in those exposed to smoke at 1:12 dilution the maximum incidence of karatinizatLon was reaehod after 28 days (¥iBure 2). TABLE 4 KERATINISATZON ON TBE MEDIAL ASPECT OF THE VOCAL COEDS FOLLOWING EXPOSURE TO 2 DILUTIONS OF SMOKE TreaCnmnt • |, Smoked Smoked Sham- Case i: 8 1:12 Smoked Control Dilution D£1ucion Number of an4m-ls show4~S k~ratlnleation* I 2/9 (22Z) O/7 (OZ) 0/6 (OZ) 0/3 (OZ) 7 117 (14X) ].19 (11%) o/6 (o:ID o13 (OZ) 1~, 3/7 (43Z) 2/7 (29Z) 0/5 (OZ) 0/3 (0~[) 2s 4IS (soz) 4/9 (44z) o/5 (oz) o13 (oz) 42 3/4 (75Z) 4/9 (A4Z) 0/6 (Og) 013 (OX) 42 (repeat phase 1/6 (17Z) only) *FiXates are number of Inimall lhowlng keratinisaCion/ number of animals in group end (that £ractlon as a parcmnCala). Exposure Period £n Days BAT Co LTD - MINNESOTA TOBACCO LITIGATION m or,,,

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-8- Lu rats exposed to vapour phase only there was no evidence of squamous meteplasia in the floor of the larynx and only a sliEht increase in thickness of ep£chel£um on the vocal cords (Table 3). Only in one rat exposed Co vapour phase was there a fully differentiated stratum corneum. This keraC£nisation has been seen, though not co~nly, in several control rats in prev£ou8 inhalation studies. Scanning Electron l~croscopy The results of this pazC of the work have been described fully in (2) end 8unmarised in FiKure 3. &ll smoke-exposed rats showed an increase in the extent of squamous epithelium on the medial aspect of the vocal cord. This increase in the ~ridth of the band of 8quamous epithelium was initially rapid and continued for up to 42 days at 8 slower rata. At each t/Jne interval over 6 weeks the 8preedinK yes ~reater in the higher smoke concentration Stoup and was also related Co the duration of exposure. Rats exposed for 6 weeks to the vapour phase also showed an increase in 8quamoue epithelium. This incre, ase , however, was less than in the Kroups exposed to whole smoke at the same d~lution (FiKure 3). The use of formalin rather than glutaraldehyde for SEM produced encouraKinK results. For this type of work at low maKn~fication, it can be used quite satimfactorilT. However, detailed work on tissue prepared in this way would not be possible since the revised technique resulted in some loss of cellular decaiI. Compaz4son of d~ferent procedures showed that t~ne'for preparation of tissues may be reduced by dahydrat£nf| material for 1 hour at IOOZ acetone rather than the acetone series previously used. BAT Co LTD - MINNESOTA TOBACCO LITIGATION r,,,o

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-9- DISCUSSION The use of two techniques for evaluating pathological changes in the rat Larynx has allowed both metaplasia (by SEN) and hyperplas£a (by I~) on the medial aspect of the vocal cords to be quantified. The meCaplasia measured by SEN was related to both smoke concentration and duration of exposure as descr£bed previously (2). Squamous me~aplas£a on the ventro-lateral surface, as earlier experiments had 8uKKested, occurred after e relatively short exposure and was found in all smoke- exposed race. £t was ms characteristic that the presence of this feature alone was sufficient for ident£ficat£on of a smoke-exposed au~mal. The occurrence of incomplete metaplasia i.e. where loci of columnar cells pers£sc ~n the ventro-lateral surface, does not appear to be smoke- related. The presence of small £oc£ of columnar cells in the in£oldings o£ the ventral surface was not sufficLent to classify a utaplasia as incomplete. Observat£ous by SEM show such cells to be wall below the general level of the epithelial surface end perhaps protected from the affect of £nhelod smoks. The deKree of hyperplas£a on the med/~l aspect o£ the vocal cord, measured by liKht m£croscopy did not follow any obv£ous trend £ollo~.~aK the initial, rapid thicken~ and could not clearly be related to durat£on of exposure or smoke d£1ut£on, at least for the particular conditions used in this study. The lesion did develop rapfdly however, and became sv4dent after only 5 days of accl~matisation plus ono day of 'full' exposure, fan indication of the rats' respiratory behaviour during exposure would greatly aid interprecatlon of these observations.) In the h~h 8moke concentration Stoups, and to loss an extent the low concenCraClon groups, meCeplaels (by scanninK electron microscopy) ~mmm~ r~ C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE .............................................................. "---'T