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COHBI:NED SCANNING ELECTRON ,I~CRDSCOFY AND LI:GHT REPORT NO. RD. I457 REST~rCTED 21.1. 1977 AUTHOR: G. Sm.i.ch ISSUED BY: S.R. Evelyn PROJECT JOB NO: 11.01.07 DISTEI.BET I0N: Dr. S.J. Green ~Js~. Z.W. Bu4Sbes Dr. E.&. Sm~ord E.M. G£bb, Esq. E.S. Wade, Esq. K.G. Nicholls, Esq. Herr H. Sottorf Dr. F. Seehofer Dr. C.J.P. de S£que£ra Dr. D.C. Felton Library Cc~7 No. 1 11 11 2 " " 3, 4 fl 11 " " 6, 7, 8 " " 9, 10 " " 11 " " 12 " " 13 " " 14 " " 15, 16 COPY NO: BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,..m ",,C>

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GS/CuU./46D Ocoup ltesmazch & Development Centre, Bricish-Amecican Tobacco Co. Ltd., SOUTHAMPTON. 21st J=nuary 1977 COH.BII~D S~J~gNT~qG RI.1ECTiON MICROSCOPY AND LIGHT HICRDSCOPY STUDY ON THE RAT LALYNX C~port ~o. RD.1457 Rast=icted) SU~qA~ Patholosical chauses observed 4. thl rat la~mx, £ollovin8 sxposuce to dilute c£sacetCe smoke, have been evaluated using light microscopy CL~) and scannins electroum~ctoscopy CSSM). The use of the two techniques has allowed both squamous hyperplasia (by LM) and sqtmmous mmCaplasia (by SEN) Co be quantified and related Co the period of exposure to, and dilution of ciKareCte smoke. Both types of lesion £ollov similar paths of development, with a rapid response.to a ralativaly short exposure to dilute smoke followad by • continued increase but mt ceduced tats. With rmsard to the applLcatlon of both techniques in any one study, the use of • coa~on fixative (formaldahyda) sLmpllfies autopm7 procedure buc it would be an advantas@ to duplicate rathmr than split animal SZ'oupI • BAT Co LTD - MINNESOTA TOBACCO LITIGATION O,,,

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-2- INTRODUCTION The development of a sqzus mecaplasLa and ke~at£nisln~ hyperplasia in the rat larynx in response to smoke inhalation may provide a basis for the rapid screening of wmoke toxicity in short-term inhalation studies. Previously, pacholosical changes in the larynx have been noted by light microscopy after a 6-weeks exposure to dilute cisarette smoke (I). However, examination of larynxes from animals subjected to less chart 6-weeks exposure 8uEgested that changes may have taken place earlier in the exposure period. Details are liven below of 8erLsi LH observations of chanses in larynKeal epithelia Ln rats exposed to smoke over 6 weeks. Corresponding SE~ observations have already hem reported for mn/Jaals treated in the same way (2). For ease of comparison, in this report the new LM details and a sumnary of the complmnentary SEM ~rk from this combined exper~nent are pretested tojtther. Using both microscopy techniques it is possible to quantify different smoke-induced changes on the medial aspect of the vocal cord; thickening of the epithellmn (squamous hyperplasia) by U4 and spreadlu@ o£ the ep~thellum (squamous metaplasia) by SEH. It is hoped that by evaluative tissue from any one study by both these methods a thorough screening of la~js~x tissue ~r~11 be possible and maximum information on observed c~anSes will be made ava£1able. It is important that in • bloassay system the preparative techniques and method of evaluatlnS are as simple, accurate and repeatable as possible. In this report the possibility of combintn8 lisht m£croscopy and acanninl electron m4croscopy techniques is examined, tolether with the £easibility of us~ comnon preparative methods, BAT Co LTD - MINNESOTA TOBACCO LITIGATION r,,o 0%

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-3- MATERIALS ~D ~TnODS ,| t The 6--pore version of the B.A.T animal exposure 8yscem yes used for all chls work. hcs were exposed co smoke from s filter ciKareCce (354) made from a scan~awd flue-cured blend of tobaccos. The Tlq4 deZivery of the c£Karecce co Ohm exposure 8ysCmm gas 18.1 mg/CiK. Ten Kroups of anlma18 were exposed co smoke ac two dilution levels, usinK the standard 5-day mcel/~aciseCion procodure in which the duration of exposure ks ~crommod to the full dose on d4y I. l~cails of the groups are given in TabIe 1. TA~-E 1 OI/YLLqE OF EXPE~~ BHOWIIqG DUR&T~ON OF EXPOSURE FOE VARIOUS TREATMENT GqLOUPS Exposure Period FolLowing 5-day Acclinacisttlou (days) 1 7 14 28 42 42 Smoke DiluC£ou (smoke:air, v/v) L 1:8 & 1:12 1:8 & 1:12 1:8 & 1:12 1:8 & 1:12 L:8 & 1:12 1:8 (vapour phase only) 1 8hma-mmokod and 1 coucrol ~coup ware k£11md on each of the 5 autopsy days. Total of 21 8~oups, 282 race. BAT Co LTD - MINNESOTA TOBACCO LITIGATION o%

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-4- There were 18 rats in each of those Stoups exposed to diluted smoke and IZ in a group exposed to vapour phase only. Corresponding sham smoked (12 animals per group) and cake control groups (6 animals per group) were included for each time period, K£vins in all 21 Stoups (282 rats). Animals were k£11ed on the day follow£ng exposure, the larynxes d£vided roughly equally for processing for either SEN or LM (see (2) for details). AS an additional check on processing technique, con~rol larynxes were fixed in buffered formalin overn~ht for scanning electron microscopy. (Gluceraldehyde is normally used but is more hasardous than formalin. ) The extent of squamou8 ep~thellum on the medial aspect of the vocal cord mas determined by SID4 (2). Larynzes for light microscopy were cut transversely ~o include the free edge of the vocal cords and the ventral depression. Hmasurmmmnts were taken of maximal uniform squAmous epithelial thickness on the medial aspect of the vocal cords, £ncludin8 the stratum corneum i£ this was present. OBSERVATIONS .Light Hicroscopy The epithelia present in the larynx sections and their relative distribution in the sham-smoked and cage control rats wire me descrLbed previously (3). In all control rats there was non-ciliated peeudostratifiad column Jr and stratified squamous epLthel£um £uvestiJag the tell of the lumen and the vocal cords. Exposure to smoke caused squamous metaplas;- of the peeudostrat£fied columnar cells in all rats (Table 2). This was often accompanied by the development of a full di£ferentiated stratum corneum which increased the BAT Co LTD - MINNESOTA TOBACCO LITIGATION r,,o

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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

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-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

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-10- and h~,perplas~t (by lijht aicroscopy) £ollowmd a s~m£1er race o£ development. For both conditions, there was a rapid response to a relatively short exposure. This was £oZ1owed by • scabil£smcion o£ the hyperplas£a and a continued increase in the mmtaplas£a though at • reduced rate. T~ the "case o£ the metaplasia the explanation for the reduced rate of development t may thus not be the presence o£ ~ types o£ colu:mar eplthelh as was oriK£nally thouKht (2). Tt may be chat maximum e,Yfect occurs a£ter only 4-6 weeks exposure irrespective of the epithelial involved. The siKni££cance o£ a meal1 increase in mean Kroup epithelial thickness £n the an~nAIs exposed to vapour phase only Ls di£££c~It to define at present. With such small numbers per Kroup, one mn/Jnal with • thickened epithelium (due to • stratum corneum) could puc an unfelt bias on the nun value for epithal/2Ll thickness. It may, however, be • 8enuine reaction bearing in mind the marked Lab£11ty oF the l•rTnKeal eplthel~s. Further york £8 needed before the contribut£on o£ vapour phase to the changes in laryngeal structure £ollovinS smoke exposure can be assessed adequately. Two di££erent techniques have been used here to evaluate chansas in the rat larynx. The study has shown thl problems of div£din8 relatively small Stoups o£ larynx smnplts i~to Chose £or SEH and LM. Would it be possible to split each larynx, one hal£ foe U4, the other £or The sa4Bittal d£vlsion o£ the l•rynx may. £n fact, be dtff£cult to s~tndaz4ise and there is • distinct llkelihood that the tissue will be damaged during dissection. With regard to hyperplas~s on the medlal aspect o£ the vocal cords, assuming •ccurate section£n8 the reaction to smoke £s somet4mes not equal on both vocal co:de. Thus // one cord o~Iy BAT Co LTD - MINNESOTA TOBACCO LITIGATION O',,

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-11- is available, 8 d£££srent set o£ measurements ~uld result as compared w£ch whole-larwnx secc£ons. The mall amount of work rattled ouc on us£ns formalin fixed tissue for SEN suKKesCs chac ac the lov maKnif£cat£ous used, ic may be possible to make use o£ a co--on £~ciCive. This ~would, for example, m~mpl~y autopsy techniques. Zt muse be noced chac derailed hish mL~fficaCion studies wouZd not however be pracc£cal on such tissue. Also preparative Cecbuiques £or SEN can be s/mpli£ied by dehydrat~ in IOOZ acetone and hoe usinK sec£es of acecone d£1uc£on~. Zn conclusion, the rat Larynx shovs chanses Ln epicheliaZ arransemancs (squmnous hyperplasla, squsmous meCaplasia) after short-term exposure co smoke. Zn general, changes are related Co both exposure duration and smoke dilution. KoCh LH and SEM techniques for assessing these responses era useful and complement each other. However, at ch£s stase :LE both are to be used on any study it mould be an advancaKe Co duplicate groups o£ au£mals co alloy processing o£ individual rathnr chart divided Zarynxes. RXFEKENCES I. 2. 3. BAT Keporc Bo. RD. 1328 RmsCr£cCed, 13.2.76. BAT Report No. KD.1400 Restricted, 11.8.76. BAT Keport No. RD.1387 Uuclass£f£ed, 30.7.76. BAT Co LTD - MINNESOTA TOBACCO LITIGATION O", r',o %0

FIB. I P,D. 1457 RF.STRIC, TED EPITHELIAL THIGKNE,~S (~m) Oi~i THE MEDIAL A~PF.LT VOCAL CORD OF THE M EAINI IIK~P T~GK~ESS ~rr~ O I "11 lii~l~l[ OILUYION • I:11. aI'IOKE OILU'TIQN X ~ a4~OKED A CA~E Gom~Q~. ~n ~Y8 ACG LI MAT ISAT ION EXPOSURE PERIOD IN DAY8 BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢1:::: O',,

FI6. 2 KERATINISATION PER 6ROUP ON THE im OF THE VOCAL COR~ R~. 145"7 RES"TRICTE£) A,SPEC.,T (!) I : II ~IOKE DILUTION • I" 12 ~MORE DILUTION RATS PER GROUP WITH FULLY DIFFmRENTIATEO S'mATUM ¢ORNEUH "/o BAT Co LTD - MINNESOTA TOBACCO LITIGATION O',, r~

FI~, ,5 RI:). 145"/ RESTRtC, T1EO VAPLIATION lhl THE WIDTH OF THE 5QUAMOU5 EP|.THEUUP~ ON THE ME~.L ASPECT OF THE VOCAL CORI). LEVEL I. t: S &I~,DKE DILU'TtON I: 12 ~OKE DILUTION X 6HAM 81vIIOHED CAGE COl~~ V V'AFIOUIR IPHAS~ ON L~I ---AREA ABOVE. LINE IrtDIC, ATES CILIA'rED EPlTHF.LIUM IN THE. *NORIVlAC RAT LAR~'NX °,'V ~TRATIFIED ~QUAMOU~ EPI'I'I-EUIJII~ WlO'TH (/.Lm) 6OO 4O0 ZOO )AYS 0 I0 10 &O 4O V AOCI,.IMATISATION EXPO6URE PERIOD IN OAY8 _._ C~ r~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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