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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

O". BAT Co LTD - MINNESOTA TOBACCO LITIGATION