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DEADLlq~- ]lATE.: I Ii II I t :DT~ZON: i|. Ol.b~ FILE 9~O.: ~L~b . Check List ~hoto~-aF~l ~o. : Reductions= T~ Pa~8: S3G ~) : Z;~t (1) : RAS (2) : RMG (I) : RSW (3) : :4nKr., P-- & V.P. Ausc. [2) : ItS (I) FS (I) : ~K--~Z': CJP de S (I) : DGF (~) : L~br=ry (2) : FiL© TAPE ~= (21i • Typin~ Check:Lr.~E o.. '0~ Sent Rood Ret~-,-ned ~e co=mock,ann (i) ~ ~ ~" ~" 77 ~-- (2) ~,~,~'~ ~ 'L, ~.,I~ ; ~ ... , • h~l ~o ~ol ~I ~tll~l~ to £~tlit&te ~n~ ~e~&ll ~he ]~'08~111 o~ GoR. & D.C. Repo~tI t]=~ ~esc:gL19~. ir~a4p9 lx1111;il thl x~=~)o=~; 'lid ~"izIi],,~r ~p]pg~o~rt~ ~ ~II~l~. The ~o EQZIa =aster after pz~paz~t£on) &t &ll mabeequen~ Ita4~m. It It be Ien~ to cent=~ ££1~=~ trllda the =amte~ when ~he ~ ie £eam.ent. 1. ~r~o~ev= posa4hlo, ~8~"~0 ~o~ ~j~ E~Ot~d be t~itten in ink. 2. ~T ~rp~n~. ~he ~es=lpt Ihould n~llly be rot'Ill.mid to the J~ho=, who vilI iubIOqUent~ ob~tn the G~up Lea~e='I and leIUe~'= &pp~81. ~. CI=~zlatlon should be ~etezm/ne~ by the Issuer. the pro $o~ aCa~urt "Prepi~e M~s~er". 5. AK%o~ 31~gLnt~, one ltx~nd oow IUmt be z~tu~ned to ~e ZemAe~ £~T ~',tn~l &~ and the Ismaer will ecAsu~e t~t one ¢o]p~ Am neon ~:r ~he M~uager. BAT Co LTD - MINNESOTA TOBACCO LITIGATIO_N_ ............. I r,.....,~ ~

ATTENTION This work was produced on the Hagnetic Card typewriter. It is imperative that all corrections, alterations, etc., ! are made clearly. E.G. penc~Iled commas, full-seops could be easily missed. A cross in the marBin, in line with the correction would draw our attention to the insertion. Would you please be kind enouBh to write 1%tEe insortions on a separate sheet of paper, and no...~c cut out portions of typed work. ~his is because the ~Snet£c Cards d~ffer from Paper Tapes. / BAT Co LTD - MINNESOTA TOBACCO LITIGATION O'-,

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3-Mo~. im~TIo. Toxzc!~ s~-oY oN xxTs CI~TE ..~Poe.z so. ~. 14;,;, ~s~zc~D " 23.3.1977 AUTHORS : Lynda V. Wilton G. Smith It. Binns ISSUED BY: S,R. EveLyn PROG. REF. : 11.01.02 DTSTR'rBUT$0N: Dr. S.J. Green Dr. I.W. Bughes Dr. R.A. Sanford R.H. Gibb, Eeq. R.S. Wade, EIq. R.G. Nicholla, Esq. Herr H.E. SotCorf Dr. F. Seehofer Dr. C.J.P. de Siquelca Dr. D.G..Felcon Library Copy No. 1 II II 2 " " 3, 4 lell tt ..~ " " 6," 7, 8 " " 9, 10 " " 11 " " 12 " " 13 " " 14 " " 15, 16 BAT Co LTD - MINNESOTA TOBACCO LITIGATION COFY NO: __ i C C:x

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V' CONTENTS ,| el SUMMARY AND CONCLUSZONS INTRODUCTION MATERIALS AND METHODS C£gara~te Type Animals Exposure System Experimental Design Oh servati.ons Te~m£nal St:udies RESULTS Monitoring of Exposure System Blood C&rboxyhae~oglobin Levels Growth Races Food Co~sump¢ion MorCali¢ies Microbiological MonLnorinK Terminal Studies Bodyvelght s OrEs= weighcs Hi• topal:ho lol;y DZSCUSSION REFERENCES PAGE NO. 1 4 4 5 5 5 6 7 8 8 9 11 12 13 17 17 18 19 20 40 51 BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢..rl k.,"l CO

• ,..',rwl~l(;S 14 6o Group Research & DeveIopme=t Centre, BrLtish-American Tobacco Co. Ltd., SOU~TON. 23rd March 1977 3-)lOFfE .INitIATION TOXICITY STUDY ON RATS EXPOSED TO ,|.~ s.o r oM a C (Report No. llD.1477 ltestricted) ~:. ~.~, V" z co.,. ..szo.s An inhalation Cox£city study has been carried out to determine how chanses An lung petbolo|Ly Lnduced in rats exposed Co smoke £or six weeks were affected by continufng exposures for up to 12 weeks and by termfnating smoke treatment. Animals were exposed to three dilut£on leve:Ls o£ smoke gram a standard £1ue-cured cigarette. Honltorfng of the smoke concencratfons to which an£mals were subjected end blood carboxybaemoglob~n levels o£ animals imned£acely agcer exposure, confirmed that animals Ln the varlous treament &coups recelve~ sf&ni£icantly di££erent smoke dosage levels. Average daiZy good consumption and growth rate were reduced in sham-smoked end particularly in smoke-exposed rats. Animals fncreased the£r food consump=£on and shoved a marked increase in growth rate when both these treatments were stopped. Hortal£ty rates were d£rectly related to the concentration o£ smoke to wh£ch animals were exposed. The pattern o£ £nd£vidual BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-2- v mor~alic£ea showed tha~ throughout the study deaths occurred more often at: the beginning of the week and became less frequent as the week progressed. Exposure to smoke did not produce any significant change in heart or lung we~ht of animals over the 12-week exposure period. Thymus weights of sham-smoked and smoke-exposed rats were reduced relative to those of cage controls. All lesions of the respiratory systa~ previously showu in rats exposed to smoke were again recorded in this experiment but their rate o£ development was shown to be di£ferent. Coblet cell hyperplasia o£ tracheal and bronchial epithelia, increased numbers of alveolar macro- phages, squamous metaplasia and hyperplasla of the larynx were seen after two weeks o£ exposure to smoke. With the exception o£ tracheal goblet cell hy~erplasla, after 6 weeks og exposure all these changes showed a maximal zesponse. These responses were either maintained (macrophages, bronchial goblet cells) or possibly reduced (laryngul hyperplasla) as exposures ~ontinued up .to 12 weeks. The frequency o£ tracheal goblet cell hyperplasia and alveolar metaplas~a ~acreussed projrssslvely throughout the study. ~hen exposure to smoke was discont£nued, all smoke induced lesions, excep~ alveolar :etaplasia, rasressed and tissues approached normality during a holding period of 6 weeks. In contrast., the frequency of the alveolar me~spLasia induced during the smoke-exposure pwr£od, comt~nued to increase even when an/~als were not being exposed to smoke. The pathology findings presented here from those animals exposed to smoke diluted T:12 with air will be compared with observations made on BAT Co LTD - MINNESOTA TOBACCO LITIGATION v'O

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-3- animals from the same experiment exposed to smoke diluted 1:8 with air, but assessed by an independent investigator. The final conclusions to be drawn from these combined results, in relation to the design of relatively short-term irritancy snudies, will be reported separately. BAT Co LTD - MINNESOTA TOBACCO LITIGATION o (..j-i o-,, -,..j

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-4- V V I N'I~, DUCTZON Eerli.er complrat~ve ~nb211eCion toxicicy studies carried out in Chess laboratories have involved the treatment of animals with smoke for approx~matel7 6 weeks (i, 2, 3), as sugiested by guidelines relating to the assessment of smoke toxicity (4). These experiments consistently produced smoke-induced changes Ln rat lunZ structure which wre related Co the duration of exposure end the desree of d~Lut~on of smoke. However, it was not kno~m vhethez an exposure period of 6 weeks was, in fact, optimal for such short-term irrltancy studies. The purpose of the experlmenC described in this report ~ts three-fold: (i) Co determine the C~ne-course of the funs pathology chanses induced over a 6 week period. (il) to determine how such changes developed as smoke-exposure was extended up to 12 weeks. and (iii) to determine the effect of stopping smoke exposure on the changes in lung structure produced by 6 weeks of treatment. ~TERZALS AND METHODS Cigarette Type Filter cigarettes (Code H3gl) used for the exper/~ent were made from a standard flue-cured blend (Blend 72). TPM delivery from this cigarette to the sn~nal exposure chamber, when smoked under standard conditions on the inhalation system, was 19.6 ms/ci2aretce. The cigarette burned to a bucc length of 25 mm in 10 puffs. BAT Co LTD - MINNESOTA TOBACCO LITIGATION c~ ................................................ --"T

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-5- Animals White albino rats (Strain CFH~) were suppl~ed by Angl£a Laboratory Antis. The average weiKhts o£ animals on arrival at the laboratory were; males 200 S, females 170 g. AC chase weights, the animals were approximately 50 days old. Animals were kept 6 per case on autoclaved wood shavinss and had free access to food end water. Exposure System In this experlmant, an/~als were subjected to frmsh, diluted smoke on the 5-port version of the B.A.T exposure sTstem (5). A clean machine, exclusively fOE the "sham-smokinK" of rats, was used to provide appropriate treatment-control animals throuehout the study. Exper imencal DesiEn The b~sic desist of the study is shown in ¥iKure I. i. Groups of animals were exposed for varylnK periods up to a total of 12 weeks (ProKrassion). Ln this part of the exper£ment, an/male were k£11ed on ~he de7 £mmed£ately foILowinK their last exposure to smoke. 2. Ocher groups of rats were exposed to smoke for 6 weeks, and then held for up to 6 weeks without further exposure to smoke Cl~sression). ~t was not possible to expose aLL the Stoups of animals simultaneously, therefore the start of the e~per£maut was staKKered. The animals in Stoups I to 7 and 19, plus those in the approprLate control Eroups, were delivered at the same time. A second delivery of animals, of slm£1ar eke and weisht tense, was made 5 weeks Later. These animals £o~med Stoups 9, iO, Ii and ~heir case conErols. One treatment Stoup (6 weeks cr~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-6- exposure) was duplicated (Group I and Group 9) so chat comparisons can be made between ohm animals of the first delivery and chose o5 the later one. For each o£ these two phases of the whole experiment, groups of animals were exposed Co smoke diluted 1 in 8 or 1 in 12 with air. One mddlcional group (Croup Ig) was exposed at the max/mum toierable smoke concencratlon (dLluc£ou Level I in 7) for g uetks. AC all chess dilution levels, animals were routinely given 2 x i0 mLnuCe exposures (4 hours between exposures) pmr day on weekdays, and I x lO minutes exposure on Saturdays and Sundays. Exposure o£ anneals to smoke began on l~th September 1975 and the experiment was compleced on lath December 1975. Each rime smoke-exposed animals were killed, corresponding sham- smoked end case conCrol cars were also Oaken. All groups comprised 6~ and 6~ rats, except the group exposed ac I in 7 dilut£on, which contained 12c~a~,d 12~ rats. In all 468 animals were used. Observations TPI4 deliveries from the cigarette to the puf£ generation unLt of the inhalation system and to the exposure chamber icsel£ were decermined gray/metrically, alter collection of particulates on Cambridge ££1cer pads. Smoke concentration within the exposure chamber was zmasured regularly £or e~ch o£ the 3 smoke dilutlon levels used in the experiment. Throughout the experi~nent, blood carboxyhaemoglobin levels of samples were measured as soon ms possible alter exposure to smoke. Blood samples £rom anaechetised animals (Ketamine hydrochloride I0 mg/lOO g bodywe£ghc) were taken [rom the orbital s£nus and analysed using an lnscrumentacion Laboratories CO-Ox~mecer. BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢...,m

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-7- Growth curves and terminal bodywe£ght data collected from previous studies have shown that exposure to smoke end sham-smoking of animals retard their rate of growth. To investigate this effect further, throuKhou~ this study average food consumption of animals was determined on alternaue days. Food hoppers on the cakes, were filled with weighed amounts (400 g) of dried, pelleted food. The food was reweiEhed on the t following day and the average food consumaptlon per rat per day calculated. Individual bodyweights of animals were routinely recorded three times per week throughout the study and the final bodyweight of each animal was determined iuusedLately before post-aortae examination. All mortalities, with appropriate details, were recorded. This study was the longest experiment to date carried out in our laboratory. Bearing in mind the potential r£ek of infection of animals held for lone periods, it was considered worthwhile to monitor the level of contamination of the air in the downstairs inhalation area of the Life Sciences Building, as an index of the general standard of cleanliness within the animal unit. To do this, 9 cm plates of blood egar were exposed for 15 minutes in the work areas. Plates were incubated at 37°C and counts of colonies made after 2 and 5 days of incubation. Te.rminal Studies Autopsy technique, histological examination and quantitative microscopy have been described (6). The weights of heart, thymus, combined InnK and trachea from each animal were recorded az pos~-mDrcem. The following sections were =ned in dar, a£l for each animal in the study: larynx (transverse to include the central depression and free edge of vocal cords), trachea (transverse to include tracheal and thyroid glands), (single left lobe including the main intrapulmonary bronchus and the maximum amount from the 3 right lobes). BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-8- In this report the results are siren of the in-house examination of tissues from those animals exposed to smoke at dilutions of 1 in 7 and 1 in 12. Tissues from rats exposed to smoke at a dilution of 1 in 8 were given to a consultant pathologist and the results of this examination will be presented separately (7). Control groups of rats were common to the two main dilution levels (1:8 and 1:12) and material from these animals was divided equally for examination by the two pathoLosls~s involved. The pathological examination of material from the" complete experiment was split for two reasons: primarily, to obtain an independent view on the treatlnent-induced changes in lung pathology seen in this relatively complicated study; secondlyp to check the consistency of observations of two observers using essentially the same semi-quantitative h£sto--pathological techniques to examine some co~n (control) material. RESULTS MonitorinE of Exposure System (i) CiBarette TPM Delivery Mean TPM delivery from the buut end of the cigarette was 24.1 mE/ cigarette. Mean ~ delivery to the exposure chamber was 19.6 n~/ciEarette. Mean TPM loss due to deposition in the puff generation unit of the inhalation 8ysta--was &. 5 ~/ctsarette. The los8 of smoke particulate within the puff Ksneration unit. before transfer of smoke to the exposure chamber itself, was 18.7Z of the TPM &enerated from the cigarette. (ii) Co,ncentrati.on of Smoke in Exposure Chambers Regular s~ling of expolnaEe ch4Lmbers throughout the course of the study gave the mean smoke concentrations to which animals were subjected (Table I). BAT Co LTD - MINNESOTA TOBACCO LITIGATION Lr'l ET~

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-9- TABLE I SMOKE CONCENTRATIONS ~ ± S.D. :) FOR EAC~I DTLUTION LEVEL USED DUEING THE EXPERIMENT Smoke Dilution Level (smoke: air/v:v) 1:7 1:8 i:12 Smoke Concentration Cms/~) 8.5 (o. 5) 6.7 (0.7) 4.7 (0.4) • Number of Observat £ons 25 65" 63 Details of the TPMmonitoring carried out durinK the experiment are shown in Figure 2. This mhows all individual concentration values for each smoke dilution level monitored throuBhout the experiment. Moat values (80Z of all samples) were within ~ IOZ of the total mean smoke concentration (dotted Zines) for each of the three dilution levels used. Whenever the values for the chamber smoke concentration fell outside these limits, checks were made to ensure that the inhalation nmchine was set properly and functloninK correctly. After any necessary adjustments had been wade, the chamber smoke concentration was re-monitored and only when this value was wlChln acceptable limits did the exposure of the animals begin. Blood Carboxyhaemoslobin Levels A summary of the blood carboxyhaemoglobin (CORb) levels in 8amptes taken after exposure co various concentrations of smoke is shown in Table 2. Mean values for pooled dace from m ale and fmmale animals are given, mince analysis showed no significant differences in COHb levels between sexes. BAT _C_Q_ LT_D - MINNESOTA TOBACCO LITIGATION

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-i0- TABLE 2 BLOOD CARBO~OGLOBIN LEVELS IN ANIMALS FOLLOWING EXPOSURE TO THREE CONCENTRATIONS OF CIGARETTE SMOKE Smoke Concentration (mE/i) and (Dilution Level) mm 8.5 6.7 4.7 (1:7) (1:8) (1:12) Blood Carb?xThaemogLobin ~Z).. "Progression" 37.9 (n) (16) "ReSts s • ion" (n) Total Mean . 37 .____99 . n = number of observations. 35.6 (39) 31.3 (49) 33.5 27.2 " (~8) 27.5 (59) 27.4 In those animals exposed to the forest concentration of smoke, all COBb values ware quite similar, at approximately 27.AZ COHb. Values were higher, though ~hey rended co be more variable, £n those samples taken from animals exposed co the two highest smoke concentrations, particularly in those Stoups (6, 7, 10 and IX) in the "Prosrmssion" section of ghe study. Mean blood carboxyhaemoglobln levels in an/male were clearly related to the concentration of smoke to which animals were exposed. A~alysis of the data did show hishly eLgn£ficant differences in COHb levels in the relatively Lares numbers of animals exposed to the t~4o dilution level• 1:8 ~ad lz12. FollowinK exposure under chase conditions, Charm were no significant differences in COHb levels in rats from the two separate batches of animals which toKether made up the complete axperLment (Append~ TabLe 2). An/mals in the Kroups exposed to different BAT Co LTD - MINNESOTA TOBACCO LITIGATION Co

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-11- smoke concentrations were clearly subjected to different smoke dosage bevels, since it has been shown Chat for this type of cigarette, blood COab Level gives an ind~catlon of relative TPM dose to the lower respiratory system (8). Full details of the analysis o£ COHb figures are g~.ven ~n the Appendix. Growth ,Rates All froth curves for the var£ous groups of smoke-exposed an/~nals p~otted against the appropr£ace contro~ groups showed a similar pattern. Examples of such curves for two of the more interesting groups of animals are shown in Figures 3 and 4, The £amilfar observation of a reduced growth ~ate in smoke-exposed rats compared with cage control anfmals, with an £ntermedlate Krowth rate £n sham-mmokers, was a consisteut gind£ng in this experiment. Generally, there was not a clear difference between growth rates o£ annuals exposed to ~ifferent dilutions of smoke (Figure 3). For those animals which were subjected to 6 weeks of exposure to smoke and subsequently held for • 6 week bold£ng period, a different @ro~h pattern was recorded (Figure 4). Exposure to smoke again caused a slower rate of growth of animals over the first 6 weeks of the exper/=ent. When smoke exposure was stopped, the grolrch rate increased markedly and the bodywe£ghe Kain was close to that og sham-smoked animals after approxJ-mately 14 days of recovery. It is not particularly useful co present the complete set of bodyweight curves for all an~uaals used in the experiment. An overall assessment of relative Krowth rates of rats in the various experiJeenCal groups has been made by a simple visual inspection of these curves and BAT Co LTD - MINNESOTA TOBACCO LITIGATION C~

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-12- is g£ven in Table 5. Further details of the effect of treatments on the bodyweish~s of rats, as assessed by analysis of the final weight of animals a~ post-mor~em, are siren below. TABLE 3 ,Asszss .z, OF zz xzvz GRo a ZAT oF FROM BO~IGHT CURVES Group N~ber 11 iO 9 7 6 I 2 3 4 5 1.18 1.18 i18 Growth Rate Males < 1/12 < sham < cage I/8 I/8 - 1/12 < sham < cage S L/12 < sham < cage < 1/12 < sham < cage = 1/12 < sham < case Females 1/I2 < 1/8 < sham < cage 118 < 1112 < sham = cage 1/8 < 1/12 < sham < cage 1/8 - 1/12 < sham < cage 118 < 1/12 < sham < cage II12 < 1/8 < sham (<cage~ 118 < 1/12 < sham < case 1/8 - 1/12 < sham < cage 1/12 s 1/8 < sham < cage 19 *caSe control as for group 3. Food Consumpclon II7 < slum < case 1112 S 118 < sham < cage 1/8 • 1112 < sham x cage 1112 s 1/8 < sham (<caSe~ 1/8 < 1112 < sham < rage 1/12 < 118 = sham < cage 1/8 < 1/12 < sham < cage 117 < sham < case In this exparlmenC, for the first c~ane, avexese food consumption of ra~s in the var£ous experlmeutal groups was measured. De~ails of food consun~tion rates for eli of the groups are not eesent£al and are not BAT Co LTD - MINNESOTA TOBACCO LITIGATION C~ Co /',O

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-13- given here. Representative curves, which show some of the commonly observed food consumption patterns are shown in Figures 5, 6, 7 and 8. These include the patterns for chose animals for which bodyweight gain curves have already been described. In male rats (Figures 5 and 6), food consumption of cage control an~Jnals showed a gradual increase from approxlmately 2~ E to 30 E/rat~day durinE the experiment. Animals which were sham-smoked throughout had a food consumption rate which wad approximately 15Z lower than that of cage controls. Animals exposed to smoke for 12 weeks showed a roughly 25Z reduction compared with the cage controls and had a daily food consumption of about 20-22 8/rat~day. There was no noticeable difference in food consumption rate between the grou~s of rats exposed to different dilutions of smoke (Figure 5). Male rats subjected to 6 weeks of sham-smokinE or smoke exposure, in comparison with cage controls, showed a reduction in food consumption. When either treatment was stopped and the en~nals then held as cage controls, food consumption rose quiCe markedly ov~T approximately 10 days to reach that of the original cage control race. Again, animals exposed to two dilutions of smoke responded in essentially the same manner (Figure 6). Food conJumpcion of all female rate was lower than ~ha~ of the males in corresponding groups. Nevertheless, trends in food consumption were generally s~nilar to chose described above for males (Figures 7 and 8). Mortalities A summary of all mortalities recorded during the complete study is given in Table &. Unless otherwLse staCed, all mortallcies occurred BAT Co LTD - MINNESOTA TOBACCO LITIGATION W O',, w

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-14- during or immediately after exposure of animals to smoke. No sham- smoked or cage control an~nals died, nor did any smoke-exposed animals ~ie during the post-exposure holdinK period of up to 6 weeks. TABLE 4 TNCIDENCE OF MORTALITIES IN GROUPS OF RATS EXPOSED TO DIFFERENT DZLUTIONS TO SMO--KE Group Number 1:7 l:B 1:12 Total pre--tsrmination deaths Male Female Male Female Male Female "~ 1 - - 0 I 0 O 2 - - 3 1 0 0 3 - - O 6 0 O 4 - - 0 3 0 O 5 - - 2 I 0 O 6 - - 1 4 0 1 7 - - 1 1 0 O 9 - - 0 2 O O 10 - - O 2 0 0 .~ II - - 1 3 O O 19 4 8 .... No. deaths/ 4/12 8/12 8/60 24/60 0/60 1160 i | L No. exposed 12/24 32/120 1/120 Total deaths/ 45/264 Total exposed Camments i rat pre&~ant: killed day 47 Male dead in cage overnight 1 rat pregnant: killed day 41 Female dead in cage overn~Khc m !m BAT Co LTD - MINNESOTA TOBACCO LITIGATION Oo 0",%

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-15- Mortality races were higher amongst the female compared with the male rats. This sex-related difference in mortality has been reported in earlier studies. The overall mortality rate Of animals exposed to smoke was low, with 45 deaths in a total of 264 animals (17Z). Of these deaths, 2 were not related to treatment with smoke. When incidence of treatment- related death is related to exposure conditions, mortality rate for both sexes was clearly proportional to the smoke concentration to which the animals were subjected (Figure 9). There was no suggestion from the mortality data that deaths tended to occur paruicularly early in the experiment(Figure I0). For those groups (i, 2, 3, 4, 5, 9, i0 and 11) subjected to smoke for up to 6 weeks only, the 24 treatment-related deaths wmre spread fairly evenly throuEhout the exposure period. In those groups (6, 7 and [9) subjected to smoke for more than 6 weeks, there were 18 treatment-related deaths, all except one of which occurred within 7 weeks of startln$ tfull' exposure. However, super~J~posed on Ehis overall pattern of a fairly steady death rate for animals over a 7 week exposure period, there was clear evidence of deaths occurring more frequently on particular days. Figure [I shows all treaement-relatadmortalities according to the day of the week on which they occurred, irrespective of the time during the exper~nent. Clearly, throushout the study more deaths occurred on ~ndays and Tuesdays than on any other days. and the number of deaths fell as the week progressed (3 of the 7 deathm which occurred on Wednesdays involved animals which reached 'full smoke' exposure on that day). BAT Co LTD - MINNESOTA TOBACCO LITIGATION O',. GO

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-16- Microbiolo&ical Muonltor£n~ (by P.C. Stauber) The results of monitoring of the air in the work areas shown in Figure 12 are siven in Table 5. In Eeneral the hiKhest recoveries of oraanisms weEe made in the anima~ rooms. The number of fungi recovered was negliKibleP and neither the numbers nor the t-~pes of bacteria (mainly Nicro~o¢cus types) were considered to represent a hazard. TABLE 5 MICROBIOLOGICAL MONITOEING OF AIR IN THE WORK AREAS USZNG BLOOD A~%R SETTLZNC PLATES 1 2 3 4 $ 6 7 8 9 I0 11 12 13 14 15 16 17 18 19 20 21 22 Humber Micro-organisms (as Colonies) per 9 cm Plate ,, J 1 Day of Incubation 20 0 43 45 15 (Spr) 44 3 20 4 O 7 2 6 6 4 (Spr) I0 4 2 5 2 1 5 Days of Incubation 21 (M) 0 45 (M) 50 (M) 16 (Spr) 44 (M) 3 22 (M) 5 I 8 5 15 21 9 (Spr) 12 4 3 5 4 2 1 (M) -mainly M~crococcus (Spr) - Spreading organism (inhibicm ErowCh of ocher coloniee) BAT Co LTD - MINNESOTA TOBACCO LITIGATION Q..m O,,, c.o r'x.a

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-17- Terminal Studies Previous experiments and the present one, have shown the effects of smoke-exposure and 8ham-smokinKs on the growth rate of race. In the earlier studies, corresponding organ weight changes in smoke-exposed rats compared with control animals have also been suggested. These chanses have most often related Co thymus, heart and combined lung and trachea weights (I, 2 and 3). There are several difglcultlms in the evaluation Of bodywe/ght and organ weight data which are worth commenting on st this stage. Because of the relatively large numbers of animals involved in smoke toxicity e~udies, and the experimental design (staggered introduction and kiliing of animals), animals may come into a study at different initial body- weights. Secondly, the treatment to which animals are subjected durin8 such studies reduces ~heir rate of weight gain compared with cage or shamosmoked control rats. Clearly such an effect, due largely to a reduced deposiclon of body fac in smoke-exposed animals, could influence the relative weight of an organ (weight of organ/lO0 g bodyweight of animal) in the absence of any real treatment-induced change in the weight of the organ icself. In an atte~pn to overcome such problems, statistical analysis of term/hal bodyveights and organ weights from this study has been based on the use of absolute values only, with an appropriate correction of .the iniclal bodyweishcs of the animals. A gunnery of the findings of this method of analysis is Elves and details of the statistical work are available if required. The correct analysis and interpretation of organ weight dace from toxicology studies BAT Co LTD - MINNESOTA TOBACCO LITIGATION O c_,--. O,,. C.D r',o r',o

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-18- V still presents several di££iculties (9, IO) and work is continuing on this aspect of our investigations. Bo d~n~e ~ght s Final bodyweights of animals killed at intervals throughout the study are shown in Figures 13, 14, 15 and 16. For both sexes, a similar gro~h pattern emerged. Weights of smoke-exposed rats ~ere s£gni£icantly lover than the corresponding control animals after 14 days of treatment, and remained so throughout the exposure period which extendedup to 12 weeks. The weights of animals (both sexes) exposed to both dilutions of smoke were never significantly different. Initially, sham-maoked and control animals had similar bodyweishts, but £or both sexes a significant reduction in body~elghC of sham-smoked animals compared vlth cage controls occurred a£ter ~2 days of shmm-amoking. Thereafter. the body~eighc of sham-smoked animals remained intermediate between ChaC of the case control and the smoke-exposed rats (Figures 13 and 1/+). In those anJ~za18 which were exposed to smoke for 6 weeks and then kept for a further 6 weeks holding period, the di££erences £n bodyweight between s~oke-exposed and controls at the end of the 6 weeks exposure period were as described above. During the boldi~ period, male rats previously exposed co smoke recovered in about 3 weeks co achieve the bodywtight of correspond~ sham-smoked animaZs. Neither of these two groups grew su~£iclenCly to reach the weiKhc of male cage control rats over the 6 weeks holding period (Figure 1S). Fmnale smoke-exposed and sham-smoked animals grew more rapidly and reached the body~eight of corresponding cage control rats between 3 to 6 weeks after the cessation of treatment (Figure 16). BAT Co LTD - MINNESOTA TOBACCO LITIGATION ~o

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-19- OrEan Weight s Thymus In all Kroups of animals, control and smoke-exposed, thymus weight fell throughout the exper~enc. After 2 weeks of smoke exposure, thymus veishts of both male and female animals were lower than those of the corresponding cage and sham-smoked controls. As the experlmenc continued this separac£on pers£sced in the male animals buc ~as less dLsc£ucc, chouKh comparable in the females. Those animals exposed =o smoke for 6 weeks showed the reduced thymus we£gh: compared wlth both sets of control animals. After smoke exposure had stopped, within 1 week thymus weights of all animals were noC 8isnif£canCly different, and remained comparable over the remainder of the post-exposure holdinK period. Heart There was no consistent effect of tTsa~nanc, either smoke exposure or 8ham-smokinK, on heart weiKht of male or female animals subjected to up co 12 weeks of exposure. Heart weiKhcs in smoke-exposed animals which were allowed a 6-week post-exposure observation period were not stKnificantly different from cot'respond~nE 8bent-smoked and caKe-control r&~s. Lunls and Trachea There was no cons£stent effect of treatment, m£ther smoke-exposure or shaw-smok£nK, on funk and trachea welg.hc of male or female animals subjected to up to 12 weeks of exposure. Lung and trachea weights in smoke-exposed animals which were allowed • 6 week post-exposure observat£on period were noc siK~/fLcancly d~fferenc from correspondlnK sham-smoked and cake control race. BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,_r C~ r',,o o-x

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-20- H£stopathology Changes seen in rJhe lungs of animals corresponded with those observed in previous inhalation studies and can be divided into the following: I. Peribronchial lymphatic tissue 2. Perivascular lymphatic tissue Non-smoke induced chanKes 3. Focal pne~oD/tie ldi,tce llaneous c~utnKe s 9 6. 7. Incipient alveolar metaplas~a 1 Alveolar macrophage activity Goblet cell byperplasia Smoke induced chan~es 1. Peribronchia!Lymphatic Tissue Concentrations of lymphocytes were found in aver7 rat especially at the junction of the bronchi and bronchiolee (Table 6). In certain cases they a~reSatad between ~he airways and adjacent blood vessels. Larse foci found in the lamina propria of the main bronchi invaded the smooth muscle layer, apparently dLvidL~ it into discrete blocks. In @xtrlme cases the bronchial aplthaLiumwas d£sruptedand there was deciliatlon and loss of Boblet cells toEether winh attenuation of the columnar epithelium cell. C~ r~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION .............................................. ---T"

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-21- TABLE 6 NUMBER OF RATS WITH PERIBRONCHIAL LYMPHATIC TISSUE v 2 11/11 Exposure to Smoke (weeks) 4 6 9 12/12 11111 12/12 12/12 12/12 11111 10/10 12112 12/12 ,,,n, 1 4 7 21 42 ~lding Period (days) After 6 Weeks of Exposure All Sham- Smoked Animals 30/30 30130 All Cage Control Animals 30130 All Sham- Smoked Animals 30/30 All CaEe Control Animals The difficulty in assessing the extant of the lymphocytes (due to variation ~n 8xact plane of section of the three right lobes) made Kroup comparison impossible and che£r frequency in 811 jroups could be regarded as within 'normalt limits, 2. Perivascular L~nphac£c Tissue In all rats, at least one vessel was assocLated with 8 focus of 'lymphocytes whLch often completely surrounded and penetrated the outer adventitial layer. The lymphatic tissue was often found between blood vessels and adjacent broncbioles. Mnltiple per£vascular cuffs (more than five blood vessels per luns section surrounded by lymphatic t£ssue) were common end their frequency was reduced by smokm exposure (Table 7). Eats exposed to smoke for 4-12 weeks had reduced 17mphatic tissue compared with those exposed to smoke for 2 weeks only end control SToups. Animals held for up to &2 days after exposure showed varying amounts of per£vascular lymphatic t£ssue apparently returninK to normal OQ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-22- values (Figuce 17). Variation £n control values however makes detailed comparison difficult. TABLE 7 |,., NUMBER OF RATS WZTH MULTIPLE PEItZVASCUI.qR CUFFS 2 411o ~3Z Exposure to Smoke (weeks) 4 0/12 OZ 6 1111 9X 9 o112 OZ 12 0/12 OZ All Sham- Smoked Animals 1313O 43Z All Cage Control Animals 8/3O 26Z 1112 2/11 0/I0 8Z 18Z OZ 1 4 7 2112 17Z 21 0112 OZ 42 Holding Period (dmye) After 6 Weeks of Exposure 8/30 26:[ All Sham- Smoked Animals 3/3O lOZ All CaKe Control Aulmals 3. Focal Pnmmonicis Pneumonitis is an tnflumation of the lung and in the rat manifests itself as lymphatic tissue invading the alveolar wall and eventually the lumen of the alveoli. There is an increase in macrophage activity and thickened walls due to infiltration of lymphocytes and/or an increase in alveolar cells. In extreme cases, the alveolar tissue is lost leavinE larse areas composed solely of inflammtr.ory cells. Focal pneumouiti8 develops as peripheral extensions of pLTibronch~al and vascular lymphatic tissue, as wall as from small discrete sub--pleural sites in the alveolar tissue. Theme sites were co~n but not recorded. BAT Co LTD - MINNESOTA TOBACCO LITIGATION o',, cz~ r',o .................................................................. -'~

BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-23- In all, 34Z of all the lungs examined showed signs of focal pneumonicis (Table 8). Of these, 51Z also had multiple perivascular cuffs. Of the 39 samples of perivascular cuffs, 31 also showed focal pneumonicLs. The variation in control values made comparison difficult bur clearly exposure to smoke greatly decreased the incidence of focal pneumoniuis in the rac lung (Figure 18). RaCe held after smoke exposure also showed decreased lymphac£e acrivit7 buc Co a lesser degree than animals continuously exposed Co smoke. D~iug the. pose- exposure periodm these changes showed signs of return/~g Co normal values • TABLE 8 N.m~SER OF RATS Wlrn~OCaL P~EUMONXT~S Exposure Co Smoke (weeks) , , e 2 4 6 6/8 3/11 2/11 75Z 27Z 18Z 9 2112 17Z 12 1/12 BZ &11 Sham- Smoked Anlmals 17130 54Z All Case Control Animals 1313O 46X 3/12 251 1 3/11 3110 [ 3/12 27Z 30Z[ 25Z 4 7 21 4/12 33Z 42 HoldinsPeriod (days) After 6 Weeks of Exposure 12/30 40Z All Sham- Smo ked Animals 12130 4OZ All Cage Con tro 1 ani,~,l s The above three leslons are cle.zrly related, there being a progressive reaction co a viral infecclon (11). This is seen as perlbronch~al infection leading to vascular and multiple perivascular cuffing whLch in turn BAT Co LTD - MINNESOTA TOBACCO LITIGATION C_m O% C~ (..-.,a

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-24- produces a pneumonitis. Such a progression appears to be age related £n 'normal' colonies. Exposure Co dilute cigarette smoke decreased substantially the reaction of rats to the respiratory infection by a virus. This is particularly noticeable when animals with both pneumonitls and multiple cuffing are compared on a group basis (Table 9, Figure 19). TABLE 9 NUMBER OF RATS WZTH BOTH MULTZPLE PERIVASCULAR CUFFS AND FOCAL PNEUMONITIS 2 4/10 40Z Exposure co Smoke (waeks) 4 6 0112 1/11 0% 1% 9 12 o112 oi12 OZ OZ A11 Sham- Smoked Animals LI/3O 36Z All Cage Contro I Animal s 7130 23Z 0112 1112 0112 2112 m,,, , ,, ' 0112 OZ IZ OX 2Z OZ 1 4 7 21 42 , ,| , _ , Holding Period (days) After 6 Weeks of Exposure 6130 20Z All Sham- Smoked Animals 3/30 IOZ All Cage Contro I Anisals 4. Mist ellannous Changes A bone ~obbec embedded in the subpleural alveolar Cissue producing no inflmmmaCo%7 rmacC£on, occurred in one raC. The evaluation for possible a~physmua was hoe a~cempCed due co variacions in lun8 inflation. In two rats mucus filled a number of alv~Imr ducts, ~n one such case there was also advanced alveolar mecmplasla. Fool of pink macrophages were nor uncommon in the parenchyma (hlstiocytosis) and blood vessels BAT Co LTD - MINNESOTA TOBACCO LITIGATION CXD O",

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-25- occasionally ve~e couSested, probably due to delay in lur~ £nfl&t£on during autopsy. 1~e presence o£ oedematous tissue is unexplained but again is probably an artifact produced by the autopsy procedure. 5. Alveolar ~tt&~l~.a£a Alvevl~ meteplas~ may inic~nlly be confused w~th focal pneumon£Cis since in both lesions there £s an increase in alveolar walI thickness and in macrophase acc£vicy. However, they can be d£stLnKuiehed by cons£dez£~K the s£te o£ occurrence coKether ~r~th the maczophase and lymphocyte populetiouus. 2n rate exposed to d£1uce smoke for 6 weeks or more, clusters of macrophaKes were £ound in ~he reKion of the teru~nal bronchioles end associated 81veolar sacs and ducts (Table IO, Figure 20). They were large, vee~olated, £rrejular in out1£ne and piKmented, often with jolden brown cy~oplaa~c inclusions. ~xp,.p- lo ~-~ER OF ~x,S Wn-~ ~n~LZ ~OCZ OF,~C~O.P~CES 2 0112 0]{ Exposure to Smak8 (yacks) , ,. , -- 4 0112 0]{ 6 3111 27]{ 9 6722" 5OZ t2 10112 83Z &11 Shmn- Smoked 0/30 OZ All Cage Control Jmizmls 0/30 OZ 3111 27Z 4 2/12' 8/12 17]{ 4AZ 67Z i | m 1 7 21 Holding Period (days) A~tez 6 Weeks 0£ Exposure 9/12 75Z &2 1/3o ,3Z All Sham- Smoked Animals 0130 OZ All CaKe Control &n~als BAT Co LTD - MINNESOTA TOBACCO LITIGATION O% Co

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-26- The number of rats showing multiple clusters of ~crophages incrQased with both cont£nuous exposure and also during the post-exposure holding period. At low magnification their distribuCion was clearly seen Co be adjacent to the alveolar ducts and termfnal bronchloles, especially in animals exposed for 12 weeks when the lungs in section had a distinctive mottled appearance. 0f the A5 rats v£th ault£ple clusters of pigmented macrophages. 32 (71Z) had thLckenad alveo3~r ~mlls. ThLs thickening appeared usually as d£sor~an£sed hyperplas£8 or =etapias£a, ~rLth the alveolar epithel£um made up og well-orlen~ated cuboldal cells. Zn one rat (exposed £or 6 weeks end with 4 days holding period) metaplasia was advanced with veil developed cuboidal cells surrounding several alveolar ducts contalning darkly pioneered macrophages. A great deal o£ the parenchyma consisted of sheets of spindle shaped calls cent=ed around alveolar ducts and teru~nal bronchi~les. Lymphocytes were often present in the lesions. The lesions shoved an increase Ln reticulin content, suggestJ.ug a possible f£broeie. Incipient mataplas~a or true metaplas~a was not observed in control =acs or those exposed to dilute smoke for 2 and & weeks. Of the remaining groups the i~cLdance of ~taplaa~a i~crQased with increased exposure. For rats held after 6 weeks exposure an /:creased proportion of the anilnals showed a develoumtent of alveolar metapl,asia even in the absence o£ further smoke exposure (Table ll, FiSurm 21). 'Homer, on average, the extent o£ the las£on in any one lun8 d£d noc increxse (Table 12, Figure 22). BAT Co LTD - MINNESOTA TOBACCO LITIGATION o',,

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-27- TABLE II m NUMBER OF RATS WITH ZNCIPXENT ALVEOLAR METAPLASZ.A 2 0/9 OZ Kxposurt co Smoke (weeks) 4 o112" OZ ,,,i 6 1111 9% 9 12 ,.m 4112 9/12 33:[ 7SZ All Sha~- Smoked Animals 0130 OZ A11 Case Control A-~,ul8 i • 0130 OZ .... d 1/12 8Z 1 2111 319 51!2 181 33Z. 42Z 4 7 21 |, 7112 48Z 42 HoldS.rig ¥erlod (days) &feet 6 Week~ of Exposuce 0130 OZ &11 Sham- Smoked An£mals 0/30 OZ All CaKe Cout:rol Anl.mals 2 0 ii Ex-posure co Smoke (weeks) l, 4 6 9 0 16 16 12 35 All Sham- Stoked AnLmLs O ..... I 18 13 12 2O 15 ,. m 1 4 7 21 42 li ii HoZd£n8 Paziod (days) After 6 Weeks o£ Exposure • | , .,| 0 &11 Sham- Smoked A~imais Al1 Case Control An~nal8 O 0 Al1 Cage Control Ani-~- ls BAT Co LTD - MINNESOTA TOBACCO LITIGATION o~ .......................................................... --T

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-28- 6. Alveolar Macrophazes LunKs vhich were reK•rded •s incompletely £nflated were not assessed. The n~nber of alveolar macrophages in a sub-pleural strip 3 uzn long (area 0.54 sq. ~) on the fnner surface o£ the left lobe was counted as an index of the macrophage population. Only cells whose greatest dimension was 10 ~m or more were counted, their shape varying from spherical to elongate. Usually • central single nucleus was present but binucleate cel~s were also observed. The macrophages were often pigmented in the smoke exposed rats (and occasionally in control rats) and either adhered to the alveolar cells or were free in the alveolar spaces. Intensity of pigmentation did not closely relate to the duration of the ezposure perfod. Although macrophage counts varied cons~dezably within any one group of animals, group mean values showed an ~ncrease to a max~numat 9-12 weeks of exposure. Animals held for up to 42 days post exposure showeda fairly rapfd return to 'normal' values during the holding period (Table 13, Figure 23). All smoke exposed rats had greater counts than their equivalent controls. BAT Co LTD - MINNESOTA TOBACCO LITIGATION C 0", CO

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-29- TABLE 13 GI~OUF MEAN COUNTS OF ~CROPI~GES (> I0 ~am~ in 0.54 sq. OF suB-PLEur~n P,.~NCHY~ (s .D. , • ,, • Exposure to Smoke (veekB) 2 9 (5~ 4 6 , i , 17 20 (93 (8) 9 12 22 22 (12) (8) All Sham- Smoked Animals All Cage Control Animals 4 .(2) 21 19 15 (16) (8) (13) 1 4 7 HoldinK Period (days) After 6 Weeks of Exposure 6 (3) 15 7 (8)(5) 2142 All ...... Sham- (2) All CaKe Control Animals 7. Bronchial Goblet Ceils There was no indlc•clon of hyperpl•sia or metaplasi• of the bronchial epithelium of smoke-exposed rats. Ther• was however • loss of soblet cells, deciliation and attenuation of the epithelium caused by perlbronchLal lymphocytes in the underlying lamina propr~a (or the agent ¢aUB~ the foc~ of lymphocyte•?). Altbaush there was some lOBS of goblet cells due to lymphatic tissue, goblet cell activity in the bronchi increased due to exposure to dilute smoke. The goblet cells in the bronchi varied considerably in appearance and sta£nin$ re•orlon. The majority vere seen only as caps of acl4 KlycoproteLn with basal $ranules of neutral SLycoproteins. Fully differentiated 'goblet' shaped cells were uncommon. Difficulty •roBe in interpretation of the nature of the secretory material due to the necessity BAT Co LTD - MINNESOTA TOBACCO LITIGATION m k.,'-I k..r'l O~ O",.

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-30- of altering the staining schedule during process£nK. For this reason, absolute quantification of bronchial goblet cells was not possible. Thus a method of gradinE was used for a fixed site between junction 1 and 2 on the main left bronchus. No adjustment was made for areas affected by lymphau£c cissuep but funks not clearly showing the area were discarded. The grading sysn*m proved Co be accurate and repeatable and a convenient means of overcominE cell variation. The soblet cell population of the bronchi increased with /ncreasin8 exposure reaching a maX',hum at 6 weeks. There was no apparent increase in numbers of fully differentiated Eoblet cells in rats exposed to dilute smoke for periods lonKer than 6 weeks (Table 14, Figure 24). Tb~se rats held for 42 days post-exposure showed a reduction in Eoblet cell activity which was gradual at first but became more marked bmtween 3rd and 5th weeks post-exposure. All smoke-exposed rats had hlgher values for Eoblet: cell population than the control Stoups. TABLE 14 ESTIMATED GOBLET CELL DENSITY IN FIXED SZTE OF LEFT ZNTRAPULHD~RY BRONCHUS 2 1.2 Exposure to Smoke Cweeks) |, ,i i m • , m A 6 2.0 2.6 9 12 , an 2.5 2.7 All Sham- Smoked Animals 0.4 All CaKe Contro 1 Animals 0.4 2,5 Z.2 2,3 2.3 1.O 1 4 7 21 42 i Holding Period (days) After 6 Weeks of Exposure All Sham- Smoked Animal s 0.4 All Case Con fro 1 Animal-. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-3~- Trachea A single transverse section of the trachea was taken near its ~unction with the lar3ntx. The tracheal lumen was lined by pseudo- stratified ciliated columnar epithelium often with loci of non-cil~ated columnar calls dorsally. These calls are seen typ£cally in sl£ghtly more anterior sections. Differences in the exact level of section or damage due to sectioning precluded the estimation of epithelial height, which varied considerably. A subjective appraisal suggested a l~ossible hyperplas£a or hypertrophy of the lining cells of the trachea from the animals exposed to smoke. The irregular distribution of cillated cells in chase animals was normal. There was no evidence to suggest ~ncreased or decreased ciliary activity. Foci of lymphocytes in the lamina propria were found in both control and exposed 6roups and their distribution was not smoke-related, although in one group of cage control rats, 33Z of the sections had foci of lymphocyces (Table 15). The epithelial cells adjacent to such loci were in some cases attenuated, uon-ciliated columnar or squamous in one or two rats. This squamous metaplasia associated with lymphatic tissue ks not common but has been noted by several authors (12, 13, 14, ~5). BAT Co LTD - MINNESOTA TOBACCO LITIGATION Co C.m .................................................. l

BAT Co LTD - MINNESOTA TOBACCO LITIGATION c...,"7 ............................................................. 'T

-32- 'v TABLE 15 N~.R OF RATS WITS FOCr Or LXMPSOC~ZS IN THE ,u az PROPRIA 2 2/12 17Z Exposure to Smoke (weeks) 4 1/11 9% 6 1110 i0% 9 o/ll 0% 12 2/12 17X All Sham- Smoked Animals 1/30 3% 2/12 0/12 17Z 0% 1 4 3/12 25% i, 7 3112 25% 21 0112 0% Holding Period (days) After 5 Weeks of Exposure 42 2/30 7% All Sham- Smoked Animals , ,,,,, All Cage Concrol Animals I0 / 30 33% 4/30 13% All Cage Control Animals Goblet cells were scarce in this re&ion, again making group comparisons difficult. In order co indicate any possible variation, a simple parameter co suggest goblet cell hyperplasia was the presence of more than five goblet, cells per section. On this basis all the smoke- exposed groups showed an increase in goblet cells compared with the control groups. The number of rats per croup showlnE hyperplasia increased with the duration of exposure over the 12 weeks of the complete study. Of the rats held after 6 weeks of exposure, the goblet cell population rama£ned stable for 21 days, and returned to normal values by the 6th week poet-exposure (Table 16, Figure 25). In most cats the tracheal mero'a~cum $1ands and ducts varied in size, distribution and secretory content and the ducts were often BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,..m C~

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-33- d£1ated. No group differences were apparent either in the extent of the glands or the nature o£ their secretory product. TABLE 16 NUMBER OF RATS SHOWING GOBLET CELL ,,EYPF.RPLASZA IN TILE , UPPER TRACHEA 3/12 25Z Exposure to Smoke (weeks) 4 6 2112 4110 17Z 40Z 9 5/12 42Z 12 7/12 58Z &11 Sham- Smoked Animals , ,m, ,! 1130 3:[ All CaKe Contro 1 Animals ,,, , • L/3o • 3Z 4113 3/10 3110 4/12 331 30Z 30~ 331 1 4 7 21 lioZding Period (days) After 6 Weeks of Exposure 42 I130 3~ Smoked Animals 1/30 3Z All Cage Centre l Animals LarynX, A transverse section was taken to include the aryteuo£d cartilage, the free edie of the vocal cords and the ventral depression. Xn this area the ~ypas of epithelia and their distribution in control animals were as daacribed prevloualy. In all the rats expoaed to dilute smoke nhers were rapid, consistent chanses in the larFnxwhich were related to both the exposure and the holdlns pgrlod. On the ventral surface, the columnar epithelium of the floor of the larynx was replaced by squamous cells, often associated wish a fully differentiated strat,-- corneum. A11 but two of the mke-exposad rata, wen those which had had only BAT Co LTD - MINN~_TA!QBACCO LITIGATION Q.m O~ oo ............................................................. l

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-34- 2 weeks of exposure Co smoke, showed siKns of squamous mecaplasie in the larynx. This chanKe was sufficient to identify a smokeoexposed animal instantly (Table 17). In several rats, loci of columnar cells persisted on the ventral surface. This is referred to as an incomplete metaplasia and in the animals exposed from 2-12 weeks its frequency was not smoke related • In the animals held for up to A2 days following Bmoke exposure there was a rapid loss of keratin and s decrease in the epithelial width on the ventral surface. The number of rats with incomplete metaplasla increased with the length of the hold~ns period. Two animals held for 21 days showed no signs of metaplasia. Of the animals held for 42 days, only one showed a complete meceplasia at the end of this time. In order to quantify further 8quamoua metaplasia in the larynx, rats were noted when the entire larynKeal lumen in section, was Lined with well d£ffersnciaced squsmou8 cells. Such • marked squamous mer~tplasia proKreseed and was maximal after 6 weeks of exposure. When smokinK stopped this particular lesion regressed quickly and was not apparent in rats held for more than one day after 6 weeks of smoke exposure (Table 18). AS before, the ventral depression showed no 8iKns of metaplaeia. BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-35- TABLE 17 NUMBER OF RATS W'£TH SqUANOUS HETAPLASZA OF THE VENTRAL EPTT'I~LIUM ('rNCOMPLETE HE Tj~LASIA) Exposure to Smoke (weeks) 2 4 11111 12112 (3) 6 _ , t 1O11O 9 11111 (1) 12 IIIIi (I) All Sham- Smoked Animals 0/30 A11 OaKe Contro 1 Animals 0/30 m 12112 1.2112 (1) (3) 1 4 ~x/xl 10112 (6) (6) 7 21 IIIii (1o) 42 Holding Period (days) After 6 Weeks of Exposure 0/30 All Sham- Smoked Animals 0/30 All CaKe Control Animals v 2 5/i1 TABLE 18 NUM.KR OF RATS WITH FULLY D~,FFE, KENTIATED SqUAMOUS ~x'rem.nm Ln~Zsc "raF. ~XmU~L U~. Exposure to Smoke (weeks) 4 6 5112 8/10 9 12 .,| 4/11 5/11 &ll Sham- Smoked Anlmals 0/30 All CaKe Control Animals 0/3 ii i= i 9/12 0112 0111 0112 0/11 1 & 7 21 42 Holding Period (days) After 6 Weeks of Exposure 0/30 All Sham- Smoked Animals O/30 All CaKe Control Animals BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-36- On the ventral, medial aspect of the vocal cords, which represents its leading edge, squamous epithelium was normally £ound and was invariably hyperplastic in smoke-exposed rats. A fully differentiated stracLuu corueum also developed. There were several examples of paraker&tos£s which was recoKnlsed by the presence o£ nuclei in the stratum corneum. Their £requency was not related to the duration o£ exposure. In order to quantify hyperplasia, the th£ckest uniform reKion o£ epithelium was measured, including the stratum cornaum, if present. The deKree of hyperplasia in the larTnx reached a umacimum ~cer s£x weeks o£ exposure and decreased sliKhtly as exposure continued up Co 12 weeks. Kacs not exposed Co smoke a£Cer 6 weeks showed a rapid return Co 'normal' values during the 42 days holding period (Table 19, Figure 26). The presence o£ a fully d£££ezentiated stratum eorneum £ollowed the pattern o£ the sqcumous hyparplas~a, but 4t relrassed ac a lascar rate wheu exposure to smoke ended (Table 20, ¥£gure 27). BAT Co LTD - MINNESOTA TOBACCO LITIGATION O 0",% ..................................................... -T

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-37- TABLE 19 ~Pz~s~ oF T~ EPrr~LI~, L~NX.G T~ ~VL~ ,sFzc~ OF ,T~ VOCAL CORD. CROUP MEAN THZCK~, SS ZN ~m,(S.D.) 2 41 (16) Exposure Co Smoke (weeks) w 4 6 39 54 (8) (t4) 9 44 (9) 12 ii 45 (4) All Sham- Smoked Animals 25 (8) A11 Case Con ~ro I Animals 22 (8) 51 (tZ) i 41 30 (7) (8) 4 7 33 (zt) 21 29 (8) 42 ~id£n8 Period (days) After 6 Weeks of Exposure 24 (8) All Sham- Smoked Animals 23 (5) i, At1 Case Cont;rol An£mals TABLE 20 NtR~EIt OF RATS WZTH. I~JLL¥ DZI~FEEENTLATED STRATIR~ CORNEI~ ON MEDIAl, ASPECT OF THE VOCAL CORDS Exposure to Smoke (weeks) 2 4/xx 36Z 4 6 8/12 10/10 67][ IOOZ 9 12 All Sham- Smoked Anlmals 2/30 7Z All tale Control Animals 0/30 O~ 10112 2/12 I/I.]L 83Z 17g 9g ,i ,,,11 ,ll 1 4 7 li~idixtg Period (days) After 6 Weeks of Exposure 3/1.2 0/1.1 25Z Og 21 42 0/30 Og 0/30 OZ a m i All CaEe Control Animals BAT Co LTD - MINNESOTA TOBACCO LITIGATION Q..m, o'x O:) O'x r,x.) ................................................... -'T

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-38- In the ventral lamina propria £oci of lympbocyces and cysts (often conca£n~ axudaca £nflmcory cells), were coa~on and observed ~.u bach control and smoke-exposed Stoups. The frequency of 1ymphaC£c C£ssue did noc appear to be affected by exposure co smoke (Table 21). TABLE 21 NUMBER OF RATS ~ITTH ¥OCI2 OF LYI41:HOCYTES VENTRAL L~lnqA PROPRLA 2 Exposure ¢o Smoke (weeks) 1/11 9Z 4 6 9 12 2/11 18Z All Sham- Smoked An~ls 2130 7Z All CaKe Control Animals 2/30 7Z z/z2 311z z/lz z112 Zlll 8Z 25Z 9Z 17: 18= • ,s, 1 4 7 21 &2 BoldLug Period (days) AJ~Cer 6 Weeks o£ Exposure 2/30 7Z Al1 Sha~- Smoked An£ma Is 4/30 13:[ AIZ Case Control An/~als The frequency of sara-mucous glands in the larynx varLed cons£derably and no group differences were discernabLe. There was a marked decrease in number of animals show'eLK mucous glands in r.he vent:al lam£na prop=i.a vhen compared v~cb prsv£ous jcudiu, due a)Jnosc cercaiJzly 1:o the sections be:Lng more poscarLor. The am:aug glands of 1:he vocal cord were the predoa/J~ant for: in Ch£s study. BAT Co LTD - MINNESOTA TOBACCO LITIGATION m (...m Go o-,,

C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-39- Rats of Group 19 were exposed to smoke at a 1:7 dilucion for 9 weeks and autopsied the day after Chair final smoke. The changes in the respiratory tract were sim£1ar in type to those found in the rats exposed to smoke for 9 weeks at a dilution of 1:12 (Group 7). TABLE 22 COMPARISON OF STAGES TN LUNG PATHOLOGY FOLLOWING EXPOSURE OF ANIIdALS TO 2 DILUTIONS OF SMOKE FOR 9 WEEKS V¸ | , ,, Peribronchial lymphatic tissue Multiple perivascular cuffs Focal pne~nlti s Perivascular cuffing + focal pneumoni tis Alveolar meraplasla Clusters of macrophagas/lung section of rats with alveolar mataplasia Alveolar macrophase activity Bronchial soblet cell index Lar~ux Sq uamous msr.aplasia Squamous byperplasia (vocal cord) Karatinisanion on the vocal cord Trachea Goblet cell hyperplasla Smoke DiluclonLevel (smoke: air) 1:7 8/8 ClOOZ) 0/7 COZ) 1/7 C14Z) 1/7 C14Z) 7/7 (looz) 42 (approx) 32 3.0 7/7 55 um 5/7 (7~Z) 2/8 (25X) I 1:12 12112 (100%) 0/12 (OX) 2/12 (17X) 0/12 (ox) &/12 (33Z) 16 22 2.5 ll/ll (1) 44 ~m 8/11 (73Z) 5/12 (&2Z) However, the observed chuq;es were senerally more severe in che hiKher dose Eroups (Table 22). In part£cular, the extent of alveolar metaplasia increased as did the severity of the lesion. In one rat from Group 19 c£11aced euboidal cells were seen lining part of the alveolar O~ C~ C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-4O- duct. Bronchlal goblet cell and alveolar mscrophage numbers, also showed a marked increase in those animals exposed to the higher concentration of smoke. Zn these animals, the larynx 81so showed more severe squamous hyperplasia, although the percentage of rats per group shoving signs of keratinisaeion was not increased. The only parameter to show an apparent decrease in those animals exposed to smoke at 1:7 dilution was tracheal goblet cell hyperplasia. DISCUSSION The TPM figures for average chamber smoke concentration were obtained by setting the exposure system to produce one of the specified dilutions of smoke, exactly as for an animal exposure. Prior to any animals being exposed in 8 morning or afternoon session~ the chamber concentration was measured and compared to the theoreulcal result which had been calculaUed assuming each cigarette had an identical TPM and than no losses occurred during sampling. The results obtained were considered satisfactory £f they fell within the range _+ lOS of the predicted value. Those results which fell o,tside of thls baml were /mmedletely notified and the person who had achieved the result was responsible for dismantling the engine in quesuion, cleaninK and reassemblinK it. The exposure of the rats to smoke did not start until the value for the chamber monitored smoke concentration was winhln fOX of the predicted value. The most conmon causes of the £nltial variation belnE incorrect.assembly of an engine or the sampling system, inaccurately set puff volumes, or the presence of small leaks in the sampling system. Bearing in mind the simple monitoring technique used to assess smoke concentration, that up co iO different techniclans were involved with the monitoring and that unselected BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,..m C~ O',, Co

¢,_,~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

-41- tiE•reties were used, chamber monitoring showed clearly that animals in the various Eroups were subjected to dlsvinctly different exposure conditions, and hence v•ryinK "doses" of smoke. Monitoring of b~ood COHb levels, which were quite different ~n animals exposed no the three smoke concentrations used, else confirmed this. Real differences in treatment of animals subjected to different exposure conditions are obviouJly • prerequisite for proper interpretation of any changes in the physiology or patholoKy which u~Kht be 8ttrlbuted to the inhalation of smoke. I~ previous experiments comparison of groups of rats receiving the sane treatment has been for Kroups from different intake batches. AlthouKh previously no consistent differences in COHb levels in such £roups have been found, any differences that did occur could have been taken to be batch dependent. This current study is the first one in which it has been possible to compare directly COHb levels in Kroups of animals from the same intake batch which have also received identical treatment. The results from this experiment indicate clearly that there may be differences in COHb levels between Kroups £rom • sinKle intake of rats, with all the animals subjected co the same treatment. (Appendix. Table I, Groups I-5; &ppendlx, Table 2. Groups 6, 7, I0 and II), which are statistically 81Kn/ficanr at levels comparable to those seen previously when between-batch ec~parisonm have been made. In fact, the COHb dace from this study and also more importantly the patholoKy findings (top of paKe 4A) show that in this experJJnent there is no difference beL3eeen batches o£ animals from the same source taken into the laboratory within a week or two of each other. 0% CO BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-42- lc is difficult to set an accurate determination of COHb levels in rats. A cause for concern are the problems associ•ted with, for example, the rapid change in COHb levels after mmoke exposure is completed, or differences in behavioural remponse of rats which may influence the amount of smoke inhaled. Because of such problems •11 blood COHb levels in studies much •s this should be taken •s • relative index rather than an absolutely reliable measure o£ smoke dosage. The familiar observation was 'again made of • reduction in growth rate of both sham-smoked =_n-d smoke-exposed animals compared with cake controls. Gro~h race was least Ln smoke-exposed rats, but no differences were seen in the weight gain of animals subjected to different concentrations of smoke. Measurements of growth rates of animals are useful in that the appearance of • recognised response Co treatment confirms that an experiment appears Co be proceeding satisfactorily. Unusual growth patterns of indivlduals or groups of animals could possibly indicate some inconsistency in •tenement. Our experience at this stake indicates chat the m~amurmRent of bodywe£ght alone is of no value ms an £ndex of relative toxicity of different dose levels of a smoke, at least within the range which we c~nly use for our work. In this study, m~asurament of food consumption showed that weight loss in sham-smoked and smoke-exposed animals is due Co • reduction in food intake which is reversed when treaemenc mc~ps. Whether the change in food consumption accounts for all of the observed changes in body- weiiht, and whether smoke exposure also causes • simultaneous reduction in fiha efficiency of food ut41ieation is not known. Overall, during the study ~ortmlity rates were higher than expected, particularly in the group of rats exposed co the highest mnok• concentration. BAT Co LTD - MINNESOTA TOBACCO LITIGATION O% C~

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-43- %_/ An important consideration is thac in this experiment, at each sesslon an~nals were exposed to smoke for I0 minutes rather than the 9 minute exposure which we have used previously. For animals experiencing a near-maximal tolerable dose of smoke, I0 rather than 9 minutes of exposuce Kay• the unusually high mortality rate in the "hish dose" group. However, this has not a£fected the general /nterpretation o£ the exper ~nenc. Another innovation in this study was that for the-first time, animals were exposed once only on Saturdays end Sundays and t~£ce on weekdays. A consequence of this red~ct£on ~n weekend dosing seems to be that animals show a reduced tolerance to the 'tfUl1" Smoking regi~e early in the £ollo~r£ng week, This is • penalty £or using the modi£ied treatment, which allows more convenient weekend work yet maintains treat3nenC £or 7 days par week. However, the use of such 8 compromise schedule does not prevent the development o£ those dose-related chanKes in lunK patholoKy which have previously been used success£ully for the bioassay o£ smoke irriCancy. The study has answered, at Least partly, the questions set ae the onset of the exper~m@nt. Patbologlcal thanK•s, partlcularly in the ~&rynx, did occur before 6 weeks, and became more severe •s exposure was continued beyond the 6 :week exposure period. The mjority o£ the induced changes regressed niter traat~nent was discontinued (sun~ar£sed in Table 23). The chanKes observed were those reported in previous studies, with the exception o£ an advanced nspindle-like" me~aplas£a in the lung of one rat. The patholoK/~•l changes induced by smoke exposure and the methods for quanti£yinK the changes were shown BAT Co LTD - MINNESOTA TOBACCO LITIGATION Co

BAT Co LTD - MINNESOTA TOBACCO LITIGATION "T"

to be reproducible to a high degree. It is worth noting that different groups with the same exposure to dilute smoke (Groups i and 9) had similar absolute values for the parameters measured. This was particularly important to establish since these two groups came from batches of tats delivered to the laboratory from the same suppliers but at d/fferent ti~es. "Normal" lesions seen in the respiratory tract of control animals were predominantly infl~atory tissue, consistin8 main/y of lymphocytes and a small number of neutrophils. The distribution and frequency of such lesions was highly variable in the normal populations examined. Their distribution in the lung but not in the la~ and trachea, was altered by exposure to smoke. In the lung, the lymphatic tissue probably ~ndicates a response to a viral infection and represents the early stages of chronic murlne pneumonia (16, 17). Its signlficance in ~11 amounts is, however. Lmcertain. Eats showing this condition do not have any visible signs of ill-health. A means of evaluating the extent of the various futures of "normaZ" lun8 pathology is a useful and sensitive method of identifyi~ early changes of chronic respiratory disease in rats. The response of the lymphatic tissue in the lung co dilute cigarette smoke was rapia, as illustrated by the comparison of group man scores for rats with multiple per£vascular cuffs plus focal pneumonitls (Table 9). After only 4 weeks exposure, very few rats showed such lesions coutpared with I0-26Z in the control groups. The response was uniform in all smoke-exposed rats, even though the extent of Zymphatic tissue in "noz~al" animals varied greatly. Zt has been suggested that the presence of lymphatic tissue may make the rat unsuitable for lone term inhalation studies on the respiratory BAT Co LTD - MINNESOTA TOBACCO LITIGATION o,,,

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-45- epithelium and parenchyma (11). Mowever, the exposure to smoke reduces the amount of lymphatic tissue, most probably by suppressing the i---unological response of the rat (18) rather than by removing the viral antigen. Th~s is compatible with more tenuous evidence from a previous study (3). This possible loss of immunological response may produce a rat less suitable for lord-term experiments not because visible lesions due to inns infection interfere with inteEprer~ation of any smoke-lnduced chanKes but because the smoke-expoeed rata amy be more susceptible to infection. Pathological changes that became evident after only 2 weeks of exposure to full smoke included squamous mecaplasia and keratinising hyperplasia in the larynx. The incidence of both these changes was related to the duration of exposure and reached a peak after 6 weeks exposure and plateaued or even regressed slightly with continued . exposure. Squamous mecaplasia and keratinisation of the larynx was most marked in rats (Groups 1 and 9) exposed to mnoke for 6 weeks and the larynxes of animals fro~ this group could be easily recosnised at low magnlfica~ions. -The distingu£shi~K feature was ~he thickness of the 8quamous epithelium, wh/ch in 80Z of these animals lined the whole la~eal l~nen of the section. All rats showed keratinisation of the epithelium on ~he vocal cord. The degree of hyperplaBia was noticeably greater than has been previously observed. Tt must be noted however that in this, as in all previous studies, the e~tent of hyperplasi8 was determined by tak£ns measurements of the squamous epithelium includinK any stratum corneum. Thus chau~es in epithelium thickness will tend to follow the degree of keratinisation (YiKures 26 and 27). BAT Co LTD - MINNESOTA TOBACCO LITIGATION <_r'l C~o

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-46- For both these features of the larynx in smoke-exposed rats (mecaplasia and k~raC£nisatio,) charm was a rapid regression when exposures stopped and 1&rynxes were apparently normal again in rats after the 42 days holding period. In those rats held after exposure there £s a clear increase in the ~ncidenee of incomplete squamous menapl•si• following complete loss of keratin after & days holding, ks in •11 previous snudies the ventral depression ~as free from smoke- induced changes. Subjective appraisal indicated • possible byperplasi•, but no group comparisons were made. Both bronchial end tracheal goblet cells were hyperpl•stic after 2 weeks exposure. As before, the problm= of evaluation was evldenc and very few fully differentiated cells were observed. The response of the epithelium in the bronchus, measured •t level I in the main lefn ~nCr•pulmonary bronchus (19), was more un£form than that in the era=had and reached a maxlmum after 12 weeks of exposure to smoke. Both tracheal and bronchial epithelium initially showed only slight signs of • reBression of the goblet cell hyperpl•sla when smok/ng stopped but after 21 days holding, both returned to near normal values. &Ithough two different methods were used to evaluate goblet ce~l hyperp1•sia, the cesults •re comparable though the siCuat£on is noC ideal. The mcrophage population in the lung p•renchyma increased slightly aft•r only 2 weeks exposure to dilute elsareCte smoke, and this response inareased to • maximum after 9 weeks. &s observed previously, with an increase £n numbers charm was • sq~nu1~aneous increase in mcrophage size and degree of pigmentation. In the rats exposed for the longer periods the macrophagee were particularly veeuoleced and often the cell outline BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,..m o-, c~

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-~7- became indistinct. The piEmentation observed is probably due to an increase in the number of lysosomes and phagolysosomes. Ul~rastructural observations have shown these organelles to contain plate-like inclusions which may be phagocytosed particulate matter (14). The pigmentation may also be phagocytosed peroxidased lipid (20) and/or iron (7, 21). There was a gradual reduction Ln the macrophage population over the A2 days holding period when smoke exposure was stopped. The most interestinK pathological changes observed in this study were related to alveolar metaplasia in the lung. ChanKes were not observed before 6 weeks exposure and were seen initially as clusters of larks pigmented macrophages £n the region of the terminal bronchioles and their alveolar ducts. It is of interest that the rats with such clusters also showed increased macrophage populations in the sub-pleural site examined. The two parameters seem to be dependent. The number of rats w~th these clusters of macrophages increased with duration of exposure end of these approximately 70Z also showed siKns of incipient alveolar metaplasia. This proBressed to a full mstaplasia in a few rats in which cuboidel cells were found linir~ the alveolar spaces. No ciliated cells were observed in these animals. The hyperplasia and metaplasla seen in smoke-exposed rats may be due to the response of a number of cell types. The increase in alveolar wall thickness could be a hyperplasia of 8quamous cells or, as is now thouEht more likely, type II alveolar cells. The type II cells may also umdergo metaplasla produc~ the cubo£dal cells. However. these could also be an extension of bron~hiolar cells into the alveolar duct i.e. bronchiolisation (15). The clusters of piKmented macrophaKes described BAT Co LTD - MINNESOTA TOBACCO LITIGATION O'x C~ rx.a

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-48- here as an indicator of incipient alveolar metaplasi• are described in the lungs of younB smokers •s respiratory bronchiolitis (22), In is postul•ted that this is • precursor of centrianclnar emphysema. It is interesting to note that respiratory bronchiolitis occurs in an irregular distribution, whereas in the rats exposed for 12 weeks in this study distribution was comparatively regular. Of importance is the increase in the number of rats per group with incipient alveolar met•pl•si• in those groups held for 6 weeks following exposure. The number of rats per 8roup showing siKns of alveolar mer~tpl•eia increased wluh the duration of the holding period, although the severity of the lesions in any one lunE did not increase. In rats exposed for up to 12 weeks the extent of the lesion in any one lung increased with continued aX'po sure. The aplndle-like cell met•pl•si• observed in one rat is •s yet unexplained. It us found together with a well differentiated cuboldel metaplasLa of the alveolar epithelial cells and mucus in the alveolar lumen. The increase in reticulin suKgests • form of fibrosins elveolitis. In conclusion, amny p•~hologic•l changes occurred after only 2 weeks of exposure to smoke. Some parameters (Table 23) increased in severity ~rfth up to 12 weeks o£ exposure whilst others appeared to reach a maximum and then plateau •t 6 weeks. Rapidly induced changes tend to regress rapidly whilst those lesions which develop more slowly regress at • slower rate. An g~porcant exception to tb/s general rule was alveolar metaplasia which, together with lymphocytic activity in the lunK, ~as the only parameter which showed no regression, in fact the metaplmsim showed signs of proKresslon durinK • non-smok~n8 period of seveE•l weeks. BAT Co LTD - MINNESOTA TOBACCO LITIGATION C.m O',, c~

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-49- TABLE 23 SUMMARY OF LUNG PATHOLOGY CHANGES OBSERVED DURING P ROG~E SS ION / REGRESS ION STUDY Multiple Perivascular Cuffs + Focal Pneumonitis Alveolar Metaplasia Alveolar Macrophages + Bronchial Goblet Cells ÷ Tracheal Goblet Calls ÷ Squamous Hyparplasia (Larynx) + Keracinisation (Larynx) + Exposure Period in Weeks (1:12 Dilution) 2 4 5 9 m o m -- -- ÷ ÷ + ÷ m ÷ m m ÷ ÷ ÷ + m ÷ 4. m 4- He idinE Period in Day s Following 6 Weeks Exposure 12 1 4 [ 7 21 42 J - - No Change - m + Progression + m 4. Regression 4. m m Regression ÷ m + Regzession - + m Regres s io n + + m Regression - - Lesion absent, 4. Lesion evident, m lesion maximal. With regard co upe~i~gntal design, this study has illustrated the need for a larger group sLze (ZO rats/group being desirable lOmale and I0 female). Inmost cases only 6 rats of any one sex par group were ava£Lable in ssmke-exposed groups and the examination of possible sex differences was not poss£ble. The s£tuaCion w&s no better in the control groups with only 3 race of each sex per group. With the marked regression of changes once smoklnE was cermlnaced, che period between the lest exposure to smoke ana autopsy should be as shore as possible. The incidence of kersCinisacion of the larynx, for example, fell from 38% co 17% within 3 days. Races of regression do vary from parameter to parameter. No defln£ce rule can be made but in general autopsies should be carried out within 24 hours of final exposure to smoke. BAT Co LTD - MINNESOTA TOBACCO LITIGATION c~ c~

BAT Co LTD - MINNESOTA TOBACCO LITIGATION C

-50- For the purposes of carrying out bioassay work with s:oke: the length of exposure may be varied depending on the parameter under investigation. Six weeks seems co ~axfmise squau~us metap~asia and kera=inising hyperplasia in the larynx ac the smoke "dosase level" used in this study, i.e. exposure ~ice per day for 10 ~nutes to a smoke concencratlon of approximately 4.7 ~/i. In fact, in ~his stud? measurement of epithelial thickness, including any keratin present, suggests that squamous hyperplasia reaches a maximum after 2 weeks exposure and neither decreases nor increases with continued exposure up to 12 weeks. The uncertainty of the validlty of the measurements includlng keratin (which can easily be sloughed off naturally) q~estions the evaluation method. Future estimations will thus include measurements of the thickest uniform areas of epithelium including and excluding any keratin present. Both sets of results will be reported. Although dosim~cry work was not included in this exper£ment, our previous studies suSgeec that chose animals exposed to smoke at 1:12 dilutiou from the cigarette used. would receive a TPM dose Co the lower respiratory system of approximately 400 ~g/g tissue/session. Under such exposure conditions, bronchial goblet cell hyperplmsia end alveolar macrophaie numbers appear co reach a ux/mum ac 7 weeks. The remaining pathological changes (alveolar ~aplasla, tracheal goblet cell hyperplasia) showed no signs of levellinE off after 12 weeks exposure to smoke at 1:12 dilution. There is thus the need for extending exposure beyond 12 weeks, particularly when considering alveolar metaplasia. BAT Co LTD - MINNESOTA TOBACCO LITIGATION m

BAT Co LTD - MINNESOTA TOBACCO LITIGATION "7-

-51- 'V P~.F~NCES 1. BAT Report No. KD.I231-R, 2.7.75. 2. BAT Keporc No. RD. 1281 Restricted, 23.10.75. 3. BAT Report No. ~.1328 Resczicced, 13.2.76. 4. First report of the Independent Co~n£ttee on SmokinK and Health (1975). Tobacco Substitutes and Additives ~n Tobacco Products. 5. BAT Report No. RD.1246-AR, 27.11.75. 6. Walker, D. (1975), Wickha~Kesearch LaboratoriesReport. BAS1, 12.9.75. 7. Walker, D. (1976), Wickha~n Research Laboratories Report. BA62 and BA63, 2L.1.76. 8. BAT Report No. RD.I&20 Restricted, 16.10.76. 9. Stevens, H.T. (1976), ToxicoIoKy, ~, 311-318. 10. Tr£eb, G., Fapprltz, G. & LUtzen, L. (1976), Tox. Appl. Pharmacol. 3_~5, 531-542. 11. Lib, D. (1975), Lab. An£m. 9, 1-8. 12. Dontenw~11, W., Chevalier, H.J., Harke, M.P., La£renz, V., Rechzeh, G. and Schneider, B. (1973), J. Nat. Cancer Inst. 5_~1, 1781-1807. 13. liay,ahi, H., Shea, 3., Fhelps, P., Holmes, L., Pochey, V., SornberKer, C. and Rubez, G. (1975), Clin. Ram. 23, $8&. 14. Brody, A.R. and CraiKhead, J.E. (1975), lab. Invese. 32, 125-132. 15. Yrasca, 3.M., Auarbach, O., Parim, V.R. and J~emon, ~.D. (1974), Exp. Hol. Pethol. 21, 3OO-312. 16. Tames, J.R.H., HcAdams, A.J. and Yv£ch, P. (I965), Amer. J. Path. 32, 1&1-159. 17. Innes, J.R.M., Garner, F.M. and SCookey, J.L. (1967). In PatholoKy o~h~racor~ Ra~e end H~ce (Edl. Cotch~n & Roe), Blackve11, Ox£ord. BAT Co LTD - MINNESOTA TOBACCO LITIGATION O',, ......................................................................................... -'T

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-52- 18. LeuchtenberBer, C. and Leuchtenberger, R. (1974). Ontology, 2_29, 122-138. 19. Lamb, D. and Reid, L. (1969). Brit. Med. J. 1, 33-35. 20. Davies, P. Personal Com=unicat:ion. 21. Mohr, U., Keckar, M.B. and Resnlk, G. ~_nn B.A.T Hamburg Progress Reporn, July-September, 1%76. 22. Niewoehner, D.E., Klelnerman, J. and Kite, D.B. (1974), New. EnKI. J. Mad. 291, 755-758. BAT Co LTD - MINNESOTA TOBACCO LITIGATION Cx~

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-53- APPENDIX TABLE I sure.my com O..A A. C G SS O." G OUPS) G¢ou~ 1. Number Heart S.D. 2. Number Mean S.D. 3. Number Mean S.D. 4. Number l~an S.D. 5. N,~ber Mean S.D. TOTAL Nund)er l~az~ S.D. Number He--, S.D. Male 6 27.1 4.6 3 32.2 1.6 6 27.7 3.9 6 33.3 5.3 4 38.5 S.7 25 31.2 5.9 1:8 1:12 i , m Female Female 5 28.7 5.8 5 30.4 4.6 5 28.0 4.4 4 35 • 1 1.6 5 35.7 8.0 24 31.4 5.9 49 31.3 5.9 )',ale 6 22.3 3.1 6 24.9 6.6 5 29.8 3.7 6 30.6, 3.0 6 30.0 8.3 29 27.4 6.1 l 6 22.2 1.6 6 29.6 2.8 6 30.3 3.6 6 25.5 3.6 6 31.3 4.9 30 27.6 4.8 59 27.5 5.4 BAT Co LTD - MINNESOTA TOBACCO LITIGATION CZ) O', ¢x:) .................................................................. "-T

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-5~- A~ALYSlS OF C0Hb DATA <"KEGKESSZ0N" GROUPS) Data 120 animals (60 male, 60 female) were randomly divided into I0 groups wlch 6 males in each and I0 groups with 6 females in each. Five groups of each sex were then allocated to two treatments, vlz. 1:8 dilution and 1:12 dilution of smoke. All zhe animals involved in this part of the experiment were from one intake. COHb measurement was taken for each animal approx/macely 4 weeks and 2 days after the smoke acclimatization. The data has been summarised in Table I. Note that group 5, 1:8, female and group 5, 1:12, male have a hiKher standard devlation than the rest of the groups, otherwise the date is fairly consistent. Analysis The da~a was created as a 2 x 2 x 5 experf4nenc with treatments and sex crossed and groups nested w£ch£n treatments and sex. Also, the effects of sex and :rea~men=s are fixed and groups are random. The analysis of variance is shown below: Source of Variation Treatment Sex Treatment x Sex Groups (within six & trut:ent) d.f. - desress of freedom SS - Sum of Squares MS - Mean Square F -- F--value Sir. - SiEnlficance level. d.f. SS MS F Sir. 1 ASL.20 451.20 5.33 95Z 1 0.02812 0.02812 <1 N.S. 1 0.50766 0.50766 <1 N.S. 16 1355.2 84.698 3.72 99.9Z BAT Co LTD - MINNESOTA TOBACCO LITIGATION O',, C~ O',,

BAT Co LTD - MINNESOTA TOBACCO LITIGATION O"-

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-55- Zt should be pointed out that the F-tests for test~nK the siKni£icence of the sources of variations are slijhtly d£f£erent from the normal tests when the erro= KS £s used as a divisor for all the other HS's. In this model, the F-tests are as eho~n belov: test Groups e£fecC by Croup MS/Error test the rest by ~nK @roup I~S as the divisor. From the Ahoy& t&ble we see that (i) there is a s£Kn~icant difference between Woups ~th~n sex and tr earnest. (£i) the=e £s no s£Kn~£aant sex x treatment and no sLKuig£~ant diference between sexes. We would have expected this from the data in Table 1. (iii) there is a siKn/~icant difference be~een treatments - 1:8 dilution produc4~[ hiKhez COKb count than 1:12 dilu:ion. the d~ta for the tire groups w~thin sex and creauaents ate analysed separately by 1-~ay AHOVA we find that: (i) for 1:8 Hsle, there £s s sLKn££icenc (99.3Z) di£ferenee he.sen Stoups. This d/~fere~ce £s maLuly accounted for by the di~feruce betweun Stoup i ~ Kroup 5, 8~oup 3 and Kroup 5. fo3 ~z8 ~emale, no si~Lf~ant d~feren~e can be detected between 8Zoups. The siK~Lficance level is 87.3X. It is feasible that the lazKe variation in K~oup 5 has £n~lated the ez~or MS, thus mak~n~ tb~ test for dete~t~ difference be~vee~ Sroups~e~e sensiti~e. ~ the ~r~abLlit7 of this Kroup was on a par with the no~mal ver~ab~li~y, then a dLfference at ~OX level of siKnificanct may have been ~ound. BAT Co LTD - MINNESOTA TOBACCO LITIGATION k.J'-t oo ............................................................. -'T

~° BAT Co LTD - MINNESOTA TOBACCO LITIGATION O ,,,O .................................................................. ~T

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-56- (iii) (iv) £o,- I:12, MaZe, ,'ha dJ.~fer~ce be,:ween 8zoupa is 8iSn£Eicaa~ ac 94.4Z level. KEain, Kroup 5 has h~gher variability than normal. £o%" 1:12, Female, =he difference between ~zoups i8 |ifnifican~ &C the 99.9Z level. BAT Co LTD - MINNESOTA TOBACCO LITIGATION ',-O r,o ................................................. ---'T

l BAT Co LTD - MINNESOTA TOBACCO LITIGATION O',,

~0 > --I o ! N Z Z > ,--I O 03 > r'-' ,H > ,H 0 Z r,-o -- tall • .-~ ¢" o-. ¢' V~ '~oo -:3-:- 1z069c 1 S01

-57- APPENDIX TABLE 2 SUMMARY OF COHb DATA ("PROGRESSZON" GROUPS) Batch Zntake Groups 11. N,--be r Mean S.D. 10. Number 7 • Number Mean S.D. 6. Number Mean S.D. , , Batch Tot:a1 Number Mean TOTAL qmmm,mmmm~ Number MeLn Treatment Total Number Mean I £n8 s Male Femal e Ha le Pemal e im i i I 2 1 2 1 2 I 2 5 40.7 2.0 4 34.5 7.0 5 36.1 6.7 6 41.0 4.0 1.I 9 38.8 37.9 6 6 6 30.5 24.9 29.1 I 3.5 3.1 2.4 4 6 6 4.1.2 30.1 29.2 3.1 6.0 4.2 5 6 6 30.0 26.5 25.5 3.7 4.1 4.4 4 6 6 30.9 27.3 25.1 2.0 3.4 5.7 LO 9 12 12 12 12 34.7 30.4 27.5 26.9 29.2 25.3 19 32.2 39 35.6 20 38.4 24 27.2 24 27.2 BAT Co LTD - MINNESOTA TOBACCO LITIGATION

INDEX HAS INDICATED GAP IN BATES RANGE HERE

-58- A.RkLYSIS OF COt~ DATA ("PROGI~ES.SION" GROUPS) Data The antis used for collecting the COHb data were the ones used in the "exposure Clme experLment'. IC is required to cast if there is a significant difference in COHb from 1:8 and 1:12 (irrespective of the time of sampling). A su~mry of the data is shown in Table 1. Treatment, Sex and Batches ace crossed and have fixed effects and groups are "nested ~riCh~n batches, sex and treatment. Group is tre~aced as a random var£abls. The A~OVA for this is shown below: Source of Variat£on d. £. SS MS F Sig. ,m TreaC~tnt 1 1496.2 1496.2 23.47 99Z Sex 1 128.65 128.65 2.02 N. S. Treatment x Sex 1 133.04 133.04 2.09 N.S. Batch 1 156.91 156.91 2.46 N.S. Treatment x batch 1 5.0162 5.0162 1 N.S. Sex x batch 1 77.531 77.531 1.22 N.S. Treatment x sex x batch 1 1.4526 1. 4526 1 N.S. Groups (within batch act) 8 510.00 63. 750 3.403 99Z N£thin groups 71 1330.O 18. 733 Note Chat the F-test for the d£fference between groups (w£thin batches etc.) is $iven by d£viding groups MS/w£th£n groups PLq but the rest of the affects are tescmd by using Groups NS as the denomlnator. BAT Co LTD - MINNESOTA TOBACCO LITIGATION %.0

'b "V r...rl O~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION ........................................................ -v--

INDEX HAS INDICATED GAP IN BATES RANGE HERE

FIG. I DIAGRAM TO SHOW OUTLINE DESIGN 3- NONTH INHALATION STUDY ii RD. 1477 RESTR! CTF..D OF GROUP II PROGRESSION - ii, I! II F"2'-2~ 14 I0 r./i////I 28 I/ / / / / / / /14~ 7 v////7",/~.//./AG~ 6 v///"//-/?//,/./;'//Ae4 , .... EXPOSURE' PERIOD (DAYS) , II REGRE5510N ' GROUP i! I I !/'//7////III Z v////'/"//,r~4 3 ~/////'//-zx~? 5 r/.////// 4z t4ZDAYr~ ID(POSUKt nHOLDING I~RIOD (DAYS), BAT Co LTD - MINNESOTA TOBACCO LITIGATION

> "-fl ('1 o [.- --} I Z Z > '-4 0 03 > N 0 t-" ,4 > 0 Z 1169c] c]01 C, ONGENTI~TION$ 9 8 ? 4 Z 0 Ik I0 (' FIG. e(' RD.I¢?7 RESTRICTED Of" TPM IN EXPOSURE GHAMBERS FOR EACH Of" THE SHO .KE DILUTION LEVEL, S ,USED IN THE STUDY 4 4O N=. oF 055ERVATIONS DURING STUDY 2O e 1:7 DILUTION • 1:8 DILUTION X I: 12 OILUTION 60 --4 5O

FIG. 3 RD. 14.77 RF.,3TRtCTED GRokrI'H CURVES FOR CONTROL RA'I'5 AND ANIMALS EXPOSED TO ;:' DLLLFF.ION$ OF ,SMOKE FOR APPROXIP'IATELY 12 WEEK,5.(IVIAI.~.E5) INC.,REASE IN BODYWEICIHT CAGE C.X)NTROL ÷ 5HAM - ,SMOKE • I: B DILUTION x I :12 DILUTION 60 7O 8O BAT Co LTD - MINNESOTA TOBACCO LITIGATION

FIG. 4- RD. 1477 RF_.STRICTED GROWTH CURVE5 FOR CONTROL RATS AND /V~IPIAI..5 EXPOSED TO ~. DILUTIONS OF SMOKE FOR 6 W~_EK5 FOLLOWED BY A POST-EXPOSURE HOLDING INCR.F_/~E IN I~ODYWEIGHT ('/o) PER, O0. , (~,u.~ 6w~./6~,~. e CAGE C, ONTROL + 5HAM" 5PIOKED • I: B DILUTION X I: IZ DILUTION BAT Co LTD - MINNESOTA TOBACCO LITIGATION PERIOD 8O

FOOD CONSUMPTION OF RAT5 FIG. 5 DURING 12 i RD. 1¢77 RESTRICTED ~/EEK5 OF EXP05URE e CAGE CONTROL 4. ,SHAH- SMOKED • 5MOKE EXPOSr:.J::) FOOD CON,.~MPTION E6 24 eo IO 16 ! I : : ,,,, , I , I 1 I , I II 0 I0 ~.0 30 40 50 &O 70 80 SO 30~ P.8 Z4., 20 18 0 I0 20 30 40 50 I'I ME (DAYS) 6O BAT Co LTD - MINNESOTA TOBACCO LITIGATION ........................................................................ "-T

FOOD CONSUMPTION FIG. 6 OF RAT5 DURING i L I I i 6- WEEK,HOLDING R,D. 14.77 G WEEK3 5HOKE PERJO,D RE, STRI C"I"ED EXP05URE AND • CAGE CONTROL + 5HAM- SMOKED • ~P1OKE EXPO,$F..D FOOD CON,$1./M PTON ( 81r T I 28 116 Ilk 1tll 18 I: IZ DILUTION I | = I 0 IO =0 I II I~ II 1 m, m 114. la p'./ sHo.~z-cx .f~soRgq, r-.~O.oZ. / /I. i=o.¢r-~~.o~=~r¢,:oo -l-- "riMe "- L..r'I BAT Co LTD - MINNESOTA TOBACCO LITIGATION

F I G. 7 RD. 1477 I~ESTRICTEO FOOD CONSUMPTION OF RATS DUPJNG 12 ~/EEKS OF EXPOSURE e r..,~E CONTROL + SHAH - 5HOKED • SMOKE EXPOSED FOOD GON,SUMPTION ( s / RATI OAY) ,,.... 2J 24,, 18 16 14. !,: 12 D!LU,T'ION (FEMALE,S) - I ,, ;, ,, , .... I , I " 0 I0 Zo ~o 4o SO 6o 7O BO SO ~.o 18 14 I" 8, DILUTION, ,(FEIMALES) BAT Co LTD - MINNESOTA TOBACCO LITIGATION

FOOD FIG. 8 CON5UHPTION OF RATS DURING G WEEKS G-WEEK HOLDING PERIOD IKD. 1477 RESTRIC.,TED SMOKE EXPOSURE AND i i , , e OAr, E. CONTROL + 5HAM- SMOKED • SMOKE EXPOSED FOOD C.,ON,SUIVIPTI ON (, / RAT/DAY) P-4' ~0 I0 IG 14 I: I?- DILUTION (F~MALBS) : I ' ' l I I ! I I ! 0 I0 Z.O 30 40 50 60 70 80 50 I:, p DILUTION .(FEMALF.3) ! " [ 14. Time (DAYS) "-.J BAT Co LTD - MINNESOTA TOBACCO LITIGATION ................................................. "T

F'tG.9 RD. 1477 RE3TRIC'I'ED THE RE.LATIONSHIP FE'TWEEN TREATMENT-RELATED MORTALITY ('F..XPRE,~ED A5 A PERCENTAGE OF AHIMAL~) AND EXPOSURE CHAMBER MEAN SMOKE CONCENTRATION MORTALITY (~'o3 80 60 4O ZO 0 O 5 4. 5 6 7 8 s~oK[ co.c~'r~r~o. (,,~/~) | BAT Co LTD - MINNESOTA TOBACCO LITIGATION oo

FIG. 10 RD. 1477 RESTRICTED ALL INDIVIDUAL TREATMENT- RELATED, DEATHS PLOTTED. (FOR ~. 5HOKE DILUTION LEVELSt bAOCORDING TO F.XPO,SURE DAY NUMBER DURING THE EXPERIMENT FEHALE 04,. HALE,5 O L I oo I I I I I I • I l • •o • 0 • • • •o• • • • ooo o• • o I I o 0 , I ! I I o • •0 • • 0 I0 ZO 30 40 50 60 70 80 SO EXPT. DAY' Ne" BAT Co LTD - MINNESOTA TOBACCO LITIGATION

FIG. II RD. 14.'77 RE3TRICTED TREATMENT- RELp,.TED DEATHS, PLOTT,,ED,ACCORDING TO THE DAY ON k/HICH THEY.OOOURED TOTAL DEATHS 14. 13 IZ ii IO 8 7 5 4 3 2 I 0 BAT Co LTD - MINNESOTA TOBACCO LITIGATION ................................................................... -1"

FIG. 12 RD. 1477 RE3TRICTED 5AHPLING P0tNT5 FOR AIR EXP05URE PLATE,S LIFE ,SC, IENC, E5 BUILDING8 IO 75 I AUTOCLAVE CAGE 5TORE C, ONDITIONING R00H ' I N~'r'RU]VlE.NT'S 14 CLEAN" @ 13 CORRIDOR I:, RE3T ROOPI •11 INHALATION 5 t el5 INHALATION 4 4. ANIHAL ROOH 4 I II m DIRTY i, I 017 ~15 I INHALATION I INHALATION @ 6 ANIHAL ROOM 3 I I I #os I 2 ANIHAL ROON ?. ! CORRI DOR INHALATION (~UARANTINE =0 l• S BAT Co LTD - MINNESOTA TOBACCO LITIGATION Lr'l o'% ',,.O r,,,j

ADJUSTED GROUP MEAN RD. 1477 REEFRICTED OF HALE ANIMAL5 ,, KILLED DURING 12 WEEK EXPOSURE PERIOD ~--- INDICATE5 NO SIGNIFICANT DIFFERENCE 8~ETWEEN THE~E TREATWIENTS. ADJUSTED ._~uP H~N ~ODYWEIGHT (s) ~J I BAT Co LTD - MINNESOTA TOBACCO LITIGATION !

ADJUSTED GROUP MEAN FIG. 14 RD. 1477 RESTRICTED BODY~VEIGHT$ OF FEIvIALE ANIMALS KILLED DURING IP. WEEK EXPO,SUR[ PERIOD "------- INDICATE5 NO SIGNIFICANT DIFFERENCE ~?WEEN THESE TREATMENT5 "JUSTED ouP ~DYWF.IQHT (s') 00, 2SO, Z80, Z'tO ZOO ZSO 24o 210 i| I I' ",,, I I 14 S8 4~. S.~ E.XPOSURE PERIOD (DAYS) BAT Co LTD - MINNESOTA TOBACCO LITIGATION O O'~ r',,.b (..M

FIG. 15 RD. 14,77 RESTRICTED ADJUSTED GROUP MEAN BODYWEIGHT5 OF HALF. RAT5 KILLED DURING 6 VEEK P05T-EXPOSURE HOLDING PERIOD m ,, ADJUSTED GROUP MEAN ~ODY WE.IGHT C9) -- iNDICATE5 NO 51CINIFI~ANT DIFFERENCE ~WEF~ THESE TREA~ENTS. ! 4- 7 Zl HOLDING PERIOD BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢.j-1

FIG. I~ RD. 14,77 I~ESTP, iCTED FINAL W'EIGHT (FEMALES) ----- INDICATE5 NO ,51GNIFIGANT DIFFERENCE BETWEEN THESE I"REATMEN'~ ADJUSTED ~'Q, OUP MEAN !~ ~DYWEIGHT r~ I @ 7 II 4= HOLDING PERJOD (DAYS) BAT Co LTD - MINNESOTA TOBACCO LITIGATION O~ ',,,c r'~ cjr'l

FIG. 17 P,D. 14.77 P, ESTRICTED No. OF RAT5 PER GROUP WITH MULTIPLE PERIVA5CULAR CUFFS (°/o) • SMOKFJ:) FULL PERIOD ® SMOKE 5TOPPED ~ 6 WEEK.5 • x ,.~HAM-$MOIKED & CAGE CONTROL 7o 40 3o I0 O 0 ZO 40 60 80 IOO N"" OF' DAYS FROM FIRST FULL SMOK[ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

FIG. 18 RD. 14.77 RESTRICTED No. OF RATS PER GROUP WITH FOCAL pNEUMONITi5 (:/:) • 5HORED FULL PERIOD ® ,SHOKE 51"0PPED AFTER 6 ~T.E]¢.5 ,~ SHAH-SHOKED A GAGE CONTROL N°. OF RATS/GROUP ~o 60 7o 50 4o ~o 2o IO / I / 0 I i I a ! O 20 40 60 No, OF' DAYS FROM FIRST FULL SP1OI'~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION ! I00 O',,

FIG. I~) RD. 1477 RESTRICTED No. OF R,6TS PER GROUP WITH BOTH MIJLTIPL.E pERIVASCULAR CUFF5 AND FOCAL PNEUMONITI5 (%) • 51'10KED FULL PERIOD e SMOKE 5TOPPED AFTER 6 WEEK,5 x SHAM - ,SMOKED A 0A~E CONTROL NQ- OF" RATS/GROUP (~.) ~o 8O 70 6o 50 4o 3O ZO IO O / I I \ i \ / I \ I \ \ /\ \. ,,./ -., 0 I I I , __ ~0 4.0 60 8O N°. OF' DAYS I:'ROH PIR,ST FULL SMOKE BAT Co LTD - MINNESOTA TOBACCO LITIGATION ! I00 c~ ",,C. oo

No. OF FLAT5 PER GROUP WITH FIG. 20 NULTIPLE FOCI RD. 14-77 RE.STRIGTED OF PIGMENTED NACROPHAGE5 (7°) • 5MOKED FULL PERIOD e 5MOKE ..~'OPPE.D AFTER 6 W/EEK5 x ,SHAH-,5 M0 KED CAGE CONTROL N" Or RAT,5/CIROUP 8,O 7O 60 5O 4O ~O tel I0 O ! o ---.4~-~---.-~ .... -~ ! I ' "' • ; : 4O 6O 80 N" OF DAYS FROM FIRST FULL SMOKE. I00 BAT Co LTD - MINNESOTA TOBACCO LITIGATION o',, r,,,a ",,c

No. OF RAT5 PER GROUP WITH FIG. 21 RD. 1477 RESTRICTED INCIPIENT ALVEOLAR NETAPLASIA (7o) • ,SHOKF..D FULL PERIOD ~KE STOPPED AFTER G WF..EK,5 x 5HAH-~HOKED A CA~E CONTROL N°" OF RAT,S/GROUP (%) 8O 70 GO 50 4.0 30 10 0 I 0 BAT Co LTD - MINNESOTA TOBACCO LITIGATION I IOO ¢,..m 0.%

HEAN FIG.ZZ No. OF HAC, ROPHAqE CLUSTERS PER P.,D. 14.77 RESTRICTED LUNG 5ECTION • SMOKED FULL PERIOD ,~>MOKE 5TOPPED/V"I'ER G WEEK,5 X 51t~,M- 5PIOKED & CAGE CONTROL HACROPHAGE C, LUS"rER5 / LUNG SECTION 40 35 3O 25 7.0 15 !0 5 0 0 -----~- 41-----~ .... --~ ZO 40 60 BO N°. OF DAYS FROM FIRST FULl. SMOKE: BAT Co LTD - MINNESOTA TOBACCO LITIGATION I I00 C~

GROUP MEAN FIG. Z3 RD. 14.77 RESTRIr~TED COUNT OF IvlACROPHAGF~ ~" I0 m IN 0"54,~. m~ OF' SUB-PLEURAL ALVEOLAR Ti$,~UE • .~IOK,ED FULL PERIOD SMOKE ,5"I'OPPF.,,D AFTER G WEEKS 51"IAN- .~"IOKED ,s CAGE CONTROL HEAN N°'OF t"IAr.,ROPHAGF~/GROUP BAT Co LTD I • H ' I I I Z0 40 GO 8o N"OF DAYS TRDM FIRST FULL SPIOKE - MINNESOTA TOBACCO LITIGATION I I0O Lr'l Lr--

F'IG. P.4 RD. 1477 RES"I'Ri GTED ESTIMATED GOBLET CELL DENSITY IN FIXED 51TE OF LEFT ill, , -,,, • im INTRAPULHONARY BRONCHUS i i ,. • E:~IOKE.D FULL PERIOD ® .SMOKE STOPPED AFTER 6 ~i/EEK;5 SHAN- 5MOKE.D CAGE CONTROL GOBLET CELL INDEX 3"0 P.-O I'O 0 O x-J~" \\_ ,.,&~" %AI"f "~--- ..~.~ , I I ' I ,, ,, : Zo 4o t~ 8O N-. OF" DAYS FR.OH FIRST FULL 5HOKF.. BAT Co LTD - MINNESOTA TOBACCO LITIGATION IOo k.jr- O",

FIG. 25 I:I,D. 14.77 RESTRICTED No. OF RAT5 PER GROUP WITH GOBLET CELL HYPERPLASIA IN THE UPPER TRACHEA (°/o.) • 5HOKED FULL PERIOD (> 5PIOKE 5TOPPED AFTER G WEEKS x 5HAM- 8MOKF..D & CAGE CONTROL No. OF RAT,S/GROUP 70 60 50 40 3O %0 I0 0 ! 0 .,___,. I I ! I 20 4O 6O 80 No, OF DAYS FROH FIRST FULL ,~HOKF.. ,, ! I00 BAT Co LTD - MINNESOTA TOBACCO LITIGATION m O',, --F-

FIG. ?..G RD. 1477 RESTRICTED HYPERPLASIA OF THE SQUANOU5 EPITHELIUM LININra MEDIAL ASPECT OF THE VOCAL CORD THE • ~NOKED FULL PERIOD e 5HOKE ,,%TOPPED AFTER EP WEEKS X 5~-IAH - SMOKED & CAGE CONTROL GROUP MEAN EPITHELIAL 6O So 40 3o P.O THIGKNE~ &--.----~..... 0 El 40 60 8O I00 N¢~ OF DAY'S TROH FIRST FULL SHOKE BAT Co LTD - MINNESOTA TOBACCO LITIGATION 0% Lrl

N.o. OF RAT5 EPI THE LUM FIG. 27 RD. 1477 RE3TRICTED PER GROUP WITH KERATINISATION OF THE 5CIU~J'IOUS ON THE MEDIAL ASPECT OF ~) No. OF RAT~/GROUP WITH STRATUM CORNEUH ( ,o1 I00 . ~o 80 '70 6o 50 41.o Zo IO 0 i=.-~_.-.J~._-T I I " I , ,, I .... ! 0 ZO 40 6O 80 No. OF' DAYS FROM FIRST FULL SMOKE BAT Co LTD - MINNESOTA TOBACCO LITIGATION • ,SMOKED FULL PERIOD ,SMOKE STOPPED AFTER G WEF.K~ ,SHAM- SPIOKED CAGE CONTROL I 100 r, jr"I (..M

INDEX HAS INDICATED GAP IN BATES RANGE HERE

BAT Co LTD - MINNESOTA TOBACCO LITIGATION