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-5- prior to autopsy produced a slight reduction Ln macrophase numbers during the post-L~posure holding period. Exposure to smoke produced a marked increase in goblet cell ~tiviCy in the left intrapulmonary bronchus (Table 6). As in the case of alveolar macrophales, the higher concentration of smoke produced the greater response. Also, at each dilution level exposure for 7 days per week produced a greater response than exposure for 5 days per week. Molding animals £or 3 days £ollo~r~ng their final exposure brought about a decrease in the number of race per group ~th goblet cell hyperplasia. The overall d£strlbutlon of goblet cells wms greater in all smoke- exposed rats with no evident d£ggerences between the varLous groups (Table 7). There yes some indication o£ £ncreased haemosideros~s (excess of iron p£gmant from the breakdown of haemoglobin) in mnokJ- exposed rats, but no group compar£sons vere ~ade. Trachea There were no marked changes in the tracheal epithelluR of smoke exposed rats except for a small but consistent increase £n epithelial thickness. Variation in e~posuze conditions did not ~fect the degree of response, except that the 3 day hold£nE prior to autopsy caused a reduction in epiChe1£al thickness (Table 8). Goblet cell activity Ln the ~d-trachea was hyparplastlc in the majority of smok~-axposed rats. The number o£ rats per Broup shoving increased activity was not clearly related to exposure conditions, although exposure for $ days a w~k at 1:12 dilut£on and also hold£ng the one Stoup £or 3 days £ollow£ng exposure save a reduced response. One control group had • comparatlvely large number o£ rats ~th incrlased goblet cell actlviCy (Table 9). BAT Co LTD - MINNESOTA TOBACCO LITIGATION O r~ r',o

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~n Larynx Squamous netaplasie in the floor of the larynx was observed in all smoke-exposed rats, irrespective of exposure conditions (Table SO). Holding animals for 3 days following exposure increased the number of cases of ~ncomplete meteplasia, and also reduced the thickness of the ventral epithelium (Table ll). The deEree of squamoue hyperplasla on the medial aspect of the vocal cords was marked, and similar, for all smoke-exposed rats (Table I2). The an;m~lS held for 3 days prior to autopsy showed a reduced respoDse. A fully di£ferent£ated 8~ratum come,u- was absent £rom all control groups but was frequently observed fn exposed animals (Table 13). The number o£ rats per group 8ho~rinK this feature followed no clear pattern related to the seve=£ty o£ exposure, boa:, the i~oups of rats exposed for § days a week at a 1:12 smoke dilution and ~ose autopsied agter a holding period of 3 days had a lover incidence of keratin£sation on the vocal cords. DISCUSSION Keduci~K the exposure to S days per week from the more usual 7 days did not agfect the mortality rate o£ the animals. As in previous studies there was the tendency for deaths to occur at the beginning o£ the week. Under the c~itlon.s used for this experiment there was no appreciable d~££erence between the 8ham-mnoked and ~he cage control ra~s. Thus from a pathology point of view one or other o£ the controls could be omitted. Preferably, shmn-moked animals should be retained since they represent the t~ treamanC control in an experJJmenc. BAT Co LTD - MINNESOTA TOBACCO LITIGATION w t..r'l.

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-7- Exposlr~ animals to dilute smoke for 5 days instead of the usual 7 days per week produced similar histological lesions in the respiratory system. The extant of the lesions did vary with the different exposure conditions used in this study, the deKree of chsnKe dependinK on the level of the respiratory tract under consideranion. A al~sary o£ the histolosical £indinss is shown in Table IA. All values in smoke-exposed Kroups were greater than their corresponding controls. The chenKes in the lunK parenchyma and bronchi were all greater in those animals exposed to the hiKher concentration of smoke. For each smoke dilution level, those rats exposed to smoke 7 days per week had consistently higher values than those exposed for only 5 days per week. This general rule applied to those raCs 8utopsied the day £ollowi~ their final exposure. Of those held for 3 days. there was usually a marked regression of lesions. This pattern of regression corresponds with previous observations (1). Zn the trachea, epithelial thickness at the level of the first tracheal ring was increased in all rats exposed to dilute smoke. Of those autopsied the day following final exposure variation in dilution and the number of days per week exposed did not affect the degree of response. HoldinK animals for 3 days prior to autopsy produced a marked reduction in the tracheal epithelium thickness. Goblet cell hyperplasia in the main incrapulmonary bronchus was more frequently observed in smoke-exposed rats. Exposure at 1:8 for both 7 and 5 days per weak, together with 7 days per week at 1-12 produced 8iJnilar results when 8n/~al8 were autopsied the day following their final exposure. Rats exposed for 5 days ac 1:8 but held for 3 days and for 5 days at 1:12 smoke dilution showed a decreased response. t~ r~ r~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-8- The larynxes of all smoke-exposed rats shoved signs of sqzus mecaplasia and hTperplasia. The chauKes were readily identified and in the rats aueopsied within 24 hours of Che£r final exposure, variation in exposure conditions did not produce any differences in response. quanci£iable changes were sim£1ar in all rats exposed to mnoke. A possible exception was the reduced frequency of keratin on the modial aspect of the vocal cords in race exposed aC the lover smoke concentration for $ days per week. Holding anLmals for 3 days caused e resolution of all laryngeal Lesions (squsmous meCaplasi.a and hyperplasia, keraCinisacion). With regard to Che exper/mencal design it is noticeable that, with the exception of alveolar metaplssia, there was resolution of all lesions. All animals must therefore be auCopsied as soon as possible a£Car, and certainly v£Ch 24 hours o£ their ££ual exposure. Dec£ding on 5 days or 7 days per week exposure over a 6 week period will depend on the reKion of the respiratory system ,mder consideration, and the aims of the project. T£ the trachea and laz~y~x are under ~nvesCiKation there ~s no d£££erence between 5 and 7 day per week exposure reS~nms. Tn the lunK 7 days per week cons£scenCly produced greater changes than 5 days per week. But £n both cases (7 and $ days per week) the Stoups exposed at different dilution levels of smoke could he d£££eranc£aCed. Thus £f max£mun response £s not nacees&~y, only Stoup separation, 5 days per week for 6 weeks, would be suitable for the &ssesmnsnc o£ comparac£ve £nhelac£on cox£cicy. Zn this connection it is worth mention£nS thac apparently more severe exposure conditions do not necessarily produce a maximal or optimal funk patholoKy response. As far as inhalation bioassay work is concernod, differences between test Stoups are more readily observed BAT Co LTD - MINNESOTA TOBACCO LITIGATION ¢,..m

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-9- when patholosY end-points lie on • simple dose-response curve than at the higher end of a ranKe. For example, we have observed lover alveolar macrophaKe counts in swoke-exposed animals compared with those in groups exposed to lower TFM concentrations of smoke (2). A possibly more familiar illustration of the same kind of affect is Kiven by the "high dose anomaly" phenomenon seen in skin paintEng experimenCs (3). Tf the 5 days per week regina is employed the number of days with two exposures per day following 2 days without exposure (usually Saturday and Sunday) may be significant. In this study, before autopsy there was a mln~ number of 3 days of "full exposure" to smoke following a weekend without exposures. Until further evidence is available this must be reKarded as the minim~n period. CONCLUS ~ONS The followlnS Kuidelines, based on the results of thls study, will be used for the conduct of the next short-term bioassay study: 1. CaEe controls may be excluded provided sham-smoked controls are used. 2. Exposure of animals to smoke Wic_.____ee daily, on 5 days pc= week for 6 weeks. 3. Animals ~ast be killed within 2A hours followinK nhe final exposure to smoke. &. &t least 3 days "full" exposure must be siren follovins • smoke-free weekend. 5. One sex only, preferably female, may be used. REFERENCES 1. • •2. 3. EAT Eeport No. RD.1477 ~stricted, 23.3.77. BAT Rmport No. RD.1281 Restricted, 23.10.75. Biolog/cal leporc B26, May 1972. BAT Co LTD - MINNESOTA TOBACCO LITIGATION r,,o

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