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taken from the animals for this purpose on a Monday morning, that is after a period of at least 64 hours without smoke exposure. •Blood carbon monoxide leveL in all smoke.ezposed and control animals were at or below the resting level, iL e. below a relative conceatra_ tion oE 2%. Hence, there is no accumulation of carbon monoxide in the blood after chronic exposure of hamsters to cigarette smoke of the coacentrationA and amounts employed. f. Htstepathological findings In lungs, tracheas and aasophsrynxes after 45 weeks of smoke ezposure - Some isolated puknonary aucrophages Pere seen in a few pf the control aniz<uls, especially In the 87. Z0 line (see Groups E. F\And FF in Table VII). However, an intense agglomeration or clnrnpiag of macrophagee, with tbe appearance of granulomas obviously occurs as a response to smoke inhalation and Is not seen in the sharn-smoked or cage.held control animals. This phenomenon is particularly pronounced in the 8-7. 2D line of inbred hamsters, where it occurs in all six anims•ls. The formation of clumps of macrophages in the lungs of smoke exposed hsmsters of the 87.20 line, thus, appears to be r distinct consequence of smoke Inhalation. It occurs after#5 weeks of exposure, and has not benn seen in the few animals that died aftar shorter periods of smoke treat:nent. It is interesting to note that through the use of inbred lines of hamsters a 100% response in one of these lines (87.20) could be achieved. Hence, this inbred liae of hamsters appears to be more susceptible to cigarette smoke than the 15. 16 line. There were other Distopathological findings In the respiratory system, and tbese are summarized in Table VIII. These findings include inflaa=ation, metaplasia and atypical cells in the bronchi, alveoli, trachea, larynx, pharynx and nasopharyax of the animals. With only one exception (two instances of bronchiohr in. fla-ation in Group C), these findings are limited to the smoke_ eYposed and sham.sa,wked groups, but in the 8?. Z0 line are muc6 more frequent In the smoke-exposed than In the sham-smoked ani. mals (Groups D and E, respectively). g. Other histopathologic findings - No distinctive features were observed in the adrenals, the pituitary and the liver of the animsls after 45 weeka on experimental or control condi- tions. CTR coNrRacTS oO5902 11225643 C ~~ R ~"~ ~"°~ 0'~ .'` .~''k 80

3. Contiauation of Experiment The low taortality of the animals after prolonged exposure to relatively 1srQe doses of cigarette smoke make it appear likely that the treatment can be continued to a duration of two years. 8ecause of the staggered beginning of the experiment, the first several lots of animals wiII have reached the 104-week ta,rget duration, when there will still be a sufficient number of animals lagging behind in the length of treatment, so that it can be decided at that time to add one or even two interrnediate sta- tions for histopathological studies bet.veen 4S and 104 weeks, s. g. at 75 and 90 weeks of exposure, provided the'mortality Will nQt have markedly increased until that time. \ After termination of this phase of the sxpcrimsnt, it would be logical to initiate another experimeat, similar in de- sign to the present one, but with the gas phase of smoke only. Depending on the results to be obtained in the two control groups of each inbred line, it might be indicated to decrease the number of animals to be included in these control groups in the future ex- periment, and to add instead an equal number of animals in the smoke.exposed groupr (gas phase only) with a higher frequency or a longer duration of treatment. Experience available now in- dicates that such increase in exposure to the gas phase of smoke will be tolerated by the animals. -10- cTR CONTRRCTS 005903 11225644 ~,., ,.. . , vrI' MI m! ! I I 1 I 0424m 8 3 w

Budget C.191 June 1, 19Tt - May 31, 1973 Salariea Supplie s ;34, 500. 00 Animala 2.600.00 Faed, bedding, other laboratory and animai room supplies 2, 980.00 Travel 600.00 Concultaats 1, 900.00 Other Books, subscriptions, photography, charting, publishing expense 650. 00 $43, 230. 00 Equipment 1,300.00 Overhead 100% of S and W 34, 500. 00 35. 800. 00 Subtotal $79, 030. 00 20% Fee 1, 580. 00 Total $80,610.00 cTR caHTRacTS 005904 11225645 L. rTR Hf j 042282

Table I Experunental Design of Inhalstion Exposure of Inbred Hamsters Number of A.nimale per Group«* Groap Identi- fication HatnstIf r Line moke Exposure SLam- Smoked Controls Cage- Held Controls A 15. 16 102t B 60# C 60# D 87. 20 :02t E F I 60s .QO$ * Males, 107 t 3 grams v+hen started. ~ In lots of six hamsters, each lot being caged aad exposed together, r 17 Lots of six. t 10 Lots of six, Ln r N N CTR coNTRACTS 005905 11225646 ....n,., CTR HN 04~~~~

Table IL Staggered negtnning of Experiuuntsl and Control Conditions by Lots of Six Hamsterso Number of Weeks Eacb Lot of Six Hamsters Ha. Seen on Experimental or Control Conditions** Total No. of Lots on F;~c- perimea Tt Group A Group B Group •C Group D Group E . Group F 69 67 69 63 58 63 69 67 68 63 53 58 67 67 67 58 42 53 -65 65 65 58 41 43 63 63 63 48 40 41 63 63 63 46 39 39 63 63 61 43 38 ~ 36 63 61 61 42 37 34 61 59 59 41 34 34 61 40 61 39 61 38 59 37 56 35 53 35 38 3t 16 9 9 16 9 9 e# t * Six 1>asneters to each lot at beginaing of experim.nt, some aniarAls have since died. Statns ae of April 3, 1972. One lot (six animals) in each group were sacriticed after about 45 weeks on ex- perimental or control conditions. cTR coNYRRCTS 005906 11225647 • {rr -l p R I I I 1 04( iM f.M' 4

Table III Suroival and Mean Body WeiBhts of Animals* Survival Group Desig, aation Mean Body Weights (Grams) Ivlean After 31 Body No. of Ani. Weeks on At Begin- Weight aulst That Expsri- ning of Ez Increase Died until mentalor 4/3/72 , parizaeat Control or Loss in 31 Conditions Weeks t A 102 6 90 6 (6. 3%) 1 07.1 105.8 _ 1.3 * B 60 6 45 9 (16. 7°,i) 1 03.9 114.5 10.6 C .60 6 46 8 (14. 89G) 1 06.3 118.8 12. 5 D 102 6 81 15 (15.6%) 1 06.6 ~ 108.1 1. 5 fi 60 6 53 1 (1. 9%) 1 07.9 146.3 38.4 F 60 6 51 3 (5, 69e) 1 09.4 145. 3 35.9 * As of April 3, 1972. i. e. after from 3Z to 69 weeks'on e:perimental or coAtrol conditions. t Percent of animals that died in parentheses. ** Body weight loss!! cTR cONTRaCTS 005907 No. of Ani. mzls at $e- giaaiag of E;eperiment No. of Ani- mals Sacri- ficed during Expe riment No. of Survivors on 4/3/72 .1 in . • /n n n . .. 11225648 ,,..nn • r LI' I R I (N 0 4 22 22 -8 i~~

Table IV Organ Wetg6ts in I5. 16 Inbred Kamsters after 45 Weeks on Ezperimental or Control Conditions Group Organ IPeighte (mg) Statiatical Significance of Differences = O i D Between Meana of Two Groups t.Values rgan es g- Stat r nation Means Deviations Group A Group B Group C Group CCt of Means ~ Lung and traebea A 967 68 5.0 - - B 1,185 76 5.0 - 3.6 C 961 219 . . - cct 918 1119 3.6 Heart -- A 401 29 - - 6.7 B 381 23 6.3 .C t 393 43 . - 4.2 CC 318 c 6.7 6.3 4.2 Liver A 4,217 299 - - - - B 4,112 313 - . - C 4.550 339 - - 3.4 CCt 4, 04Z 150 3.4 Kidneys A 949 60 - . 5.2 B 946 31 - - 7.0 C t 976 86 - - 4.6 CC 800 37 5.2 7.0 4.6 Adseazls A 11.1 2.7 4.3 - - D 17.2 1.7 4.3 - - C 15.8 3.7 - - - CCt 14.8 2. 6 Spleen A 77 19 - • - B 76 3 - - 5.3 G 95 14 . - 5.6 CCt 58 7 5.3 5.6 Brain A 981 43 - • 3.5 B 852 119 - . - C 928 97 - - - CCt 881 57 3.5 Pituitary A . Z 1.16 - - - B 2.6 0.92 - - - C 3.7 1.63 - - - CCt 2.2 0.39 - ontinue on neat page CTR coNTRaCTS 005908 11225649 t.Tr.7•(1 ,cc' ' ') '; o••, -i• •T •nrr •na t..r Tf"r i f i 7 0'-T 2286

Table IV - Continued Footnotea ~ t_Values of less than 2.7 indicate the absence of statistiul significasce of the difference, and these values are not tntered in the table. t Lnitial control animals without felt collsrs of 107 t 3 grams body weights. ', CTR coNTRaCTS 005909 11225650 '7 "` •t ' l1^(` 'nu 4T I. 7- i c . • '7 : 9., C w°R H~~ 04~2.81 "

Tsble V Organ Weights in 87.20 Inbred Hamsters after 45 Weeks on Experimental or Control Coaditions Organ Weights (m41 5~4stical Signif'iesnce of Differeaces * o G Setween Means of Two Groups t. Values r up Organ Desig- Standard t nation Means Deviatioas Group D Group E Group F Group FF of Meaas ~ L.ung and traches D 1,237 116.4 - - 4. Z E 1,310 181.6 - ` . 4,2 F 1, Z55 229.5 - - 3.0 FFt 929 139.4 4.2 4.2 3. 0 Heart - D 473 61.3 . . 4.3 E 526 38.7 . - 9.7 F 483 23.0 . - 10.7 FFT 361 15.7 4.3 9.7 10.7 Liver D 4, 825 5C4 2.9 Z. 9 - E 5,657 541 2.9 . 5.0 F 5,967 826 2.9 - 4.5 ~ FFt 4,187 513 S.0 4.5 Xidneys D 1,087 93. S 2.9 3.6 7.2 E 1, 225 78.7 2.9 - 12, 7 F 1,342 146.1 3.6 - 8.9 FFt 804 19.2 7.2 12.7 8.9 Adrenals D 12.3 2.06 3.4 - 3.2 E 2011 5.13 3.4 . - F 13.8 4.66 - . - FFt 15.3 0, 99 3. 2 - Spleen D 62 15.9 - 4.8 - E 96 36.5 - - - F 124 27.6 4.8 - 4.6 FFt 68 10. 5 4.6 Bra.in D 985 112.8 - - - E ' 1,046 207. 6 F t 995 88 - - - FF 897 4S Pituitary D 2. 0.81 E 4.1 2.32 F t 4.9 1.94 .- - - Fl:' 2. S 1.01 Continued on next page. cTR CaNTRacTS 005910 11225651 ,,. , , . I , , . , . ., CTR H~`'" 0'~~ ~ ~ ~ +

. Table V - Continued Footnotes t ~ t_Valnes of less than 2. 7 indicate the absence of sta. tistical significance of the dMerence, and theee values are not entered in the table. Initial control animsls without felt collera of 107 t 3 grams body weights, ~ CTR CONTRRCTS 005911 11225652 - CTR HN 0422--