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MICROBIOLOGICAL ASSOCIATES MAry Ann Montg ' September 1, 1978 - August 31, 1979 Prepared By C. J. Henry, Ph.D. R. E. Kourl, Ph.D. L. M. Schechtman, Ph.D. R. D. Curren, Ph.D. M. Dinowitz, Sc.D. B. Bhooshan, Ph.D. August 24, 1978 Director of Co Administration , ' Proposed Studies For CTR 0030 - Smoke Inhalation Carcinogenesis Studtes in Mice 6 (o4&1 ery ,S Richard E. Kourl, Ph.D. tract v Director of Research CTR CaNTRRCTS 027833 11247583 f CTR VIN 0442..

PROPOSED STUDIES Contract CTR-0030 TABLE OF CONTENTS ABBREViATiONS 1. CHRONIC INHALATION STUDIES A. •Introduction B. CTR-101A. Chronic exposure of BC3F1/Cum mice to cigarette smoke. CTR-1018. Chronic exposure of C3H/Anf-Cum mice to cigarette smoke. CTR-101C. Chronic exposure of DBA/2J mice to 1 cigarette smoke. i ii. SUPPORT SERVICES A. Dosimetry 1. CTR-102. 2. CTR-103. 3. CTR-104. 4. CTR-105. 10 aerosol. 11 B. Distribution of Cigarette Smoke Constituents 1. CTR-106. Deposition, dosimetry and chronic smoke exposure using 2R1 cigarettes compared to 2Al cigarettes in BC3F1/Cum mice. 8 Evaluation of SEM 11 animal containment capacity using 14C-DTC-2R1 cigarettes.• 10 Deposition and dosimetry of 14C-DTC-3A1 cigarette smoke in BC3F1/Cum mice. Comparative evaluation of deposition and dosimetrv after exposure to • B-naphthylamine or catechol in a smoke Autoradiographic analysis of lung tissue after exposure of mice to 2111 cigarette smoke. 12 2. CTR-107. 3H-Thymidine labeling index (LI) in lung,-liver, bladder, or kidney after exposure to smoke, or treatment with carcinogen. (Dr. R. Rasmussen). 14 3. CTR-108. The effect of cigarette smoke from different types of ci yarettes on pulmonary mixed function oxidases (MFO) levels in three strains of mice. 15 C. Short-Term 4ssays Relating to Possible Initiation Events 1. CTR-96. Inhibition of pulmonary DNA repair capacity after exposure to cigarette smoke. (Or. R. Rasmussen). 18 CTR coNTRacTs a2-7a34 11247584 CTR 1111N 04•42'2'9B

PROPOSED STUDIES Contract CTR-0030 2. CTR-8?A. •3. CTR-109. 4. CTR-110. Characterization of pulmonary cytochromes involved in s.-.,oke associated MFO induction. (Dr. 1. Wang). 19 Alterations in the immune response ' after exposure to cigarette smoke. (Dr. H. Herscowitz). 20 Evaluation of adaptation and stress in the inbred•strains of mice exposed to cigarette smoke. (Dr. W. Essman). 22 D. Short-Term Feasibility Assays Relating to Possible Promoting•Events 1.' CTR-111. Comparison of smoke induced ODC in three strains of mice and discrimination between ODC i•nduction and.AHH induction by cigarette smoke. 24 2. CTR-112. Evaluation of feasibility of a focus enhancement assay. 27 E. Aerosol Studies 1. Introduction 2. CTR-113. Acute toxicity of aerosolized TPA in the Inbred strains of mice. 3. CTR-114. Deposition and retention of aerosolized TPA. 29 30 30 4. CTR-115. Feasibility study of biochemical response to aerosolized chemicals. 31 F. Budget 32 CTR CQNTRRCTS 027835 11247585 CTR MN 04429013*

PROPOSED STUDIES Contract CTR-0030 > A(3BREVIATIONS AHH aryl h drocarbon hydroxylase BA benz(a~anthracene BaP, BP benzo(a)pyrene COHb carboxyhemoglobin CSC cigarette smoke condensate ConA concanavalin A DMBA 1,12-dimethylbenz(a)anthracene DTC, 14C-DTC doctriacontane EH epoxide hydrase ETR ethoxyresorufin FA flucinolone acetonide HA hemagglutinating Hb hemogTobin 3H-TdR 3H-thymidine IT intratracheal IgG immune gammaglobulin LI labeling index LPS lipopolysaccharide MCA 3-me th.y 1 cho 1 an th rene MFO mixed function oxidase ODC o*rnithine decarboxylase ORNL Oak Ridge Nattonal Laboratory PAG polyacrylamide gel PFC plaque forming cells PHA phytohemagglutinin SEM Smoke Exposure Machine SRBC sheep red blood cells TPA tetradecanbylphorbol acetate TPM total particulate matter WHSM Walton Horizontal Smoking Machine CTR CQNTRRCT6 027836 11247586 ~ ~~ ~-I~>~ 044,300

PROPOSED STUDIES Con t ract CTR-0030 I. CHRONIC INHALATION STUDIES A. Introduction. These studies proposed will describe those conditions necessary for maintenanc,e, utilization and implementation of a state-of-the-art inhalation facility for the determination of the potential biological effects of whole cigarette smoke in the inbred strains of mice. In order to carry out successful chronic cigarette smoke inhalation studies, it is required that: 1) the facility must be capable of generating and characterizing large quantities of cigarette smoke; 2) the facility must be capable not only of documenting and quantitatina the level of smoke generated, but also documenting the smoke deposition patterns in the test animal; 3) the facility must be capable of utilizing large numbers of animals in chronic studies; and 4) the facility must be capable of evaluating and characterizing the potential biological response of the test animal to these exposure conditions. The inhalation facility at M.A. meets these rather stringent conditions. The following proposed studies should ensure a continuation of the state-of-the-art inhalation facility. B. CTR-101A. Chronic exposure of BC3F1/Cum mice to cigarette smo e. CTR-101B. Chronic exposure of C3H/Anf-Cum mice to ci arette smo e. CTR-101C. Chronic exoosure of OBA/2J mice to - cigarette smoke. The chronic inhalation studies are designed to evaluate chronic exposure of rJiole cigarette smoke as a potential com- plete carcinogen in.three strains of mice (BC3F1/Cum, C3H/Anf-Cum, and DBA/2J).Data to date suggest that cigarette smoke at best, possesses weak biological potency to man. Because of this low biological potential and because of the conditions under which man is exposed to cigarette smoke, it is a necessary•requirement that any smoke exposure study use conditions whereby: a) the smoke deposition and distribution parallels that of human smokers b) the length of time of exposure parallels that of human smokers, and c) the animal system employed is capable of - expressing those types of biological lesions associated with cigarette smoking in man. 1. Rationale.. Several parameters must be considered for this experiment including: 1) estimated lifetime of the animals under these test conditions; 2) numbers of mice required initially in order to insure a significant number of animals during the time when lung lesions may be expected; 3) an assumption as to the incidence and latency of lung lesions -1- CTR CONTRRCTS 027837 11247587 CTR I-IN 0'44301

PROPOSED STUDIES Contract CTR-0030 4) the type of cigarette employed; 5) dose of cigarette employed and 6) equipment capacity for smoke exposure. The experiment will be scheduled so that mice are exposed to smoke for their "lifetime". In an attempt to deter- mine what the lifetime is for each strain, the following data were assembled. Table 1 Mean Life Span Strain (LD50) References i3C3F1/Cum 30 Months Peter, et al (Mech. A ing Develop. +T~c'251, 1975? C3H/Anf-Cum 28-30 Months Peterson (Fersonal Com- munication) DBA/2J 22 Months Myers (Birth Defects 14:41, 1978) Realistic estimates for the lifetimes of the mice are difficult to predict because of variations in laboratory con- ditions and because of the treatment conditions employed. Data from our laboratory'(CTR-i100) suggest that BC3F1/Cum mice exposed to 2A1 cigarettes at a dose level of 10 per day yield an LD5o (50% of the animals are dead) of about 44 weeks. The machine control animals also yield an LD50 of about 44 weeks. However, less than 1% of the uninoculated control , animals have died during this observation period. The factors causing this high death rate include, human and equipment associated accidents, stress, toxicity of treatment and a combination of these factors. The accidental deaths have decreased dramatically during the past four months mainly' for two•reasons: 1) the technical staff has become experienced in the day-to-day handling of the animals, and 2) equipment malfunctions have been an increasingly rare occurrence. The death rates in the machine control groups have decreased significantly as the result of the installation of flow meters at the end of the smoke line in the animal containment units. These flow meters insure adequate flow of air and/or cigarette smoke and have significantly decreased the numbers of animals dying on the smoke exposure machines. Because.of these latter changes, we now assume'that the LD50 for BC3F1/Cum mice for uninoculated controls, machine controls, and smoke exposed animals will be approximately 30 months, 15 months, and 12 months, respectively. Although not directly comparable, results from experiments CO-91:and CO-94 tend to corroborate these assumptions.that is,handling animals every 4-5 days throughout their lifetime has resulted in at least 20% survival at 118 weeks of age. Therefore,• it would seem for the BC3F1/Cum, C3H/Anf-Cum, and DBA/2J strains that the initial numbers of mice should be 1600, 800, and 400 for smoke exposed, machine controls, and uninoculated controls, respectively. A tentative schedule for the bioassay of the effects of whole cigarette -2- CTR CONTRRCTS 027838 11247588 CTR NN 0443022

I ~E 1 . 1 It BC3F1 9/78 C311/f 7/79 0BA/2J. 3/80 TABLE 2 SCHEDULE FOR CHRONIC INHALATION STUDIES -•CTR-0030 8/21/78 ESTIMATED NUMBERS OF SURVIVORS 1978 I • 1979 I 1980 1981 Strain Group S 0 N D J F M A M J J A S 0 N D J F M A.M J J A S 0 N D 1 J F M A M J J A S ~ k 1 1 1600 :1200 800 400 200 ^-0 I . Smo e BC3F 2. Mach. ~00 • 550 380 • 200 NO j. Uninoc. 00 400 350 200 nv0 4. BPs .~ Smoke 200 150 100 ...0 5. BP t i Mach. 200 1S0 . 100 ti 0 ~ 6. 8P 100 100 100 ~ 0 1 TOTAL SI:OKE 1800 1350 9oo 600 400 200 C3N/f Cum I. Smoke 1600 1200 ' 800 600 400 2. Mach. 800 550 380 3. Uninoc. 400' 400 350 ~ 4: BP + Smoke 200 150 100 '~0 N 5. BP • I 100 L• 0 ~ Mach. 200 i50 v (7 , 6. BP 100 100 100 A,0 0Cn0 t0 TOTAL SMOKE ~ 1800 . 1350 9oo 600 4 # (~ OBA/2J 1. Smoke 1600 1200 600 350 2. Mach. 800 550 300 250 , 3. Untreat. 400 400 275 250 4. BP + Smoke 200 150 100 .v O 5. BP• ~ Mach. 200 150 ' 100 100 / 0 O rw. 6. BP 100 1UO 0. TOTAL SMOKE ~ 1800. 1350 700 .~ ~ OVERALL SMOKE Ln IOTAL SPACE 0 Pres.. Equlp. 1800 2000 1350 2000 2700 2000 . 3750 2000 2650 2000 1500 2000 750 ~ OVERALL 'AVAIL. NJ SPACE 200 650 -700 -1750 -650 +500 ~ CD TOTAL SPACES. a 4 4000 ~ 051. Pres. Equip. 4000 000 W AVAIL. SPACES 1300 250 1350 the equipment capacity will be doubled at this time. aIt Is proposed that

PROPOSED STUDIES Contract CTR-0030 smoke from the 2R1 cigarette on BC3F1/Cum, C3H/Anf-Cum, and DBA/2J strains is given in Table 2. The schedule was made with the assumption that approxi- mately 200 animals would be alive during the time at which the LD50 Qccurred to the time at which the L090 occurred.. This is the minimal number of animals which must be evaluated in order to insure that an assumed 2% squamous cell carcinoma incidence In the smoke exposed group and a zero incidence in the control groups will be statistically significant. A 2% .inc.i.dence was based on the extrapolation of the data of Hammond, et al (Origins of Human Cancer, =d. Hiatt, Watson, Winsten, Co1U-Spring Harbor Laboratory, 101-1t.2, 1977). on the inci.dence of lung cancers in humans exposed to high levels of cigarette-smoke (approximately 2 packs/day) for extended periods of time (>40 years). These proposed studies will use the 2R1 reference cigarette. A comparison of its total particulate matter (TPM), nicotine, and puff characterisitics relative to the 2A1, 3A1, and 1R3 cigarettes are given in table 3. Cigarette Type Table 3 TPM m /Ci .) Nicotine m /cl .) Puffs (#1 Cig. ) 2A1 40 0.51 10 3A1 43 0.35 11 2111 50 2.81 12 1R3 26 1.25 8 Recent studies in our lab have shown that this relatively "high" nicotine cigarette can be used If given for 20 second exposure times, eleven puffs/cigarette, and a total of 5 cigarettes per day. it is assumed that this exposure regimen will result _ (n the deposition of about 25 mg TPM/kg/day. (A dosimetry experiment with the 2R1 cigarette has been proposed (CTR-102) to quantitate this deposition.) Such a dose is equivalent to a human smoking approximately 3.5 packs of cigarettes per day. Also presented in Table 2 Is the schedule of an experiment to study the potential co-carcinogenicity of ciga- rette smoke in BaP Induced lung cancers. The rationale for this study is as follows: 1) previous studies have shown that IT instillation of cigarette smoke condensate materials enhanced BaP induced lung tumorigenesis (see progress report, 1978), and b) this enhanced tumorigenicity was observed within 1.0 year afte.r BaP treatment. This pa-rt of the chronic inhalation study would be expected to end by 12-18 months. ' The schedule in Table 2 shoais that the BC3F1/Cum strain will be put on test September, 1978 and in order to keep to the scheduled initiation dates of Sept.1979 (for C3H/Anf-Cum) -4- CTR CONTRACTS Q27B4'Q 11247590 CTR MN 044304

PROPOSED STUDIES Contract CTR-0030 and March, 1980 (for ')EA/?J), the capacity for animal exposure must be doubled. We propose to evaluate two alter- native arrangements of animal containment units that should double :he number of anim?ls that could be exposed per day. The alternatives are a tandem (one behind another) arrangement o" animal containment units, or the doubling the number of smoke •lines coming from the SEM. These alternatives will be evaluated by testing for total flow rate, smoke particulate deposition and levels of CO/CO~, in line. These details are presented in proposed study CTft-103. Table 4 Schedule for Mice Exposed to 2R1 or 3A1 Cigarettes (12 puffs, 12 min) Time- Smoke Exposed Dos # Ci e Machine as. Exposure Dose # Ci E gs. auiv. Run # # Mice . Run # = Mice 6 a.m. (~ 7 8 (S-1) 480 ~ 5a (M-1) 480 5 9 10 (S-2) 480 5 '(M-2) 480 5 11 ,~ Clean Machine 12 QS-3) 480 5 (M-3) 480b 5 2 1-4) 48ob 5 Dismantled & Clea n 3 4 Set up for next day 5 p.m. a. Each 2R1 or 3A1 cigarette requires 12 min to burn to the 23 mrn butt. A rest period of 12 min then follows. Thus, approximately 120 min are required for a dose level of• 5 cigarettes. Loading and unloading 480 mice requires an additional 20-30 min. b. Total Capacity: a) 19?0 mice ex posed to 5 cigarettes, or 960 mice exp osed to 10 cigarettes b) 1440 mice ex posed to 5 . cigarettes equivalent (machine controls). -5- CTR CONTRRCTS 027641 11247591 CTR t-IN 044,~OE;

PROPOSED STUDIES Contract CTR-0030 2. Procedure. The following groups of BC3FI/Cum, C3H/Anf-Cum, and DBA/?J mice will be used at the scheduled times. (see Table 2) No. of G rouo pS i ce T rea tmen t 1 1600 Smoke exposure 2 800 Machine control 3 400 Uninoculated 4 200 BaP and smoke exposure 5 200 BaP - machine control 6 100 BaP control The procedures followed will be those used and developed under 1-100 and the daily schedule is presented In Table 4. a. Mice will be 8-12 weeks old when ear-tagged and put on test. Each mouse will be individually marked. b. Observation records•will be kept on Individual mice. Weekly observations will be recorded in all groups and will become part of the permanent record for the experiment. c. All mice will be weighed monthly, at the same time of day, two days after cages have been changed. For the smoke exposed and machine control mice, weights will be obtained at least two hours after exposure. d. Smoke and machine exposure will be initiated gradually until exposure reaches 5 cigarettes/day and will continue 5 day/week for the lifetime of the animal. e. Carcinogen treatment of the designated groups will be given at weekly intervals and be completed before the first smoke exposure. The dose will be 3 x 1.2.mg BaP/0.02 ml gel-saline. f. Urine will be routinely collected from each group of animals and assayed for mutagenic components according to procedures described. g. Carboxyhemoglobin, hemoglobin and methemoglobin determinations will be made for mice from each group at monthly intervals. h. Several assays will be used to monitor this long-term study: OOC, AHH, and pulmonary DNA repair capacity. These will be performed periodically on 3-4 mice and compared to known controls. i. Mice will be taken off test when moribund and the following tissues examined histopathologically: lung, trachea, larynx, head, and any other abnormal tissue. -6- CTR coNTRaCTS 027842 11247592 CTR HN 044,306