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I H T®012000! A CAPABILITIES STATEMENT FOR INHALATION TOXICOLOGY STUDIES USING A NOSE-ONLY INHALATION SYSTEM Prepared by Carol J. Henry. Ph.D. Ken K. Kanagalingam, Ph.D. William C. Hall, V.M.D., Ph.D. Richard E. Kouri, Ph.D. March 18, 1981 Microbiological Associates 5221 River Road Bethesda, Maryland 20016 (301) 654-3400 !Ka . ,1g~+~btiolo~iC~d :~5s0C1a~ -q'.M-S

a H TM01 20010 I. INTRODUCTION C The human health effects of exposure to chemicals, both alone and in conjunction with other environmental and occupa- tional agents. continues to be of major public concern. Today, some 30 goverranent agencies and many industrial and research organizations are actively working to identify and char3cterize potentially hazardous substances and to understa,zd the human health implications of exposure to these compounds. A significant route of exposure for introducing toxic and other hazardous substances to the body is through the respiratory tract. Each day, adults respire 10,000 to 20.000 liters of air containing particulates such as occupational and atmospheric dusts, commercial aerosol products, smokes, radioactive parti- cles, and other noxious agents. In addition, a large portion of the population voluntarily inhales one particular complex aero- sol. cigarette smoke. Respirable aerosols are deposited along the entire respiratory tract, and probably play a significant role in initiating or exacerbating a variety of pulmonary, car- diovascular, or infectious diseases. The mechanisnt by which these agents exert such wide ranging toxicological and/or carcinogenic effects is not known. These processes have been postulated to occur in multiple steps (Figure 1), many of which have been studied in detail (18)- Given the complexity of the biologic response, an important aspect of experimental inhalation studies concerns limiting the route of exposure to or.ly that which is inhaled and avoiding dermal or oral exposures. Microbiological Associates (MA) has evclved a model system for conducting inhalation toxicology stud3es by uhich rodents, which are obligate nose-breathers, are exposed to aerosolized materials via oEIX the nasal orifice. MA has implemented these studies under a program of stringent ani.roal health requirements, thereby min:mizing unknown interactions orr synergistic effects, so that the endpoints of these studies are not compromised. ' .1'IiC1obi01"icswl Usocha 1 r_._~..,:..-

11. OF3JECTIVES MT®0120011 MA is now in a position to make available its "state of the art" approach to inhalation toxicology. During the last 8 years MA has been the prime contractor for the development, implementa- tion and characterization of a pulmonary toxicology bioassay in rodents. This has enabled us to a) develop specialized equipment not only for the generation, delivery and monitoring of a variety of aerosolized materials, but also develop a rapid, safe, and effective animal containment system faciltating nose-only expo- sure to these aerosols b) develop and characterize an animal model system for pulmonary carcinogensis, and c) institute a com- prehensive animal diagnostic laboratory in order to preclude utilization of diseased animals for this research as well as pre- vent introduction of infectious agents into the factlity. This background enabLes MA to address several existing problems in inhalation toxicology at a level of understanding which could not be considered in the past, and which we feel has great potential for the future. III. CAPABILITIES C A. Nose Only Exposure The animal containment system developed for nose-only expo- sure is shown in Figure 2. Groups of five animals are placed in a"stock-like" holder usina a combination of a neck slot and a restraining spring (Figure 3). A chin rest insures that the nose of the animal is aligned with the cone shaped opening on the e--posure modules. The nose of each animal passes through a dental rubber dam diaphragm, which forms a seal that prevents exposure of the animal body to the aerosol. Air circulates freely around the animals, preventtng temperature build up which often occurs when whole body tubes are used. In adc:ition, fecal and urine contamination is prevented by the plastic coated grid which supports the animal in the tray. Urine and feces are easily collected from the tray when required. Daily restraint for up to three hours (twice per day) results in no mortality or obvious ill effects to the animals. B. Aerosol Generation The animal containment unit which provides "nose-only" exposure of the animals is compatible with any aerosol genera- tion system which might be required. Four applications with which MA has considerable expertise are detailed in thr follow- ing sections. 1. 13ebulizer A modified Kay nebulizer (17) has been employed for aerosol generation of specific chemicals, such as catechol (1.2- .~YCl~Ob1D~CA1 ~S50C3:~ 2 2 m ~

I HiFS G12L(;i2_ r dihydroxybenzene, a major constituent of cigarette smoke and , shown to be a tumor promoter on mouse skin, 5) and 12-0-tetrade- canoylphorbol-13-acetate (TPA, the active component in croton oil and the most potent tumor promoter known. 25). Chemicals soluble in ethanol or water have been used in this system to successfully generate particulate aerosols of particle size -0.5 um geometric mean diameter. The particulate deposition in mice exposed to these aerosols has been shown to be primarily in the lung (60- 70%) and total respiratory tract ('80%). 2. Dust-generator of this system is in progress. A Timbrell dust generator has been recently employed with this system to generate aerosols of UICC chrvsotile asbes- tos. Flnoloying a vertical elutriatorr in the asbestos aerosol stream resulted in particle sizes ranging from 0.2-10.0 um, mass median diameter. An Anderson cascade impactor was used to with- draw particle samples from an animal exposure hole to charac- terize th e particle size range offered to the animal in the aerosol stream. Complete characterization of the potential use 3. Cigarette Smoke l Whole cigarette smoke has been generated and charac- terized using two different generation systems: a dynamic expo- sure system (Figure 4) and a static or standing exposure system (Figure 5). Specific details of these two systems may be found in the references (4, 10, 12) and in Appendix A. Smoke was generated under standard conditions for puff duration. puff volume and puff frequency, and particle sizas characterized. The deposition and internal distribution of the total particulate matter of the smoke was determined in mice after exposure to a wide range of experimental conditions (10, 12 and Section IV of this document). 4. Vapors or Gases Vapors, gases or gas phase constituents of complex aero- sols can be generated and used in this system. Carbon.monoxide and carbon dioxide have been used as positive controls for cer- tain cigarette smoke exposures and an assay has been developed to routinely detect carboxyhemoglobin in mice (9). C. Aerosol Monitoring System A monitoring system was developed for continuous measurement of particulate aerosols (see Figure 6) and certain gases. The aerosols are monitored with an optical-type scattering detector (13). Each exposure is documented on a strip chart recorder. which provides a permanent record of aerosol concentration and exposure period. In the case of cigarette smoke, this monitor provides both a puff by puff profile of each smoke exposure and a 3

I • NT00120013 r cunulative value for the total particulate matter from each l individual exposure. The mcaitoring system also allows for con- tinuous measurenent of carbon monoxide and carbon dioxide, either as single constituents or when present in complex aerosols generated during combustion proce« es. Sampling ports are available for collecting particulates or gases for more detailed chemical or microscopic anaJyses. Samples can be collected immediately preceeding the animals (Figuros 2 and 6), or from any point along the exposure module. D. Animal Health Health, quality, and conaistency of experimental animals are absolutely essenti al to the production of accurate and repro- ducible test results. Infectious agents can cause clinical disease of laboratory animals resulting in mortality. Such disease frequently can reduce the numbers of subjects on test below an acceptable minLnum, will significantly effect the sur- viving animals' response to the research proLocol, and will result in expensive, time-consuming delays in the intended pro- ject. Not as obvious, but of equal ly serious concern, is the effect of subclinical infections with a variety of agents which can alter the animal host's response to numerous toxicological procedures. Disease diagnosis is an essential part of the disease con- trol program in operation at MA, an approach which MA nas helped to pioneer. A11 animals are quarant3_ned and screened for adven- titious agents, as outlined in Figure 7. Specif ically for long term inhalation studies MA has used the mouse because of the availability of colonies f-ee of two lung viruses, Sendai and pneumonia virus of mic4. With the availability of other colonies which are freQ of these two lung pathogens, other species can be routinely used. As further protection during long-term studies, a vaccine against Sendai virus has been developed within this program The utility of this vaccine has been recently demon- stratzd (1, 23). IV. RELEVANT BACKGROUND STUDIES A. Dosimetry and Distribution . 1. Cigarette Smoke Farticulates Jsing standard smoke exposure condi-tions, the deposition and internal distribution of the total particulate matter from cigarette smoke has been determined in various strains of mice (10. 11, 12). Results showr: a) smoke exposure conditions can be varied so :iat deposition from 30 ug to 200 ug total particulate matv'leP/lung can be obtained, b) 80-90% of the deposition of the total par:iculate matter is found in the respiratory tissues. c) the mouse-to-mouse variaticn for particulate deposition in pulmonary =issue is -20$, d) similar deposition ana distribution 4 /.~lo M....~ical :As5ocia

H TM0120014 of particulates are observed in both male and female mice, and e) deposition and distribution of particulates are not altered in mice exposed to smoke on a daily basis for over 6 months. Depo- sition, distribution and clearance of a specific constituent of smoke are dependent upon the metabolic fate and chemical class of constituent used. The pharmacokinetics of four particulate phase constituents of cigarette smoke have been studied in our labora- tories (11 15, 16). Measurements of carboxyhemoglobin levels reflect the uptake of the gas phase smoke and are highly corre- lated with pulmonary deposition of total particulate matter (9). 2. Particulate Aerosols The deposition and internal distribution of the phorbolester, TPA has been determined after exposure of mice to 3B-TPA aerosols. Approximately 60-70% of the total initial radioactivity was found in the pulmonary tissues immediately after the exposure period, with rapid loss of radioactivity from the lung ( tIs -30 minutes ) and total body ( t.4 .-24 hours ). This exposure system with TPA has been used in our laboratories to specifically induce pulmonary ornithine decarboxvlase, an enzyme which is rate Limiting in polyamine biosynthesis and thought to be a marker for tumor promotion (3, 25). Similar aerosol experiments are in progress with catechol (15, 16). B. End-Points for Chemically Induced Lung Cancer 1. Pathology Well defined histopathologic criteria have been estab- lished for both acute and chronic exposure to model chemicals and whole cigarette smoke. These criteria have resulted from micro- scopic analysis of tissues using Light microscopy, transmission electron microscopy and scanning electron microscupy. The inbred strains of iaice have been used as an animal model for lung cancer with model chemicals and have been shown to express those types of lung cancer normaLly associated with cigarette smohing in humans (e.g., squamous cell carcinomas, alveolar adenocarcinomas, adenosquamous carcinomas and poorly differentiated carcinomas, 2. 6, 7). As has been suggested in humans, susceptibility to lung cancer in mice seems be genetically regulated, and is linked to a higher capacity to metabolize certain chemical carcir.ogens (18, 19, 20, 21). Morphologic criteria have been rigorously defined for preneoplastic and neoplastic lung lesions in mice treated with model chemicals, thereby demonstrating the potential of the system for the evaluation of the biological effects of unknown chemicals or treatments (2, 6, 7). Experimental conditions can be designed so that 80-90% of the mice die from pulmonary carcinomas ',H. 7). T%aus a model system is available to study the induction, expression and pro- gression of lung tumors under conditions where a large proportion of the animals express a neoplastic response. 5 ~-J° .r~a~+or,iooogica~ ~ocia

2. Short Term Markers HT20120015 Several short-term markers have been determined for the evaluation of the biological effects of pulmonary exposure to intratracheally instilled chemicals, aerosols, and whole ciga- rette smoke. These include: a) induction of microsomal mono- oxygenases in the lung and liver (20), b) induct ion of pulmonary ornithine decarboxylase (3, 8), c) determination of levels of DNA repair capacity of lung tissue (24) d) determination of the extent of DNA damage in pulmonary tissue, e) determination of rates of pulmonary macromolpcular synthesis (i e., DNA, RNA, protein and collagen), f) separation and identification of speci- fic metabolites using high performance liquid chromatography, and g) physiologic response in the lung such as the formation and persistence of aberrant cell types as measured by both light and electron microscopy. V. FACII,ITIES Smoke inhalation experiments are carried out in our inhala- tion facility, which occupies 8000 square feet and employs 25 individuals. The facility has restricted access to ensure the health of the animals. A gas powered generator provides emer- gency power in the event of power failure. Fresh, filtered air, regulated for temperature and humidity, is provided to the animal rooms. Air to the animal rooms is not recirculated and is changed at the rate of 10-12 room changes of air per hour Rooms are monitored 24 hours per day for changes in te,nperature by an alarm system, which notifies responsible personnel in the event of a malfunction. A cage washer and autoclave are avail- • able tor sterilization of food, bedding, and cages. Chemical fume hoods and Type II, Class II hoods are equipped with charcoal and HEPA filters. Specialized laboratory equipment available for the inhalation fac:ility includea Instrumentation Laboratory CO-oxirrater, a Packard Tissue Oxidizer, Leitz Ortho- plan binocular compound microscope, Psrkin-zlmer Gas Chromato- graph, Beckman Infra-rad ultraviolet./visible Spectrophotometer, Aminco-8owman Spectrophotofluorometers, Beckman Scintillation Counters, and Sartorius remote control, balance interlaced to a Hewlett Packard terminal for automatic animal weighing. 0 .1~i~bi010~ :~ia ~6 m ~,

vI . PERSONN'..L N iC-012 0 016 ~.. The seven Ph. D.' s = nvolved in these studies have a wide range of technical expertise in the biological and chemical sciences. Their combined experience has resulted in significant contributions to the fields of tcxicology, carcicogenesis, and biochemisty, resulting ia over 100 publicationr This research team is aided by a well qualified and trained technical staff (M. S., B. S., and AALAS certified ) and is involved in ongoing col- laborative efforts with colleagues at such institutions as NCI; .LCRD NIA"D; =Br .1IAID; NIVCDS; Oak Ridge aational Labo- ratory; University of California, Irvine; University of Call- fornia, San Francisco: Cl:ildren's dospital, Los Angeles: 3aylor College of Medicine; Georgetown University: University of South Carol:nar and University of Maryland. MA also has extensive expertise in the areas of veterinary care, veterinary pathology, histology, and data processing. The Animal Disease Diagnostic ±es:.ir.g Laboratory is under the super- vision of a veterinarian, board certiEied in animal pathology. Four of the 7 member staff in the histology department are cert=fied in histotechnology. The Data Processing depar t-ment is domprised of 6 professionals with expe_ience rang:nS from compu- ter science to automated statistical applications. \ 7 }W f ° 1iie:vbiol4cal Uw.'

H T®01 20017 ( APPENDIX A SMOKE-GMtERATION SYSTEMS I. Small Capacity, Static System The Walton Horizontal Smoke Exposure Machine is shown in text Figures 5a and Sb. This machine is designed to expose a small number of experimental animals (12-20 mice, 12 rats or hamsters) to cigarette smoke under the conditions of a 2-sec puff, 35 ml puff volume and one puff per minute. The features of this machine have been described elsewhere (6, 11). The Walton provides exposure to standing or static smoke from a single puff that is uniformly mixed by a rotating fan. The animals breathe the smoke for a preselected time ranging from S to 45 sec. The particle size for of the smoke aerosol has a a geometric mean diameter of 0.40 to 0.63 um during a 30 sec exposure period (14). The chamber is flushed with fresh air between puffs. The smoke concentration can be varied by changing either the chamber volume (between 384 and 1150 ml) or the number of cigarettes (up to 3) simultaneously smoked. It. Large Capacity, Dynamic System The Smoke Exposure Machine (SEM II) is a dynamic exposure system which has the capacity to expose up to 480 mice to smoke at any one time. The SEM-II shown in text Figure 4, operates in a manner simulating human smoking and automatically and seque n- tially loads. lights, puffs, ejects, and extinguishes a series of 30 cigarettes. The operatiag principles of this machine are pre- sented elsewhere (22). Tt:e standard smoking conditions employed are: puff frecuency (one/minute), puff A»-ation (2 secor.us), average puff volume (35 ml) and cigarotte butt length (23 mm average). These conditions are malntained independently of the aaimal containment system. Variable amounts of dilution air can be introduced at the butt end of the cigarette allowing a range of smoke concentrations from 5-100$ (w/v) to be offered to the test animals. A diagram of the smoke generation system ar.d animal contairunenrt unit is presented in text Figure 7. Smoke can Le alternated with breathing air for various intervals*within a one minute cycle. Air is provided to all channels in the absence of smoke. Cigarette smoke is delivered to the test animals with- in 2 seconds after generation at the cigarette. Previous studies have shown that smoke particle size was log normally distributed with a geometric mean diameter of 0.34 microns and geometric standard deviation of 1.35 microns (14). 8 s . ~ . . ~ ` •~~~

HT®0120018 REFERENCES 1. Bealmear, P.M., Lahuada, C.P. Osmani, B., Freimark, B. Seeger, T., Mirand, E.A., Holterman, O.A. Amelioration of the Immunosuppressive Effects of Sendai Virus on the Mouse by Vaccination. Proc. Am. Assoc. for Lab. Animal Sci. #90A, (1980). 2. Billups, L.H., Henry, C.J., Whitmire, C.E. Kouri, R.E. Pathogenesis of Chemically Induced Lung Lesions in Mice. Proc. Am. Assoc. Can. Res. 19 (1978) 142. 3. Dinowitz, M., Nims, R., Bhooshan, B., Kouri, R.E., Henry, C.J., Induction of Ornithine Decarboxylase (ODC) by 12-0- Tetradecanoylphorbol-13-acetate (TPA) in Pulmonary Tissue: A Model System for Tumor Promotion in Mouse Lungs. Proc. Am. Assoc. Cancer Res. 21 (1980) 31. 4. Guerin, M.R., Stokely, J.R., Higgins, C.E., Moneyhun, J.H., and Holmberg, R.W. Inhalation Bioassay Chemistry -- Walton Horizontal Smoking Machine for Inhalation Exposure of Rod- ents to Cigarette Smoke. J. Nazl. Cancer Inst. 63 (1979) 441. 5. Hecht, S S., Carmella, 5., Mori. H.. Hoffmann D. A Study of Tobacco Carcinogen X4C. Role of Catechol as a Major Co- carcinogen in the Weakly Acidic Fraction of Smoke Conden- sate. J. Natl. Cancer Inst. 66 (1981) 163. 6. Henry, C.J. Billups, L.H., A:•ery, M.D. Dansie, D.R.., Lopez, A., Breth, L.A., Rude, 'C.. Kouri, R.E. Lung Cancer Model Systems in Inbred Strains of Mice Proc. Am. Assoc. Can. Res. 20 (1979) 242. 7. Henry, C.J., Billups, L.H., Avqry, M.D , Rude, T., Dansie, D.R., Lopez, A., Sass, B., Whit.zirE., C.E., :Couri, R.E. A Lung Cancer Model System Using Lzbred Strains of Mice Submitted (1981). S. Henry, C.J., Billups, L.H., Dinowitz M. Rasmussen, R.E., Avery, M.D., Dansie, D.R., Lopez, A., Breth, L.A.', Mullinax, H.D., Kouri, R.E The Effect of Exposure to Whole Cigarette Smoke on Pulmonary Mixed Function Oxidase, Ornithine Decar- boxylase, DNA Repair Capacity and on 3-Methylcholaiitbrene (MCA) Induced Lung Tumors in BC3F1/Cum Mice. In: Symposium on Cocarcinogenesis and Biological Effects of Tumor Pro- moters. October 13-16, 1980. Castle of Elmau, Bavaria, Federal Republic of Germany p. 56. 9. Henry, C.J., Breth, L.A., Gerhart, J.M., Dansie, D.R., +tullinax, H.D., Whitmire C.E., icouri, R.E. Carboxyhemo- globin Levels as a,leasure of Cic;arette Smoke Exposure in Mice. In preparation. (1981). 9

HT®012U1.i9 10. Henry, C.J., Caton, J.E., Stokely, J.R., Guerin, M.R., Lopez. A., Avery, M.D.. Dansie, D.R., Henderson G.M., Gayle, T. Whitmire, C.E., Kouri, R.E. Deposition and Distribution of the Total Particulate Matter of Cigarette Smoke in Mice using a Large Capacity Smoke Exposure System. Toxicol. Appl.Pharmacol. (in press) (1981). 11. Henry, C.J., Lopez, A., Dansie, D.R., Avery, M.D.. Whitmire, C.E., Caton, J.E., Stokely, J.R. Guerin, M•R., Curren, R.D., Kouri, R.E. Distribution and Clearance of Three Cigarette Smoke Constituents, Dotriacontane (DTC), Nicotine (NIC), and Benzo(a)oyrene (BP), after Exposure of Mice to Whole Cigarette Smoke. The Toxicologist. 1(1981) 139. 12. Henry, C.J., Whitmire, C.E., Lopez, A., Dansie, D.R., Avery, M.D., Caton, J.E., Stokely, J.R. Holmberg, R.W. Guerin, M.R., Kouri, R.E. The Dosimetry and Distribution of Whole Cigarette Smoke Particulates in Inbred Strains of Mice: Comparison of a Large Smoke-Exposure Machine (SEM) with a Small-Capacity Smoke-Exposure Machine (Walton). In: Pulmonary Toxicology of Respirable Particles, C.L. Sanders, F.T. Cross, G.E. Dagle and J.A. Mahafley, Eds. Technical Information Center, U.S. Department of Energy, NTIS, 1980, 177-192. 13. Higgins, C.E., Gayle, T.M., Stokely, J.R. Sensor for Detection of Tobacco Smoke Particulates in Inhalation Expo- sure System. Beitr. Tabakforsch. 9(1.978) 185. 14. Holmberg, R.W. Determination of Particle Size in Tobacco Smoke Inhalation Devices Using ::=thylcyanoacrylate Fixation and Scanning Microscopy. In: Tobacco Smoke Inhalation Bio- assay Chemistry. M.R. Guerin, J.R. Stokely, and C.E. Higgins, Eds., DOE Report ORN'L-5422, Oak Ridge Natic•nal Laboratory. NTIS, 1979. 15. Hwang, K.K., Bhooshan, B., Kouri, R.E., Henry, C.J. Synthesis of Tritium Labelled Catechol. J. of Labelled Compd. and Radiopharmaceuticals. (in press) 1981. 16. Bwang, K.K., Sonko, 0., Dansie D.R., Kouri,. R.E.. Henry , C.J. Studies in the Deposition and Distribution of Catechol from Whole Cigarette Smoke in SC3F1/Cum Mice After Exposure to Smoke. In preparation. 1981. 17. Kay, K.R. The Collision vebuliz=- Description, Perfor- mance, and Application. J. Aerosol Sci. 4 (1973) 235. 18. Kouri, R E. Genetic Differences in Chemical Carcinogene•sis. CRC Press, Boca Raton, Fla. 1980. 10 ~'f ' .`IiCrOltemlOgiCa1 .~' l"~ .W~°~

.._~.~r ~ . ...._.-._.. ~u}..r^.~ _Y 111 h01 2GG20 C 19. Kouri, R.E., Nebert. D W. Genetic Regulation of Suscepti- bility to Polycycl.ic-hydrocarbon-induceC Tumors in the Mouse. in: Origins of Human Cancer Book A, Hiatt, H H.. Watson, J.D., and Winsten, J.A., Eds., Cold Spring aarbor Laboratory, New York, 1977, 811. 20. Kouri, R.E.. Rude, T.H., Curren, R. D., Brandt, K. R. , Sosnowski, R.G., Schechtman, L.M., Benedict, W.F., Henry, C.J. Biologic:ai Activity of Tobacco Smoke and Tobacco Smoke- Related Chemicals. Environm. Blth. Perspect. 29 (1979) 63. 21. Kouri, R.E., Schechtman. L.M., Nebert, D.W. Genetic Control of Carcinogen Metabolism. In: Genetic Differences in Chemical Carcinogenesis. R.E. Kouri, Ed., CRC Press, Boca Raton, Fla., 1980,21-66. 22. Moneyhun, J.H., Stokely, J.R., Florant, L. Process and Instruments Corporation Automatic Smoke Exposure Machine -- SEM II. In: Tobacco Smoke Inhalation Bioassay Chemistry . M.R. Guerin, J.R. Stokely, and C.E. Higgins, Eds., DOE Report ORNL-5422, Oak Ridge National Laboratory, NTIS, 1979. 23. Parker, J.C. The Possibilities and Limitations of Virus Control in Laboratory Animals. In; 7th ICLAS Symposium, Utrecht 1979, Gustav Fischer Verlag, Stuttgart, .Tew York. 1980. 162-172. 24. Rasmussen, R.E., Boyd C.H., Dansie, D.R., Kouri, R.E. Henry, C.J. DNA Replication and Unscheduled DNA Synthesis in Lungs of Mice Exposed to Cigarette Smoke. Cancer Res. (In press) (1981). 25. Verma, A.K.. Shapasl B.F., Rice H.M , Boutwell R.K Correlation of the inhibition by Retino:,ds of Tumor Promoter-Induced Mouse Epidermal Ornithine Decarboxylase Activity and of Skin Tumor Promotion. Can. Res. 39 (1979) 419. 11 )C__- .1tiCiobiO:AsSOCib.

1-• i-. Figure 1. STAGES IN CARCINOGENESIS . ('.fM .AfiCPK~GENE9iS ~- INITIATION somommmo norw~r~a~ son HYnn.wry R.E. Kouri, C.J. Henry, , ., >+--BIOCB-IEMICAL PROMOTION-- A-% CELLULAR PROMOTION FACTaRS N TllMOii GF'iOVVrH .a•aaE staos pens4a Ym...CaWlancw inLe.wpp..won ~ FACTOM N CELL PROI.RRA170N E~ooaer.ous ew W.moo newwoa p`9 LxOCfnOUS M•41M fAsmL W

HT®0120022 \ \ Figure 2. SEM II animal containment system. One side of the unit is shown, with ~he capacity for exposing 240 mice per side (480 mice total) Rubber stoppers are used in the exposure holes when less than the full capacity of mice are to be exposed. ~a .*aadaww~C~ ~

( .( Figuse 2. H TQ01 20023 ~

4 Figure 3a,b. Animal restraint system, showing "stock-like H"T®0120024 r holder.

Figure 3a. ° ~~aausrmetirsLoar Fi;ure 3b.

HT®0120026 Figure 4. The SEM II with front and side panels open to show electronic componets. Cigarettes (C) are loaded from a hopper (EO) into a rotating drum holder (DH). The lighter (L) automatically ignites the cigarette and puff air is forced through each cigarette in turn. Flow is caused by a constantly held differential pressure in the dome (DO) between the ignited end and the btitt end of the cigarettes. Dilution air (DA) is introduced at the butt end of the cigarettes. Side stream smoke is removed through the vent (V). Ciga- ettes are removed by the automatic ejector (E) and discarded in the butt chute (S).

( Fig. 5(a) Wsiton Hoeisontal Smoideg %IaehEae. Mice• ue shoaa teeQaiaed in the cyiindttcai eube& ~

t Fig. 5(b) Waltaa expoetue eh-uaber. One sde plate haa been temo.ed to show the inside bt espwuse chamber tCH) with the canft¢Uyhaped exposuse holes (Zi) visible. Dunag an eaposute the dgaeette- dome (p) moves foraraed and eutomaticallq igmtes the dgetens IC). and 35 ml of puff air in fereed through the ciguette into the- chamber. A contiouous+Iq rotating [an (F) is t*qnired to enmssea rapid and unifotse mixiug of the smoke. At thb end of an eslosuae iatetval, smoke is vented through the eshamt port (E). H 11 I ...'r11 •r ~U~~r L.9

e n. Ftcjure 6 Aw4ao1 CoMalnnrang end Smolso Flow Syatern ClGAqETTES ~ T1 ~ AtR aurometk i~f(Jon, jnm~P pull ojoctlWe d ~rqnt =1t® OPTICAI. QISTRIBUTiON SENSOG VAI-VE r .r--, Soct~ ol Pd~eplw~w~o Mwlulo CGUes STOCK TYPE IIEAp IIESTRAINT ANIMAL CONTAIPjb1ENT 1Ploee up Viaw ot Anbnal 1lsecralna /n /iolalion lo PdYaal+onslo MmW1o IIEAD RESTRAINT NOSE SEAL

\ Figure 7. COMPREHENSIVE LA8ORATOttY ANIM1#L DIAGNOST,IC AA1D MANAGEMENT SERVICE IyDIVIDUAL ANIMAL DIAGNOSTIC REPORT Client: Date Received: Species/Strain: Date Necropsied Group Designation: Date Completed: Accession No.: Histology No.: CLINICAL E:{AMINATION PARASITOLOGY External Parasites Fecal Flotation Direct (Cecal Contents) Intestinal Wet Mount Trichosomoides BACTERIOLOGY Oropharyngeal Culture Feca]. Culture Liver Culture Other VIRAL AND MYCOPLASMA SEROLOGY M. pulmonis '(ELISA) LCM ECTROMELIA PVK KRV Reo3 MVM SENDAI MHV (ELISA) K Virus POLY PCV (ELISA) GD-VII H-1 ADENO SVS G'stOSS NECROPSY FINDINGS H 100120031 X6.0iiai~ba01f~3c81 As61cocia! .-W"Mft

HT10120032 Figure 7. COMPREHENSIVE LABORATORY ANIMAL DIAGNOSTIC AND MANAGEMENT SERVICE MICROSCOPIC DIAGNOSES OTHER COMMENT William C. Hall, V.M.D., Ph.D. Veterinary Pathologist

Figure 7. HT®0120033 \ COMPREHENSIVE LABORATORY ANIMAL DIAGNOSTIC AND MANAGEMENT SERVICE INDIVIDUAL ANIMAL DIAGNOSTIC REPORT Client: Species/Strain: Group Designation: Accession No.: CLINICAL EXAMINATION Date Received: Date Necropsied Date Completed: Histology No.: PARASITOL•OGY l External Parasites Fecal Flotation Direct (Cecal Contents) Intestinal Wet Mount Trichosomoides BACPE?ZIOLOGY Oropharyngeal Culture Fecal Culture Liver Culture other VIRAL AND MYCOPI,ASMA SEROLOGY M. pulmonis (ELISA) LCM ECTI:ONELIe1 PVNi KRV Reo3 MVM SENDAI MSV (ELISA) K Virus POLY PCV ( E:.ISA ) Gh-VII s-1 ADENO SV 5 GROSS NECROPSY FINDINGS m ~'~•'CIoh10k0cal AwK3a1 ~ ~ ~

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