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t NON-CANCER ENDPOINTS There are perhaps three considerations that distinguish the health risk evaluation process for cancer endpoints from that for non-cancer endpoints. I. While carcinogenic effects are thought to be linear with dose all the way to zero dose, for non- cancer endpoints there exists a threshold dose level below which no adverse health effects occur. This level is typically called the reference dose (RfD), allowable intake chronic (AIC), or no observed adverse effect level (NOAEL). II. The contrast between target tissues and the rest of the body is generally more sharply drawn than in carcinogenesis. That is, with non-cancer endpoints the target tissue f organ is often exquisitely susceptible to harm in comparison to other body tissues. Calculation of health effects Often calls for the use of physiologically-based pharmacokinetics (PB-PK), so that dose to target tissues can be more closely estimated. III. Non-c-mcer endpoints of injury are much more widely varied and toxin-specific than in cancer, where we believe there is primarily one endpoint, genetic damage, and one outcome, death, that we seek to avoid. Because of the diversity in non-cancer endpoints, it would be impossible to present an overall survey, and one example will be discussed in some depth. Many of the principles can be extrapolated to other organ systems. Assessment of Risk for Inhaled Airborne Material There are many methods available to assess the toxicity of inhaled agents. As summarized below, these tests ra.nge from studies in human populations, to measures of lung function in whole animals and histopathological studies of lungs from exposed animals, to in vitro measures of pulmonary macrophage fimction (phagocytosis, viability), etc. The following outline describes various categories of lung injury and types of assays for indicating onset of tissue- damage. I. Inhalation toxicology data development A. Air fronitoring and characterization of collected dusts. B. Epidemiologic studies of previously-exposed populations. C. Ctin.cal trials using controlled exposures of humans. D. Animals, chronic lifetime studies. E. Short term animal bioassays. F. In vit)-o tests on mammalian or non-mammalian cells. G. In vitro examination of molecular interactions with phospholipids, enzymes, nucleic acids, etc.

2 II. Mechanisms of lung injury As a consequence of inhaling toxic gases and particles, a number of pathological processes may be set into motion. None are specific to the lung, but their expression and consequences depend on the unique architecture and physiological role of the respiratory system. Major pathological mechanisms to be discussed are: A Pulmonary edema: Transudation of fluid, altered alveolar stability, impaired gas exchange, and respiratory distress B. Inf7ammation: Irritation leading to mucosal edema, increased mucus production and bron- chitis, appearance of neutrophils and inflammatory mediators, increased cell renewal C. Immunologic reactions: Asthma, hypersensitivity lung disease, extrinsic allergic alveolitis, anaphylaxis - D. Altered susceptibility to infection: Cytotoxic and competitive effects on macrophage func- tion, altered mucociliary transport because of changes in cilia or the quantity or rheological character of mucus E. Infection: Bacterial, viral, or fungal pneumonia F. Proteolysis: Destruction of elastin and collagen leading to emphysema, obstructive lung disease G. Fibrosis: Increased connective tissue scarring, excessive collagen, restrictive lung disease H. Degenerative changes: Necrosis, calcification, and autolysis I. "Pulmonary carcinogenesis: bronchogenic carcinoma, oat cell carcinoma, adenocarcinoma, me:sothelioma" III. Measwre.ment of lung injury If the lr.ing is injured by inhaled toxic gases and particles, how can the lung injury be detected and quantified? What repertoire of approaches can be used? Approaches and Parameters or Methods: A. Mechanical properties (pulmonary function) 1. Resistance 2. Compliance: pressure-volume curves 3. Lu:ng volumes: VC (spirometry), TLC, RV, and FRC (measured by helium dilution, Boyle's law) 4. FEV1 o and Full or Partial flow-volume curves B. Gas exchange, Adequacy of ventilation, Distribution of ventilation and perfusion 1. Alveolar gas tensions (PAC42, PA®2) 2. Arterial paC42, Pa02 3. Ventilation homogeneity: N2 washout 4. Ventilation (133~ce) or Perfusion (67Ga) scans

3 5, a-A concentration gradients 6. Diffusing capacity (carbon monoxide uptake) C. Measurement of pathology by radiologic techniques 1. Atelectasis 2. lFibrosis, emphysema, etc. 3. l3ronchography (Tantalum) 4. Focal lesions D. M:ucociliary transport (in vitro and in vivo) 1. Nasal 2. Asirways 3. Mucus studies 4. Cilia studies E. Luni; lavage parameters 1. Surfactant: quantity, composition 2. Cell numbers, appearance, and viability 3. Cell differential counts: RBC's, PMN's, monocytes, macrophages, lymphocytes 4. Proliferation: production of colony-forming units (CFU's) by lavaged cells, uptake of tri- t.iated thymidine 5. Mucus constituents 6. Biochemistry: albumin, hemoglobin, hydroxyproline, elastase, collagenase, LDH, myelo- peroxidase, antiproteases, lysosomal enzymes, active oxygen species, chemotaxins, proli- ferative factors, and inflammatory mediators (histamine, prostaglandins, leukotrienes) 7. In vitro functional assays of macrophage activity: trypan blue dye exclusion, oxygen con- sumption, ATP levels, lactate production, migration, chemotactic responsiveness, phago- cytosis, killing of microorganisms, release of mediators F. Morphology 1. Gough sections 2. Reid index 3. Mo:rphometric approaches: airway and alveolar dimensions 4. Cell types: connective tissue, inflammatory, neoplastic 5. Proliferation and cell turnover measures 6. Vascular changes G. Renewal of lung constituents observed in tissue sections 1. Metaphase counts - colchicine 2. Uptake of tritiated thymidine 3. Collagen and elastin breakdown and synthesis H. Lung clearance

4 ].. DTPA-measured lung epithelial permeability 2. Clearance of radioactively-labelled inhaled particles 3. Clearance of magnetic inhaled particles 4. Macrophage motile activity measured by inhaled magnetic particles I. Microbicidal activity 1. Recognizable experimental pulmonary infections (morbidity and mortality studies) 2. Bacterial aerosol models, in vivo models 3. rn vitro killing 4. Phagocytosis: in vitro and in vivo J. Identifying pulmonary carcinogens 1. E xperimental pulmonary carcinogenesis (Saffiotti model) 2. t:hromosome abnormalities 3. ~~mes mutagenesis assay IV. Bioassays for measuring toxicity of particles and components of particles A. Whole animals B. In 1,vitro cell culture systems C. Ce1I homogenates V. Questiors s to be considered in the interpretation of data A. Species extrapolation. Are human and animal toxicities equivalent ? B. Dose extrapolation. Are the doses given to animals comparable to human exposures ? C. Time extrapolation. At what stage is the injury being measured, and how does it compare to the time course of disease development in humans ? D. Correlation of disease mechanism with bioassay result E. Spe<:ificity of bioassay result: Is result unique to the agent tested ? Is the result generaliz- able to a class of agents ? If the agent is a complex mixture, what are the active com- poner,:ts ? How does the bioassay result agree with disease outcomes in cases where human data are avaiIable ? N ~ N BIBLIOGRAPHY ~ f I. Mechanisms w1d Measurement of Lung Injury ~ 1. Allison, A.C:. Mechanisms of macrophage damage in relation to the pathogenesis of some lung diseases. In: ~ Respiratory Defense Mechanisms. (Lung Biology in Health and Disease., Monograph 5). Brain, J.D., Proctor, ~ D.F., Reid, L., Eds. Marcel Dekker. New York. 1977. 1075-1102. w~ 2. Brain, J.D. :Macrophage damage in relation to the pathogenesis of lung diseases. Environ. Health Perspectives. IPA 35:21-28, 1980.

5 3. Brain, J.D. Toxicological aspects of alterations of pulmonary macrophage function. Ann. Rev. Pharmacol. Tazzcol. 26:547-565, 1986. 4. Doull, J., Klaassen, C.D., and Amdur, M.O. Taricology: The Basic Science of Poisons. New York: MacMillan, 1980. See particularly Chapter 12, "Toxic Responses of the Respiratory System," by D.B. Menzel and R.O. McClellan, and Chapter 24, "Air Pollutants," by M.O. Amdur. 5. Fishm:art, A.P. Pulmonary Diseases and Disorders., volumes 1 and 2. New York: McGraw Hill, 1980. 6. Gadek, T.E., Fells, GA., Crystal, R. Cigarette smoking induces functional antiprotease deficiency in the lower respiratory tract of humans. Science. 206: 1315-1316, 1979. 7. Harington, J.S., Allison, A.C. Tissue and cellular reactions to particles, fibers, and aerosols retained after inhala- tion. I:n: Handbook of Physiology, Section 9: Reactions of Environmental Agents., Falk, H.L., Murphy, S.D., Eds. Bethesda: American Physiological Society, 1977, pp. 263-283. 8. Harris, C.C. Pathogenesis and Therapy of Lung Cancer. New York: Marcel Dekker, 1978. 9. Janoff, .A., Carp, H., Lee, D.K., Drew, R.T. Cigarette smoke inhalation decreases alpha-l-antitrypsin activity in rat lung. Science. 206: 1314-1315, 1979. 10. Kirkpatrick, C.H., Reynolds, H.Y., eds. Immunologic and Infectious Reactions in the Lung. ( LungBiology in Health and Disease., Monograph 1). New York: Marcel Dekker, 1976. 11. Kuhn, C,1II, Senior, R.M. The role of elastase in the development of emphysema. Lung. 155:185-197, 1978. 12. Litwin, S.D., Ed. Genetic Determinants of Pulmonary Disease. New York: Marcel Dekker, 1978. 13. Snider, G.L., Lucey, E.C., and Stone, P.J. Animal models of Emphysema. Am. Rev. Respir. Dis. 133:149-169,1986. 14. Turino, G.M., Rodriquez, J.R., Greenbaum, L.M., Mandl, I. Mechanisms of pulmonary injury. Am. J. Med. 57:493-.505,1974. 15. Wahl, L.P1, et al. Collagenase production by lymphokine-activated macrophages. Science. 187: 261-263, 1975. 16. White, R., P,in, H.S., Kuhn, C. III. Elastase secretion by peritoneal exudative and alveolar macrophages. J. Exp. Med. 146: 802-808, 1977. 17. West, J.B, j?ulmonary Patltophysiology: 77ie Essentials. Baltimore: Williams & Wilkins, 2nd edition, 1982. IL Occupational Lung Diseases 18. Brooks, S.M., Lockey, J.E., Harber, P., eds. Clinics in Chest Medicine: Occupational Lung Diseases I. Philadelphia: W.B. Saunders Company, 1981, 19. Brooks, S.:M., Lockey, J.E., Harber, P., eds. Clinics in Chest Medicine: Occupational Lung Diseases II. Philadel- phia: W.:B. Saunders Company, 1981. 20. Dosman, JA., and Cotton, D.J., eds. Occupational Pulmonary Disease: Focus on Grain Dust and Health. New York: Academic Press, 1977. 21. Key, M.M., et al. eds. Occupational Diseases.•A Guide to TheirRecognition. Dept. of Health, Education, and Wel- fare Publication No. 77-181, Washington, D.C., U.S. Government Printing Office, 1978. esp. Chapter V, "Diseases of the Airways," by W. Keith, C. Morgan, and N. LeRoy Lapp. 22. Kusnetz, S., Hutchinson, M.K., eds. A Guide to the Work-Relatedness of Disease. Dept. of Health, Education, and Welfare Pulblication No. 79-116, Washington, D.C., U.S. Government Printing Office, 1979. 23. Morgan, W.K.C., Seaton, A. Occupational Lung Diseases. Philadelphia: W.B. Saunders Co., 2nd edition, 1984. 24. Parks, W.R. t)ccupational Lung Disorders (2nd ed.). London: Butterworths, 1982. ' 25. Clayton, C.D.,, and Clayton, F.E. Patty's Industrial Hygiene and Toazcology. Vols. 2A, 2B, 2C.• To.zzcology., 3rd Rev. Ed. New'York: John Wiley and Sons, 1982.9. Wagner, W.L., Rom, WA., Merchant, JA., eds. Health Issues Related to Metal and Nonmetallic Mining. Boston: Butterworth Publishers, 1983. 26. Wolf, A.F. Occupational Disease of the Lungs. Part I. Ann. Allergy 35:1-6, 1975. 27. Wolf, A.F. Occupational Diseases of the Lungs. Part II. Inhalation diseases due to inorganic dust. Ann. Allergy. 35:87-92, 1975.

6 28. Wolf, A.F. Occupational Diseases of the Lungs. Part III. Pulmonary disease due to inhalation of noxious gases, aerosols, or fumes. Ann. Allergy. 35:165-171,1975. III. PL~lmonary Bioassays 29. Hende:rson, R.F., E.G. Damon, and T.R. Henderson. Early damage indicators in the lungs: Lactate dehydrogenase activity in the airways. Toxicol. Appt. Pharmacol. 44:291-297, 1978. 30. Kavet, RI., and Brain, J.D. Methods to quantify endocytosis: a review. J. Reticuloendothel. Soc. 27:201-221, 1980. 31. Beck, E.D., Brain, J.D., and Bohannon, D.E. An in vivo hamster bioassay to assess the toxicity of particulates for the lungs: Toxicol. Appl. Phannacol. 66:9-29, 1982. 32. Smith, 'C T., Beck, B.D., Brain, J.D., Hinds, W.C., Baron, S.G., and Weil, L. Prediction of pneumoconiosis risk by bioassays of particulates from occupational exposures. In: Inhaled Particles, V, Walton, W.H. ed., Oxford: Per- gamon ]Press, pp. 163-176, 1982. Also in Ann. Occup. Hyg. 86:163-176,1982. 33. Beck, B„D., Gerson, B., Feldman, HA., and Brain, J.D. Lactic dehydrogenase isoenzymes in hamster lung lavage fluid after lung injury. Toxicol. Appl. Pharmacol. 71:59-71, 1983. 34. Henderso:a, R.F. The use of bronchoalveolar lavage to detect lung damage. Environ. Health Perspect. 56: 115-129, 1984. 35. Brain, J.D., and Beck, B.D. Broncboalveolar lavage. In: Toxicology of Inhaled Materfais, Handbook of Experimen- tal Pharr,7acology, Vol. 75, Witchi, H. and Brain, J.D., eds. Berlin: Springer Verlag, pp. 203-226, 1985. 36. Brain, J.D, and B.D. Beck. Bioassays for mineral dusts and other particulates. In: In Vitro Effects of Mineral Dusts., Beck, E.G. and Bignon, J., eds. NATO ASI Series Vol. G3. Berlin: Springer Verlag, pp. 3?3-335,1985. 37. Henderson, R.F., J.M. Benson, F.F. Hahn, C.H. Hobbs, R.K Jones, J.L. Mauderly, R.O. McClellan, and JA.Pickrell. New approaches for the evaluation of pulmonary toxicity: Bronchoalveolar lavage fluid analysis. Fund f!p,nl. Toxicol. 5: 451-458, 1985. 38. Beck, B.D., E.J. CIabrese, and P.D. Anderson. The use of toxicology in the regulatory process. Principles and Methodc of Toxicology, 2nd Edition. (A.W. Hayes, Editor), Raven Press Ltd., New York, pp. 1-28. 1989.

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