Document 2jGZVb5m09dBdZ2xjKmzpgK36

UXICOLOGY a n d a p p l ie d p h a r m a c o l o g y 99, 377-383 (1989) CONTEMPORARY ISSUES IN TOXICOLOGY Establishing Aerosol Exposure Concentrations for Inhalation Toxicity Studies T r e n t R . L e w i s .* P a u l E. M o r r o w ,f R o g e r O . M c C l e l l a n ,:): O t t o G . R a a b e , G e r a l d L. K e n n e d y ,11B e r n a r d A. Sc h w e t z , 1 T h o m a s J. G o e h l / J o s e p h H. R o y c r o f t , 1 a n d R a j e n d r a S. C h h a b r a 1 *Division o f Biomedical and Behavioral Science. National Institutefor Occupational Safety and Health. 4676 Columbia Parkway. Cincinnati. Ohio 452276; t Environmental Health Science Center. University o f Rochester. Rochester. N Y 14642: ^Lovelace Inhalation Toxicology Research Institute. Albuquerque. New Mexico 87185; ^Laboratoryfor Energy-Related Health Research. University o f California. Davis. California 95616: "Haskell Laboratory. E.l duPont de Nemours & Co.. Newark. Delaware 19711: a n d 1National Toxicology Program. National Institute o f Environmental Health Sciences. Research Triangle Park. North Carolina 27709 Received October 15. 1988: accepted March 28. 1989 Establishing Aerosol Exposure Concentralions for Inhalaiion Toxicity Studies. L e w is . T. R.. M o r r o w . P. e .. M c C l e l l a n . R. O.. R a a b e . O. G.. K e n n e d y . G. L . Sc h w e t z . B. a .. G o e h l . T. J,, R o y c r o f t . J. H. (1989). a n d C h h a b r a . R. S. Toxicol Appl. Pharmacol 99, 377-383. Criteria for the selection of aerosol concentrations to be used in inhalaiion studies assessing the toxicity and carcinogenicity of chemical substances were discussed by the authors in a meeting sponsored by the National Toxicology Program. Concepts in the design of aerosol inhalation studies emerged from that meeting and are being communicated through this publication. Inha lation studies assessing the toxicity and carcinogenicity of aerosols have often used maximum exposure levels on the basis of technological feasibility. Evidence has now accumulated that the amount of pulmonary burden o f deposited panicles impacts on panicle clearance above some as yet not well-defined exposure concentration. The sequelae are such that lung clearance de creases with increased particulate burden to the point of approaching complete cessation. This paper focuses on the major determinants in establishing maximal aerosol concentrations for use in inhalation toxicity studies with special emphasis on experimental design features to assess lung retention. The subject matter of this paper is a rapidly developing area in terms of knowl edge. Accordingly, the contents of this article are intended as guidelines and not as absolute rules for the conduct and interpretation of inhalation exposure studies. Previous guidelines or recommendations for ihe conduct of inhalation toxicology and car cinogenesis studies have not fully addressed the subject o f the maximal aerosol exposure concentrations to be employed (EPA, I982ab; OECD, 1981; NTP, 1987). As part fits mandate to address issues related to the design of toxicologic studies, the Toxicology Design Review Committee (TRDC) o f the National Toxicology Program (NTP) con vened a meeting to address this particular ,0Pic. The nine participants (four non-NTP national experts in inhalation toxicology rep resenting academe, national laboratories and industry, and five NTP scientists) focused their attention on the major determinants to establishing maximum aerosol concentra tions in inhalation toxicity studies. Special emphasis was placed on substances which are relatively insoluble and of low order of sys temic and respiratory toxicity. It was not ex pected, however, that the discussants would resolve all the issues inherent to the design and conduct of prechronic and chronic aero- 377 0041-008X/89 $3.00 378 LE W IS ET AL. sol inhalation toxicity studies. The para mount considerations in the design, conduct, and interpretation o f inhalation studies of aerosols discussed were the interrelationships among exposure concentrations, dose to the respiratory tract, and the subsequent toxico logic responses in the test subjects. Emphasis was placed on in vivo physiologically based pharmacokinetic factors as determinants of dose delivered to a specific tissue site and the resultant biologic responses. Issues related to the mechanics o f aerosol generation and physicochemical modifiers of the aerosol concentrations within the exposure cham bers such as. aerosol losses in the exposure system or to the animals' fur and contamina tion o f the test atmosphere by products o f an imal excreta, were not addressed. DOSE TO THE RESPIRATORY TRACT The conduct of inhalation studies employ ing aerosols will involve substances that vary widely in their pulmonary retention. Those with high dissolution rates in tissue fluids (quite soluble) will usually be rapidly cleared from respiratory tract tissues and be translo cated to other tissues or excreted. In this pa per, pulmonary clearance is defined as a de fensive function o f the lung involved with the removal o f foreign substances via structures as the mucociliary system, alevolar macro phages and the lymphatic system and is de pendent upon such factors as absorption, sol ubility and particle size. Even with chronic exposure, respiratory tract tissues may never accumulate large concentrations. In such cases, respiratory tract toxicity may or may not be evident. Many particles that are quite soluble will also have appreciable absorption from the gastrointestinal tract for that por tion of the inhaled material that is cleared from the respiratory tract to the oropharynx and swallowed. Establishment o f exposure concentrations and the resultant doses to tis sues in chronic aerosol inhalation studies is inherently less complex when systemic effects are dominant and can be related to differing dosage regimens. Other substances may be quite insoluble n tissue fluids and are thus cleared reiativeh rapidly from the nasopharynx and tracheo bronchial regions by mechanical processes and cleared slowly from the pulmonary com partment o f the respiratory tract by several processes, including dissolution. For such compounds, the pulmonary burden of parti, cles increases with exposure concentration and duration o f exposure. Test protocols for aerosols that are to be employed in sub chronic and chronic inhalation exposure, toxicologic, and carcinogenic studies should take into consideration the expected lung burdens with increased exposure concentra tion or duration. Accordingly, lung burdens o f a test aerosol should be estimated, if not measured, in inhalation studies. This is espe cially important in chronic studies in which the test chemical is relatively nontoxic but high-level exposures may overwhelm pulmo nary clearance mechanisms. ASSESSMENT OF PULMONARY DUST RETENTION AND METABOLIC OVERLOAD Interpretation o f the results o f inhalation toxicity studies, and especially o f the presence or absence o f pulmonary pathology, requires a knowledge o f the lung burden of inhaled test material throughout the exposure and postexposure observation periods in the test species. For most inhalation studies, the test species are the rat and the mouse. The burden o f relatively insoluble materials in the respira tory tract, and in particular in the lung, may be greater than predicted at higher levels of chronic exposure because o f impairment of the pulmonary clearance processes. The use o f brief exposure to a radiolabeled particulate probe to monitor lung clearance rates can be useful in determining the lung burden o f a test material which produces mg pi brief e at van Studie: Wolff INHALATION TOXICOLOGY AEROSOL LEVELS 379 c effects liffering lublein lativeh racheoocesscs y comseveral r such f partitration :ols for n subwsurc. should i lung :entraurdens if not s espcwhich ic but ulmo- :y lation sencc quirehaled i and e test e test jrden spirama' els oi nt ot overloading. Exposure to the aerosol probe j :an be used to help determine and interpret qudy conditions which avoid this problem. I There is no one radiolabeled particulate iracerthat may be applicable to all aerosol in halation studies. It is particularly useful that i the probe and test aerosol be o f equivalent size. An excellent monitoring dust candidate is radiolabeled iron oxide due to its ready availability and low cost, the stability o f the j label and the ease o f generation o f specific j particle sizes; radiolabeled polystyrene or I fused aluminosilicate particles are alternate candidates. Such particles, when inhaled briefly, at low concentrations, have very re I producible pulmonary retention over a wide range of lung burdens and may be useful in assessing changes in the ability o f the lung to clear particles. Serial measurements o f the re j tention of the radiolabeled particles can be made rapidly with relatively inexpensive I equipment. Such measurements may pro vide valuable insight into the presence or ab sence of impaired clearance. Appropriately sized iron oxide aerosols are easy to prepare and with 59Fe providing a surrogate com pound subject to noninvasive external mea surement. These radiolabeled particles are largely (>90%) removed slowly from the !ungs by macrophage-mediated clearance 'Gibb and Morrow, 1962; Lehnert and Morrow, 1985). Suitable radiolabeled polytiyrene particles are commercially available, eg.. 3M. J Several laboratories are currently measur Ing pulmonary dust retention following a brief exposure to a radiolabeled test aerosol ^ various times during prechronic or chronic . studies o f inhaled dust (Muhle el al.. 1988; Wolff al., 1987). f STUDY DESIGN FEATURES beled -ance lung luce- : A minimal o f three concentrations o f the j a|rborne test aerosol should be used in 'Tronic inhalation studies to establish dose- response relationship of the test substance. In general, the highest concentration o f test ma terial used should produce only minimal in terference with the lung defense mechanisms as judged by impaired particulate clearance. Optimally, the lower exposure concentra tions should not result in altered patterns of particle clearance and accumulation. The fractional deposition o f an aerosol in various regions o f the respiratory tract o f any species is dependent upon the aerosol size dis tribution and species differences in anatomi cal and physiological characteristics. Aero sols with a mass median aerodynamic diame ter (MMAD) o f less than 2 zzm and a geometric standard deviation o f less than 2.0 will have the highest fractional deposition in the lower respiratory tract o f rodent species (Raabe el al.. 1977). Aerosols with a larger MMAD will have increasingly larger frac tions o f the material deposited in the upper respiratory tract and, correspondingly, less deposited in the lower respiratory tract. Aerosol exposure concentrations in the chronic study can be selected on the basis o f at least two considerations, i.e., modeling and experimental determination. For relatively insoluble materials, the lung burden may be predicted using mathematical models based on experimental studies of pulmonary depo sition and clearance for individual laboratory animal species and aerosol sizes (Raabe ei al.. 1977; Raabe ei al.. 1988; Snipes el al.. 1983; Snipes 1986). In addition, prechronic testing car, supply both deposition and retention data. Prudency dictates that the prechronic lung burden data be checked to ensure the ac curacy o f the models. Collectively, these methods provide a reasonable capability to the investigator for estimating the outcome (lung burden) o f exposure levels chosen for a chronic (2-year) study. For a steady-state lung burden concentra tion o f />, e.g.. 20 mg particulate material/g lung tissue (Chan e ta i. 1984; Lee el a/.. 1986; Muhle et al.. 1988; Wolff el a/.. 1987; Snipes. 1989). the maximum aerosol exposure con centration could be estimated (with proper dimensional corrections) from the equation 380 LEW IS ET AL. maximum aerosol concentration = - X lnf 2 X ,'unS P ass x --1-- x --L /1 T \ Dep MV where / i s the average fraction o f time dedi cated to inhalation exposures. In,,2 is the nat ural logarithm of 2, T \ is the nominal lung retention half-time for the deposited parti cles, Dep is the fraction o f inhaled particles that deposit in the deep lung (pulmonary re gion) and MV is the normal resting minute volume of respiration. With the possible ex ception of b which can be assumed to be the same for all species when dose-effects are equated, all o f the parameters in the equation are a function of species. For example, in the case o f the rat let T \ = 100 days or 144.000 min; fractional depo sition = 0.068 for 2 un MMAD aerosol (G SD=2) (Raabe el ai. 1987); lung mass = 2 g; b = 20 mg/g; / = 0.1786 (6 hr/day. 5 day/ week 24 hr. 7 day-week or 30/168. and min ute volume = 0.15 liter/min. r^ max 20 mg/g X 0.693 X 2g 0.1786 1.44 X I05 min 0.068 0 . 15 liter/min _ 27,72 mg max 0.026 X 104 liter = 1066 mg/104 liter or 106.6 mg/nv' Thus, to achieve a steady-state lung con centration of 20 mg/g in the rat requires an insoluble aerosol concentration o f 106.6 mg/ m3. It should be noted that the calculation of a high steady-state lung burden (> 2 -5 mg particle/g lung) will almost always lead to a reduction in particle clearance and a change in lung buildup that comes closer and closer to unity. Pragmatically, the steady-state model will overestimate both the time and test material needed. Thus, the actual lung burden will be in excess o f the computed steady-state burden. In addition to this exam ple, Morrow has proposed other models for exposure level estimations (Morrow, i9gf, Morrow and Mermelstein. 1988). For inhalation toxicity evaluations with ro. dents, the test aerosols should typically ha\e a MMAD of 3 or less with a geometric standard deviation of no greater than three to maximize alveolar deposition. Aerosols wineven a smaller MMAD o f 1 to 2 /m and j tighter size distribution, i.e., a geometric stan dard deviation o f less than two, would he preferable. However, this is sometimes not possible due to the nature o f the material re quired in large quantity for chronic toxico logic study. For hygroscopic aerosols a smaller size, approximately 1 MMAD. i<, preferred recognizing that the particle will en large in the humid environment of the respi ratory tract. If "enrichment" of small panicle sizes is required to increase the respirabilitv oi the test dust aerosol, the respective respirable fractions of the test and modified test dust should be measured to reconcile the exposure levels used in chronic toxicity testing with those expected or documented under typical conditions o f use. Some materials for which toxicity or carcinogenicity testing evaluations are desired may normally occur as panicles with sizes greater than a few micrometers and perhaps as large as several hundred microme ters. Such powders may be physically altered to produce aerosols with MMAD's of a few micrometers to facilitate the experimental study o f their toxicity. Since such approaches increase the deposition, dose, and possibility o f effects o f the material in relation to those expected under normal conditions, a knowl edge of the differences in respirabilities be tween the enriched and the normal dusts in both the test animal and man is necessitated A minimum of two years o f exposure is recommended for chronic inhalation studies in both rats and mice. In the case of rats, they could be held for an additional 6 months without exposure prior to the scheduled kill or until 20% survival is reached for the pur pose o f maximizing incidences of tumors. The primary basis for the extended holding period for rats is that results from recent stud- INHALATION TOXICOLOGY AEROSOL LEVELS 381 1986: ,es with Fischer-344 rats exposed to a range ,if concentrations of diesel exhaust demon- th ro llavo netric ree to ; with md a stanld be s not al rexico>ls a D. is j sirated an excess of tumors only at the highest I ixposure concentrations and near the end of iherat's life span (Mauderly c/ al.. 1987). The vast majority (approximately 80%) o f the ex posure-related lung tumors were observed in rats killed or that died after 24 months from the initiation o f the 5-day per-week expo sures. These results emphasize the value of observations beyond 2 years for detecting pulmonary carcinogenesis. However, exten sion of the duration of an inhalation toxicol ogy study might not be appropriate/possible forall chemicals or with all strains o f rats due 11enespi- iovariations in life spans or for other species commonly used in chronic inhalation toxic rticlo itystudies, e.g.. mice and hamster, whose life ityof spansdo not permit a 24-month exposure du rablo I ration and postexposure follow-up. dust I Arelated question is the exposure duration isure 1 that is warranted if observations o f lifespan with shortening diseases such as pulmonary fibro sisor other indications o f chronic toxicity are hicn made in addition to. or in lieu of. tumorige- ions nicity. If tumors are detected between the icles 15th and 20th month o f exposure, one ap and proach might be to maintain the rats for 30 me- | months following initiation o f exposure or ored until a 20% survival is reached. Alternatively, few during the post 24-month exposure period ntal animals could be killed periodically to show ;hes other progression o f the lesion or regenera ilit> tive repair, since one or the other usually oc lose curs. It is implicit that if life span shortening >wl- ^curs, it is important that adjustments be be made to ensure that useful histopathologic s in data are obtained. Jed. A single set o f exposure levels cannot be e is ^commended that will be applicable to all lies i chronic inhalation studies. For aerosols not he> causing impairment o f clearance in a 90-day iths s,udy, one must look for a lack o f proportion kill alitY between lung burdens and exposure ur- I concentrations over the range o f concentrairs l|ons of the test aerosol. Such observations mg | can be made by plotting lung burden versus jd- of exposure and noting changes in slope to detect increased lung burden of the test aerosol or by constructing a matrix using air concentration (exposure level) and duration o f exposure, e.g., 30, 60, and 90 days, in which one enters the ratio of the lung burden to the exposure level for each box denoting a specific time period and exposure level. The higher the ratio for an exposure level, the more disproportionate is the lung burden at one or more durations o f exposure. In pre chronic studies, when animals that are killed lung burden data should be obtained. These determinations should be coordinated time wise with other serial kills programmed for determination of toxicologic endpoints. The lung burdens can be obtained by determining the mass o f test material per gram o f lung by chemical or other appropriate means. These results may then be compared with model predictions to determine if lung burdens in excess o f those predicted from kinetics at lower levels and overwhelming o f clearance mechanisms are occurring. Within a given study, the relationship between lung burden and exposure concentration can be com pared for the several exposure concentrations to give an indication o f altered kinetics at the higher exposure levels relative to the lower levels (Wolff cl al.. 1987). Depending on the physical/toxic proper ties o f the test aerosol, both 14-day and 3month studies can provide varying degrees of useful toxicologic information for planning and interpreting chronic exposure studies. Such studies also provide the opportunity for rehearsing all methods and fully evaluating aerosol generation and characterization tech niques before initiating the more expensive chronic studies. In many cases, results from as many as five exposure levels plus control for both 14- and 90-day studies are valuable in selecting exposure levels for the chronic study. A range o f exposure concentrations should be selected to assure inclusion o f one level that potentially suppresses pulmonary clearance and results in pathology in addition to one lower level which is free o f these effects. 4 382 LEW IS ET AL. Testing should not be done at the highest exposure concentrations that are technologi cally feasible. The exposure concentrations should be selected so they produce an opti mal range of lung burdens. With exposure to aerosols o f MMAD of less than 3.0 m and a geometric standard deviation of less than 3.0, acute studies using aerosol concentrations o f greater than 1,000 m g/m 3 or long-term stud ies using aerosol concentrations in excess of 100 mg/m3 are of limited utility in assessing human health posed by inhalation o f aerosols unless it is known that human exposures at or near these levels actually occur. DISCUSSION In the course of conducting inhalation tox icology studies, one must consider issues spe cific to administration o f the test material by inhalation as compared to those of other routes o f administration. Exposure by the lat ter means is often a case of administering a specific quantity o f the test chemical whereby the dose to the target organ(s) is rapidly achieved. Conversely, when insoluble materi als are aerosolized, it is much more difficult to quantify the dose of the test substance and steady-state lung burden may take months to achieve or may never be achievable as in the case where "critical masses'" of the test sub stances in the lung markedly impair pulmo nary clearance mechanisms. Thus, there are important biologic differences which distin guish establishing maximum aerosol concen trations in inhalation studies from maximum tolerated doses using the oral and parenteral means of administering varying amounts of a test chemical to experimental subjects. This paper has attempted to highlight the importance of understanding the relation ships among exposure concentrations and durations, the particle size and species depen dency of fractional deposition o f an aerosol in various regions of the respiratory tract, dose (lung burden), and toxicologic response as they relate to inhalation toxicology studies. Cogent details on these critical issues werc MM provided as well as guidelines and reeom mendations for conducting contemporar inhalation toxicologic and carcinogen^ studies. ... PER,-\ha .hro' ;ii8- Ui'RR' Researchers who conduct inhalation tox,. .ure cology studies must be cognizant that thc inn' topic o f " Maximal Aerosol Exposure Con Xk'R1 Cirri centrations in Inhalation Studies" isa rapid!-, l pitl'3 developing area in terms of knowledge. Ac ! t-rpi cordingly. the contents of this article are in | tended as guidelines and not as absolute rules . \U llt- The key point that is made is that all toxieit. j Ore I ,ure studies conducted with inhaled particulate VI. material must involve a determination of the j NJliOI actual amount of test material in the lung as j era! a function of time. The measurement of lunt \ t an burden is appropriate at all exposure levels at i DH Hea 3. 6, 12. 18. 24 and 30 months after initiation ; . irgan of exposure in a chronic bioassay. Temporal mer determinations of lung aerosol retention ' t Vu must also be incorporated into the prechronk ! R ixB' studies to adequately plan a chronic bioassav j Rai mot For additional information on this com plex topic, the reader is also referred to other papers on this subject (Morrow, 1986; Mor row and Mermelstein. l988;Snipes, 1989). REFERENCES C h a n . T. L.. L e e . P. S.. a n d H e r r in g . W. E. (198-1' Pulmonary reteniion of inhaled diesel particles aft prolonged exposure to diesel exhaust. Fundam. App, Tdxieol. 4. 624-63 I. Environmental Protection Agency (EPA)( 1982a). Peu; eide issessment (iuidelines Subdivision F. Hazan; Evaluation: Human anti Domestic Animals. ST IS 540/9/82-025. Spnngtield. VA. Environmental Protection Agency (EPA) (1982bi Health Ellens Test (iuidelines. NTIS 560/6-82-001 Springfield. VA. G ib b . F. R.. a n d M o r r o w . P. E. ( 1962). Alveolar clear ance in dogs following inhalation of an iron-59 oxide aerosol. / Appi Physiol 17,429-432. Lee. K. P.. Henry. N. W III. T rochimowicz. H. J . and Reinhardt. C. F. (1986). Pulmonary response to impaired lung clearance in rats following excessive TiO- dust deposition. Environ Res. 41. 144-167. Lehnert. B. E.. and Morrow . P. E. (1985). Associa tion of wiron oxide with alveolar macrophages dunng alveolar clearance. Exp Limy. Res. 9, 1-16. INHALATION TOXICOLOGY AEROSOL LEVELS 383 es were recomiporarx logenie >n toxihat the e ConrapidU ge. Ac are in te rules, toxicits ticulate n of the lung as of lune evels at itiation mporai tention ;hronn oassas n o older j; Mor989). Mauderly, J. L.. Jones. R. K... G riffith. W. C.. Hen . derson, R. F . and McC lellan. R. O. (1987). Diesel I exhaust is a pulmonary carcinogen in rats exposed i chronically by inhalation. Fundam. Appl. Toxicol. 9, I 208-221. Morrow. P. E. (1986). The setting of particulate expo sure levels for chronic inhalation toxicity studies. J inter. Coll. Toxicol. 5, 533-544. Morrow, P. E.. a n d M e r m e l s t e in , R. (1988). I Chronic Inhalation Toxicity Studies: Protocols and Pitfalls. In Inhalation Toxicology: The design and in I terpreialion o f inhalation studies and their use in risk assessment, pp. 103-1 17. Spnnger-Verlag. New York. Mlh le, H,, B e l l m a n . N. B,. a n d H e in r ic h . U. (1988). Overloading of lung clearance during chronic expo sure ofexperimental animals to particles. Inhaled Part VI. Ann. Occup. Hyg. 32. Suppl. I. 141-147. National Toxicology Program (NTP) (April 1987). Gen eralStatement of Work for the Conduct o f Toxicity and Carcinogenicity Studies in Laboratory Animals DHHS, Public Health Service. National Institutes of Health. Organization for Economic Co-operation and Develop ment (OECD) (1981). OECD Guidelines for Testing Chemical. Pans. France. Rvxbe. O. G.. Al-Bayati. M A.. T eagus. S. V.. a n d Rasolt, A. (1988). Regional disposition of inhaled monodisperse coarse and fine particles of aerosol in small laboratory animals. Inhaled Part VI Ann. Occup. Hyg. 32, Suppl. I. 53-63. Raabe, O. G., Yeh, H. C., Newton. G. J., Phalen, R F,, and Velasquez, D. J. (1977). Deposition of inhaled monodisperse aerosols in small rodents. In In haled Particles (W. H. Walton. Ed.), Vol. IV, Part I. pp. 1-21. Pergamon. Oxford. United Kingdom. S n ip e s . M. G. (1989). Species comparisons for pulmo nary retention of inhaled particles. In Concepts in In halation Toxicology R. O. McClellan and R. F. Hen derson. Eds.), Chap. 7. pp. 193-227. Hemisphere. Corp.. New York. Snipes. M. G. (1986). Comparative simulated lung bur dens of dust in rats, guinea pigs and dogs with chronic exposure. In Inhalation Toxicology Research Institute Annual Report. LM G-115. DOE Research and Devel opment Report, pp. 96-100. National Toxicology In formation Service. Springfield. VA. S n ip e s . M. B.. Bo e c k e r . B. B . a n d M c C l e l l a n , R. O. (1983). Retention of monodisperse or polydis perse aluminosilicate particles inhaled by dogs, rats and mice. Toxicol. Appl. Pharmacol. 69, 345-362. W o l f f . R. K.. H e n d e r s o n . R. F . S n ip e s . M .G .. G r if f it h . W. C.. M a u d e r l y . J. L.. Cu d d ih y . R. G.. a n d M cC l e l l a n . R. O. (1987). Alterations in particle ac cumulation and clearance in lungs of rats chronically exposed to diesel exhaust. Fundam Appl. Toxicol. 9, 154- 166. . (198-1 ;les alls ' rrt. I/V a). Pi'' Hu:s. s i i ' (198-T .-82AH' lar clear 59 o'lT- Z. H > respi'1' " xctfy-'- 67. A ssy 's due's