Document KR0k399ZzgoGV0L1wGEK89mL6

Toxicology Long-Term Retention and Clearance of Particles Inhaled by Mammalian Species Morris B. Snipes I. INTRODUCTION Inhalation is a common route of exposure of humans to hazardous materials in the workplace and environment. For vapors and aerosols of hazardous materials, inhalation repre sents the most significant route of exposure. The adult human inhales about 20,000 1of air daily, and the gases and panicles in the inhaled air have the opportunity to interact with about 70 m2of respiratory tract epithelium. Depending on their chem ical composition and physical characteristics, which influence retention and tissue exposure patterns, inhaled materials may constitute an immediate or long-term threat to the health of the individual. The magnitude of the threat depends on temporal associations between tissues and the inhaled material; these associations are dictated by deposition, clearance, and, ulti mately, retention patterns. Physical and chemical descriptions of the exposure at mospheres, amounts inhaled, and the tissue retention patterns for the deposited materials are generally not available for ac cidental human inhalation exposures. Of necessity, studies with laboratory animals provide the exposure, dose, and biological effects data needed to evaluate the inhalation toxicity of haz ardous materials potentially inhaled by humans. Scientific understanding of the mechanisms and processes involved in deposition of particles in the respiratory tract is relatively advanced. Patterns of initial deposition and early clearance for inhaled materials have been extensively studied in humans and in several species of laboratory animals. How ever, only a few well-documented cases of human inhalation exposures that provide the kinds of long-term retention and clearance data required for direct risk assessments are available. The mechanisms associated with retention and clearance of particles deposited in the respiratory tract are not well under stood. Retention is a convenient expression for the time-de pendent distribution patterns for materials, or their reaction products, that have deposited in the respiratory tract and have not yet been cleared. Clearance is removal of deposited ma terials or their reaction products from the respiratory tract. Clearance is discussed in terms of physical processes that trans port particles from one place to another and dissolution-ab sorption processes. Some particles appear to be retained for extended periods in phagocytic cells in the alveoli Other par ticles are found in cellular constituents of the interstitium. Most, or all, of the particles in the interstitium appear to be in mobile cells that have the options of remaining in the in terstitium, moving to the mucociliary escalator, moving to lung-associated lymph nodes, or moving into the lymphatic or circulatory systems. Our current understanding of the factors that control or influence these options within and among species is not adequate; the options for retention and clearance must be the focus of additional research if we are to develop a betterunderstanding of the processes involved and of the bass for species similarities and differences in these processes. Inhalation exposures can be single events involving in halation of only a small mass of material. Alternatively, ex posures can be intermittent or chronic and may involve inhalation of substantial amounts of material over a long time period. Repeated, or chronic, inhalation exposures may produce sig nificant cumulative lung burdens that, because of physical and/ or chemical damage to tissue, can disrupt lung function and produce long-term pathological effects. Small or moderate par ticle burdens in lung may not represent a threat to health or respiratory tract function when relatively nontoxic materials are inhaled. However, even nontoxic materials can be prob lematic if large masses of the particles are accumulated in the lung. Few human data are available on long-term retention and tissue exposure patterns for inhaled materials. In the absence of these data, scientists and regulators must rely on data from experiments with laboratory animals to provide the bases for decisions relative to human inhalation exposures. Unfortu nately, differences between species make it difficult to directly extrapolate the results of animal studies to humans. Some of the more obvious similarities and differences among species relate to anatomy. Most mammalian species have the same anatomical components of the respiratory tract. The anatomical differences that influence deposition patterns include size and configuration of airways, e.g., degree of convolution of the nasal turbinates, lengths and diameters of the conducting air ways, numbers of branches, branching angles, and branching patterns in the conducting airways. Other factors that influence deposition patterns include respiratory parameters such as tidal volume and respiratory frequency. The factors that result in species differences in retention of particles in the lung have not been defined, but probably include physical and functional differences among the cellular constituents of lung tissues, especially pulmonary alveolar macrophages. This review is not intended to provide a complete discus sion of deposition, retention, and clearance for all types of materials. Emphasis is on long-term retention and clearance of inhaled particles ranging from the moderately soluble to the M. B. Snipes received his B.S. and M S. from the University of New Mexico. Albuquerque; his Ph D. was earned at Cornell University. Ithaca. New York. Dr. Snipes is a Physical Biologist in the Patho physiology Group at the Lovelace Biomedical and Environmental Re search Institute, Albuquerque. New Mexico. 1989 175 Critical Reviews In latively insoluble. These types of particles can have the most significant impacts on human health because they can be re tained in the respiratory tract for long times after a single exposure or accumulate with repeated exposures. This review has two major focuses: (1) long-term retention and clearance patterns for particles inhaled during acute exposures and (2) models for retention and clearance of particles inhaled during repeated exposures. Human data are discussed and included in the modeling projections where available and appropriate. II. DESCRIPTION OF THE RESPIRATORY TRACT A. Anatomic and Functional Differentiation Into Regions The respiratory tracts of all mammalian species can be defined in terms of three anatomical-functional regions. The three regions are identified as (1) the nasopharyngeal (N-P) region, (2) the tracheobronchial (T-B) region, and (3) the pul monary (P) region. Figure 1 schematically depicts these three regions for the human. The anatomical divisions between the three regions of the respiratory tract are arbitrary and difficult to clearly distinguish. However, by definition, the N-P region begins at the anterior nares and includes head airways that extend to the larynx or epiglottis. The primary functions of the isal airways can be defined as (1) olfaction, (2) heating and humidification of inhaled air, (3) filtration of airborne particles, and (4) metabolism or detoxification of deposited materials. The nasal passages are convoluted as a result of folds in the nasal turbinates. The folds represent a relatively large surface area with which inhaled air can interact. The basic structural components and function of the N-P region are the same for all mammalian species. However, there are substantial differ ences among species in physical sizes, shapes, and dimensions of the components of the N-P region.' The nasal cavity is also a dynamic structure that can change dimensions during inspi ration and expiration; these changes can result in higher or lower particle deposition in the N-P region, depending on Row air flow patterns and velocities are influenced. In addition to dimensional changes during nasal breathing that appear to be normal, tissue reactions to inhaled irritants may cause inflam matory responses that further alter the dimensions and shape of the nasal passages. These factors make it difficult to define a standardized N-P region for humans or for any laboratory animal species. The T-B region is distal to the N-P region. It begins at the larynx and includes the trachea and other ciliated airways, ending with the terminal bronchioles. This represents the duct ing for transport of inhaled air and aerosols into and out of the pulmonary region. In all mammals, the trachea divides into 'o major branches (bronchi) that supply ducting to the left uid right lungs. The bronchi further divide into the smaller ducts that comprise the remainder of the T-B region. RESPIRATORY TRACT COMPARTMENTS NASOPHARYNGEAL (NP). LARYNX trachea TRACHEOBRONCHIAL (T B ) TERMINAL AONCHIOLES PULMONARY (P ) RIGHT LUNG LEFT LUNG FIGURE 1. Schematic representation of the human nasopharyngeal (N-P), tracheobronchial (T-B), and pulmonary (P) regions. The major bronchi are lined with ciliated epithelium and mucous-secreting goblet cells. Mucous glands and serous glands are also present in the major bronchi and contribute to the mucous lining of the airways. Mucus is a discontinuous blanket of viscous fluid that performs at least two important functions in the T-B region. First, inhaled materials may be trapped in the mucus and thus would have a smaller potential to cause damage to the epithelial cells or to become phagocytized. Sec ond, the mucus overlies the ciliated bronchial epithelium and is moved toward the oropharynx by wave motion of the cilia. This movement serves to move dead cells, particles, and other debris out of the airway system, to be swallowed. Distal to the T-B region is the P region, which has the largest surface area of the three major regions of the respiratory tract. The P region includes the respiratory bronchioles, al veolar ducts, alveolar sacs, atria, alveoli, interstitial tissues, alveolar capillaries, and pulmonary lymphatics. This is the nonciliated, gas-exchange region of the respiratory tract and is the region that dominates long-term retention of inhaled materials. By definition, the P region begins with the respi ratory bronchioles and ends with the lung parenchyma. Con siderable species variation in the morphology of respiratory bronchioles exists and may be the major reason for the vari ability observed among species in retention and clearance of inhaled materials and in biological responses to the materials.' There a r e substantial species differences in gross and m i croscopic morphology of the respiratory tract.3'6 Differences among species include lengths of airways, branching p a i t e m s , branching angles, numbers and branching generations of res piratory bronchioles and alveolar ducts, cell types, and size of alveoli. Anatomical differences are clearly important factors in initial deposition patterns for inhaled materials. H o w e v e r , 176 Volume 20, Issue 3 Toxicology there are no anatomical differences that can be used to easily explain the basis for species differences in retention and clear ance patterns for inhaled materials, except that the retention and fate of inhaled materials may be influenced by the initial sites of deposition. A continued effort devoted to comparative functional relationships among inhaled materials and the var ious structures and cell types of the respiratory tract is needed to evaluate the bases for species differences in retention and clearance of inhaled materials. B. Cell Kinetics The dosimetry associated with long-term retention of par ticles in lung tissue is dependent upon ( 1) the types of cells or structures the particles are associated with and (2) the length of time the particles can interact with the cells or other tissue constituents. For example, if an individual cell retaining a particle is replaced or replicates during the residence time of the particle, the exposure time will be uncertain and exposure to the particle may be shared among several cells. To under stand relationships between retained particles and cell damage, it is important to determine the cell types associated with par ticle retention and the rate(s) at which those cells are normally replaced. Another important reason to study cell turnover times is that they may be influenced by exposures, and changes in cell turnover times may be directly linked to damage processes. Bowden7 provided a summary (Table 1) of published in formation relating to cell turnover in the lungs of rats and mice. This summary represents data from Bowden's laboratory and from other sources.8' 13 The results summarized in Table 1 are for nonmigratory cells and macrophages. Because alveolar macrophages are migratory cells, their turnover times are more properly interpreted as times for transit through the tissues of the respiratory tract. The presence of particles in the lung may influence the numbers and residence times of alveolar mac rophages in the lungs.14-16 Thus, caution has to be exercised when interpreting results of studies attempting to define the turnover times for pulmonary alveolar macrophages. Table 1 Summary of Experimentally Determined Turnover Times for Selected Cells of Respiratory Tracts of Rats and Mice Tissue Turnover time (days) Rat Mouse Tracheal epithelium Bronchial epithelium Bronchiolar epithelium Alveolar epithelium Alveolar macrophage 7-- 48 8-- 27 -- 29 8-- 35 2-- 20 2-- 21 10-- 59 28-- 35 6-- 21 Data from References 7 to 13. Another migratory cell important to normal lung function is the polymorphonuclear leukocyte (PMN)' This cell accu mulates in significant numbers in the lung in response to in flammatory stimuli. Some inhalation exposures result in the influx of PMN into alveoli, and PMN have been clearly im plicated in physical translocation of particles.17 Thus, when evaluating turnover times of pulmonary phagocytic cells in the lung, it is important to include PMNs. It is clear from reviewing the literature on cell turnover studies that considerable resources need to be committed to research on cell kinetics of respiratory tract tissues. This area represents an important key to understanding retention and clearance of inhaled materials. Understanding species similar ities and differences for cell turnover rates in the respiratory tract, especially for phagocytic cells, is vital to a complete understanding of the bases for species similarities and differ ences in long-term retention of inhaled particles. III. DEPOSITION PATTERNS FOR INHALED PARTICLES A. General Deposition refers to the amount of inhaled material that deposits in the respiratory tract during an inhalation exposure. Deposition can occur during any part of the respiratory cycle. Therefore, particles not deposited during inspiration are avail able for deposition during exhalation. Deposition is defined in two general ways -- total depo sition and regional deposition. The total amount of inhaled material deposited in the combined N-P, T-B, and P regions of the respiratory tract constitutes " total deposition" . It is expressed as the ratio of the amount of material retained in the respiratory tract immediately after an inhalation exposure to the amount of material inhaled. Total deposition can range between 0 and 100% of the amount of material inhaled. How ever, deposition is rarely 100% because some portion of the inhaled material generally remains suspended in the air and is exhaled. Figure 2 represents predicted deposition fractions, as a function of particle size, for the three regions of the respi ratory tract of nosebreathing humans. Similar deposition pat terns exist for other species, with differences related to respiratory patterns and anatomical factors, as discussed later. Regional deposition defines the initial patterns of deposi tion in terms of relative amounts of deposited materials present in each of the three functional regions of the respiratory tract immediately after an inhalation exposure. The deposition for each region can range between 0 and 100% and the sum for deposition in the N-P, T-B, and P regions is defined as 100%. Numerical values for regional deposition are useful for inter preting relationships between particle size and deposition, de fining tissues and organs potentially at risk from inhaled materials, and evaluating species similarities and differences with respect to initial deposition patterns for inhaled materials. 1989 177 Critical Reviews In FIGURE 2. Regional deposition fractions for aerosols inhaled by humans. Solid lines are estimates based on Reference 19; dashed lines are estimates based on Reference 18. This figure presents generalizations for regional dep osition patterns in all mammalian species. Total and regional deposition are both dependent upon the particle size of the inhaled materials and upon the species. The 'onships among particle size, total deposition, and regional u -^ sitio n in the respiratory tract have been described in detail, with considerable discussion devoted to species similarities and differences for deposition in the N-P, T-B, and P regions of the respiratory tract. The reader is referred to References 19 to 32 as definitive reviews and summaries of the information available on this topic. A broad range of particle sizes and types can be encoun tered in the home, workplace, and environment. Sizes of aer osol constituents can range from molecular dimensions to tens of micrometers in diameter. The probability of deposition of inhaled particles in the respiratory tract depends on the physical characteristics of the particles, the most important character* istics being aerodynamic equivalent size for micron-sized par ticles and diffusion equivalent diameter for ultrafine particles.1920 The site of deposition is influenced by size of the particles; anatomical features of the respiratory tract, such as diameters and lengths of airways; and breathing patterns. As a generalization, particles larger than 10 p,m have a very small probability of passing the N-P region in any mammalian species that breathes through its nose. Mouthbreathing humans can deposit particles 10 p.m and larger in their lungs, but most laboratory animals species are obligatory nosebreathers and cannot. In many cases, an inhaled material may be irritating to the re'-'iratory tract or may have unusual physical characteristics i nfluence its deposition. While generalizations about dep osition can be made for most aerosols, caution has to be ex ercised for others, and it may be prudent to determine the deposition patterns for each material experimentally. B. Factors That Modify Deposition Most deposition data for laboratory animals come from inhalation studies in which no control of respiratory parameters was attempted. Control of respiratory parameters in animals is possible, but requires unusual conditions, such as controlled levels of sedation or the use of mechanically assisted venti lation. In contrast, it is possible to train human subjects and thus conduct inhalation exposures using carefully controlled breathing patterns. This leads to more uniformity in experi mental results and allows better evaluation of inter-subject var iability in deposition. Breathing rate and tidal volume determine the flow patterns and volume of aerosol inhaled per respiratory cycle and per unit of time and also determine the relative amounts of inhaled material available for deposition in the N-P, T-B, and P regions of the respiratory tract. Table 2 presents a summary of the significant respiratory parameters for selected laboratory ani mals and humans, along with body weights and lung weights. There are clear differences among these species in size and relative respiratory minute volumes. Smaller animals have higher minute respiratory volumes per unit of body weight to supply their relatively larger requirements for oxygen.3334 The larger minute volume per unit body weight in small animals means that they inhale larger amounts of aerosol per unit body weight compared with larger animals or with humans. Thus, respi ratory tract ventilation rate is an important factor contributing to the different lung burdens achieved by different species exposed to the same aerosol. A factor that markedly influences deposition patterns is the mode of breathing. During nosebreathing, deposition of inhaled particles in the head airways can be an effective way for the respiratory system to preferentially filter large and very small particles out of the inspired air. With mouthbreathing, a potentially larger regional deposition of inhaled particles can occur in the P region. The relationships among particle size and pulmonary deposition have not been clearly established for mouthbreathing humans. Svartengren et al.35 conducted a study using 24 nonsmoking, male human volunteers to deter mine the deposition efficiency for mouthbreathing of large `" In-labeled Teflon particles. Alveolar regional deposition av eraged 15% for 8.2-p,m particles, 4% for 11.5- and 13.7-p.m particles, and 1% for 16.4-p.m particles. Few of these particles would have reached the pulmonary region in nosebreathing humans. Snipes et al.36 exposed rats and guinea pigs to 3-, 9-, or 15-p.m polystyrene latex microspheres. The 9- and 15-p.m mi crospheres did not reach the pulmonary region in either species. Comparable results might be expected for most or all mam malian species exposed to these particle sizes during nosebreathing. 178 Volume 20, Issue 3 Toxicology Table 2 Body Weights, Lung Weights, and Respiratory Parameters* for Selected Laboratory Animal Species and Humans Species Body weight (kg) Lung weight (g) Respiratory minute volume (1) Respiratory minute volume per kg (I/min/kg) Mouse Rat Guinea pig Rabbit Monkey Cat Dog Human1' 0.03 0.25 0.70 2.4 2.4 3.7 10 70 0.20 1.5 4.0 9.1 22 20 110 1000 0.04 0.20 0.46 0.62 0.70 0.96 3.6 20 1.30 0.80 0.66 0.26 0.29 0.26 0.36 0.29 Approximations of body weight, minute volume, and lung weight for laboratory animals were adapted from Phalen2 and the cumulative pub lished and unpublished data from the Lovelace Inhalation Toxicology Research Institute. These approximations were assumed reasonable for comparative purposes, but may vary under different conditions of ac tivity, sedation, anesthesia, or confinement. 6 Adapted from References 32 and 236 (adult man, "light activity"). Changes in minute volume can be expected to result in proportional changes in the amount of aerosol entering the respiratory system. An example of the effects of altered res piratory parameters on deposition can be seen with anesthetized animals. Hamsters under general anesthesia inhaled and de posited less of an aerosol composed of insoluble particles hav ing an activity median aerodynamic diameter of 0.45 p.m than did unanesthetized hamsters.37 The anesthesia reduced respi ratory minute volume and thereby reduced the amount of aer osol drawn into the respiratory tract during the exposure. If increased respiratory minute volumes are induced, the effect is to draw more aerosol into the respiratory tract and increase deposition. This was demonstrated in one study where the authors used C 0 2 to cause the experimental animals to breathe more deeply than normal, thereby increasing aerosol intake and deposition.38 Respiratory parameters are a major source of variability. Lippmann39 reviewed the literature available for human re gional deposition and concluded that a considerable amount of inter-subject variability exists for deposition fraction, as a func tion of inhaled particle size, in all regions of the respiratory tract. Most of the variability was ascribed to variations in tidal volumes, flow rates, and functional residual capacity. The res piratory pattern can markedly influence regional deposition. Shallow, rapid breathing or deep, slow breathing can produce the same respiratory minute volume. However, the shallow, rapid breathing (small tidal volume) results in increased dep osition in the N-P and T-B regions and decreased deposition in the P region. Slow, deep breathing results in a larger fraction of the inhaled volume reaching the P region, and thereby en hances deposition in the P region. Increased flow rates through respiratory airways cause increased deposition due to impac tion, especially at locations where sharp angles are encountered by the inhaled particles. Small functional residual capacity allows relatively more inhaled air to mix within the pulmonary air spaces, thereby causing increased deposition in the P region; the opposite occurs with large functional residual capacity. Inhalation of irritant substances can alter deposition pat terns by changing the activity level of the subject. To some extent, this might reflect responses to physiological stress. Alarie40 discussed this subject in detail and described ways to measure and compare the irritating properties of inhaled materials. Bruce et al.41 reported effects of 10 ppm ozone exposure on respiratory parameters, metabolic rate, and rectal temper ature in mice. Soon after initiation of the 90-min exposures to ozone, these parameters all decreased, and the decreased respiratory parameters would have resulted in decreased dep osition of aerosols inhaled along with the ozone. In a similar study, Silver at al.42 exposed mice by inhalation to acrylate esters, which are respiratory tract irritants. Minute volume quickly dropped to one third of normal resting values and body temperature decreased. Chang et al.43 observed depressed min ute volume in rats and mice exposed to formaldehyde; Medinsky et al.44 noted the same result for methyl bromide inhalation by rats. These changes in respiratory parameters would have altered deposition of particles inhaled during the exposures to the irritant substances. A number of studies relating inhalation of irritant sub stances or stimulants to deposition have been reported in recent years. An important result of these studies is that they have provided a data base demonstrating that deposition patterns are sensitive to factors that change respiratory patterns. Knowing this, investigators can use inhalation exposures to specific ma terials to enhance or decrease deposition of other materials in specific large regions of the respiratory tract. In addition, the information from these studies is useful for helping to predict deposition patterns for simultaneous exposures to irritants and particles. In addition to variability caused by respiratory parameters, some variability was ascribed to genetically related differences in airway and airspace morphometry. Deposition may therefore not be constant even for the same subject measured at different times, and any factor that influences anatomy or causes res piratory parameters to change with time will influence respi ratory tract deposition patterns. C. Intra- and Inter-Species Variability There is considerable variability in deposition of inhaled particles within and among species. Literature sources of spe cific data on species similarities and differences in deposition patterns for inhaled particles include References 23, 26, 29, 32, 39, and 45 to 49. 1989 179 Critical Reviews In Based on deposition studies with humans, in which res piratory parameters could be controlled and the main variables influencing deposition could be limited to anatomical differ ences, Heyder et al.50 concluded that differences in airway dimensions are likely to be the primary factors responsible for inter-individual variability in deposition within a species. Air way length and diameter are important factors because they influence deposition due to sedimentation of diffusion; the time required for particle deposition to occur by both mechanisms is proportional to the distance the particle must travel. Airway length can also influence local deposition patterns in the lung. Particles that have a short path length to travel between the trachea and terminal bronchioles have a higher probability of being deposited in the P region. This may be the basis for the higher relative deposition generally observed in the apical lung lobes of experimental animals. While Heyder's dat^ came from human deposition studies, the same degree of inter-individual variability may exist in other species. An important consideration in evaluating tissue exposure patterns for inhaled materials and in relating the exposure pat terns to biological responses is that the variability among sub jects may be large. Within a population of exposed individuals, the average exposure will be representative of that occurring to only a small portion of the population. If only the highest exposures produce biological responses, study results may be icorrectly interpreted. Cuddihy et al.31 discussed this topic in retail and presented results for Beagle dogs and humans. The authors considered inter-subject variability for lung weight, deposition, and uptake or retention of an inhaled test material. Figure 3 demonstrates the relative distribution of doses to the lung or other internal organs for Beagle dogs or humans ex posed to the same test material. The distribution of doses was approximately log-normal and was typical of what might be observed for exposures to a variety of respirable materials. A small fraction of the exposed population would receive a dose that would be much less than the average. It is significant that about 2% of the population would receive more than three times the arithmetic average dose for the group. Cuddihy,et al.51 concluded that, because of the variability in exposures among individuals exposed to the same material, it would be prudent (1) to allow a factor of 10 to account for the low-tohigh exposure in a group of subjects, and (2) to quantitate individual exposures whenever possible to reduce the uncer tainties associated with making judgments about the toxicity of test substances. The same considerations apply to groups of animals exposed to a test aerosol to determine deposition pat terns. Within the population of test animals, there will be sufficient variability so that, if possible, each individual should be evaluated separately to determine its deposition patterns. Estimates for amounts of deposited material are normally made for each animal individually if the animals are exposed le at a time. These estimates can be made by using several methods or combinations of methods, including external mon- N O R M A L IZ E D V A L U E S ( x/ x ) FIGURE 3. Projected internal organ dose distribution for Beagle dogs ex posed to inhaled aerosols. Normalized values are the observed values divided by the group average. (Adapted from Cuddihy, R, G ., McClellan, R. 0 ., and Griffith, W. C ., Toxicol. Appl. Pharmacol.. 49, 179, 1979. With permission.) itors, aerosol particle counters, and information about air con centrations of the exposure material, minute respiratory volumes of the subject, and exposure duration. If the exposure material contains a label, e.g., a radiolabel, the amount of material deposited may be determined during the exposure by using a collimated chest monitor or immediately after the exposure by counting the subjects in a suitable counting system. In many studies, the exposure material cannot be readily measured, and assumptions must be made about the amounts of the material inhaled and deposited. In such studies, when a group of animals is simultaneously exposed to the same aerosol, the assumption is made that all of the animals received the same average exposure and have the same amount of exposure material de posited internally. Deposition variability is not the same for single and re peated exposures. Single, short-term exposures of groups of animals result in a wide range of internal burdens of inhaled material. Repeated exposures tend to reduce the variability in deposition among subjects. As one example, Lundgren et al.32 exposed Syrian hamsters once (n = 311) or seven times (n = 217) to aerosols of ,44C e 0 2. The coefficients of variation for lung deposition of the test material were 32 and 21%, respec tively, for the single and repeated exposures. This result, where variability associated with the accumulated lung burdens exists for any single exposure but the repeated exposures provide more uniformity by averaging the results for each individual exposure, may be typical for inhalation exposures. 180 Volume 20, Issue 3 Toxicology Variability in human inhalation studies can be reduced by conducting the study under rigidly controlled conditions. Aer osols, breathing mode, and respiratory patterns can be carefully monitored and controlled. With laboratory animals, the ex perimental conditions cannot be as carefully controlled. It is relatively easy to control aerosol concentrations and the chem ical and physical attributes of exposure materials during animal inhalation exposures, but it is not as easy to control the res piratory parameters of the animals. Another factor relevant to comparisons between single and repeated exposures is physiological stress, a poorly defined concept relating the physiological status of a human or animal to its environmental conditions. In essence, almost any envi ronmental change will influence body functions of the exper imental subject and thus influence the experimental results of the study. Placing experimental subjects in aerosol exposure systems may cause altered respiratory patterns and hyperactiv ity. Animal handling or animals' reactions to irritant or toxic aerosols may therefore influence deposition patterns and be an important source of variability among species. Conditioning to handling and to the exposure environment prior to actual aer osol exposures may reduce the magnitude of the animal's phys iological responses. The adaptation inherent in a series of repeated exposures, combined with the averaging effect on deposition resulting from several individual exposures, tends to bring about more uniformity in respiratory tract deposition for repeated exposures compared with single exposures. IV. DISPOSITION OF PARTICLES AFrER DEPOSITION IN THE PULMONARY REGION A. General When particles are inhaled and deposited in the P region, the initial alveolar deposition appears to be relatively random. However, several studies have indicated that this is not so. Because of the physical forces that influence particle dep osition patterns, larger amounts of inhaled material are gen erally deposited near bifurcations and the entrances to alveolar ducts.53'56 Therefore, even from the time of initial deposition, the distribution of particles in the lung is relatively nonuniform. These initial deposition patterns may vary substantially among species and influence subsequent distribution and clearance patterns, as well as biological responses to the deposited materials. After deposition in the pulmonary region, particles are subjected to processes that seem to be similar among all mam malian species. The particles are rapidly phagocytized by pul monary alveolar macrophages or other phagocytic cells. 1757 77 Chemotactic responses are one mechanism for attracting phag ocytic cells to particles deposited in the respiratory tract. If exposure levels are high, or the exposure material produces an inflammatory response, large numbers of PMNs appear in the lung and can also phagocytize particles. The fate of specific phagocytic cells after they ingest par ticles can vary, depending on the physical/chemical nature of the material ingested. Some particles may be toxic to phago cytic cells; cells that ingest toxic particles may die and become debris for other phagocytes to ingest. Migration and grouping of phagocytes containing particles can lead to redistribution of the deposited particles. Understanding the interactions between phagocytes and particles is important to elucidating the mechanism(s) involved in particle retention and clearance. Also, the functional abilities, longevity, and migration and retention pat terns for alveolar macrophages may be different among species and may represent the basis for differences among species in retention and clearance of inhaled materials. There are indications that particles deposited in lung are not retained in a static mode; the particles appear to redistribute over time due to dynamic processes occurring in the lung tissue. Migration and grouping of particle-laden cells can lead to re distribution of particles in the lung and a continuously changing exposure or injury pattern that may be different among animal species and humans. There are also indications of different clearance rates from different alveolar regions within the same lung that may contribute to variation within and among species for pulmonary retention of particles.55 Mechanisms responsible for long-term retention of parti cles or their constituents after deposition in the respiratory tract are probably common to all mammalian species, including humans. Physiological conditions unique to the species, such as cell kinetics and metabolic rates, could result in differences among species in retention and clearance of materials deposited in the lung. B. Pulmonary Alveolar Macrophages Pulmonary alveolar macrophages are an important cell type involved in retention and accumulation of particles in the lung, as well as in clearance of particles from the lung. These are relatively large, nucleated phagocytic cells that appear to move freely in the lung. Three types of macrophages are recognized in the lung: (1) alveolar, (2) interstitial, and (3) airway.71 These types of macrophages appear to have the same origin and are only different with regard to function. Defining macrophages in this way provides recognition of some mode(s) of com munication with and among phagocytic cells in the lung that influences their dispersion patterns and function. The movement of macrophages is like that of amoebae, they can change their shape and use diapedesis to move through small openings between cells and within spaces in lung tissue. The ability of macrophages to migrate through lung tissue or along airways improves their chances to encounter and phag ocytize dead cells, inhaled particles, bacteria, and other foreign materials. Phagocytized materials can accumulate in macro phages in substantial amounts. The process of phagocytosis is a means of isolating the material in a way that makes it possible to transport the material out of the system, digest the material. 1989 181 Critical Reviews In dissolve it and make it available for clearance via the dis solution-absorption process. The numbers of macrophages in lungs of different animal species and humans have not been clearly determined. In one study, Blusse van Oud Alblas and van Furth78 demonstrated that the total pulmonary macrophage population in SPF Swiss mice was about 2 million, of which 93% were pulmonary alveolar macrophages and about 7% were pulmonary interstitial macrophages. Macrophages normally comprise about 3% of the total alveolar cells in healthy, nonsmoking humans; a sim ilar fraction is present in other mammalian species.79 The numbers of macrophages that can be washed from the lung by lavage is also quite similar among mammalian species. Brain80 reported that the combined yields of 12 washes of the lungs of dogs, rabbits, guinea pigs, hamsters, rats, and cats provided averages of 3 to 15 million cells per gram of lung. This suggests that many mammalian species have about the same relative numbers of macrophages. While macrophages are always present in the lung, the actual number of macro phages present at any given time appears to be influenced by the presence of other phagocytes, foreign materials, or cell debris. Major influencing factors appear to be the numbers578182 and the types of particles deposited in the lungs. Small numbers of deposited particles do not appear to cause an increase in -'acrophage numbers, but when particle numbers are increased, icrophage numbers increase to maximum values.7llt4'87 Ini tially, this increase in cells is due to recruitment of blood monocytes, whose maturation is associated with migration through the pulmonary interstitium into the alveoli. However, as more and more particles deposit in the lung, the recruitment of macrophages may be supplemented by proliferation of mononuclear cells in the pulmonary interstitium. An increase in pulmonary macrophages appears to be a generalized response to instilled84 or inhaled particles, but the magnitude of the response depends upon particle composi tion,85 number, and mass. However, the magnitude of the increase appears to be related more to the number of particles than to the total mass of particles. Therefore, equivalent masses of the same material may not produce the same macrophage response if the two masses are associated with particles having different physical sizes.88 Bingham et al.89 exposed rats by inhalation to 2 mg coal dust per cubic meter for up to 4 months. The numbers of macrophages that could be lavaged from the rats were not elevated. The authors suggested that the results of most earlier studies demonstrating increased numbers of macrophages were influenced by the intratracheal instillation procedures used to produce the lung burdens. In a similar study by Castranova et al.,90 rats were exposed by inhalation to coal dust and diesel exhaust, alone or in combination, for 2 years at air concentra- ns of 2 mg/m3. None of the exposures affected macrophage .lability. Exposures to diesel exhaust had no effect on numbers of macrophages recoverable from the lung by lavage, whereas the coal dust exposures did cause an elevation in the numbers of macrophages. The particle size of the coal dust was larger than that of the diesel exhaust particles, so there would have been a substantially larger number of diesel exhaust particles deposited in the lungs of these rats for the same mass of par ticles inhaled. If the number of particles was the most important parameter relative to changes in the macrophage response, then a larger change should have been associated with the diesel exhaust exposures than with the coal dust exposures. The op posite was observed, suggesting that size of inhaled particles is more important than numbers of particles in eliciting re cruitment of macrophages. These results emphasize the points that experimental findings can be heavily influenced by ex posure procedures and that biological responses are difficult to extrapolate from one material to another. The responses are clearly associated with factors that include numbers and com position of particles. It is also possible that some inhaled particles cause a de crease in production of phagocytic cells or that there may be an inadequate number of phagocytic cells present to effectively transport the deposited particles away from the lung.91 This may be a major factor in the ability of particles to penetrate the pulmonary epithelium and become incorporated into con stituents of the pulmonary interstitium. Phagocytic cells can engulf a broad range of particle types and sizes ranging up to tens of micrometers in diameter. The rate and efficiency of phagocytosis appear to depend on prop erties of the deposited particles that include size, chemical composition, shape, mass, and degree of opsonization. In one study with spherical aluminosilicate particles in the size range 0.3 to 2.2 |xm, macrophages phagocytized larger particles more rapidly and in greater numbers than they phagocytized smaller particles; the investigators suggested that 2-p.m particles of this type were near the optimum size for phagocytosis by pulmonary alveolar macrophages.92 In an earlier in vitro study, fewer 6p.m particles were phagocytized than 3- or 1,5-p.m particles.93 Results of these two studies suggest that the optimum size of particles for phagocytosis by pulmonary alveolar macrophages may be in the range of 1.5- to 3-p.m geometric diameter. The physical attributes and chemical composition of par ticle surfaces appear to be important for the rate of phagocy tosis. Lundborg and Holma94 compared rates of phagocytosis for fungal spores and polystyrene particles having about the same physical size. The in vitro rate of phagocytosis for the fungal spores was greater than for the polystyrene particles. Camner et al.6364 and Camner and Lundborg95 found differ ences in vitro in the ability of alveolar macrophages to phagocytize 4-p.m Teflon particles coated with carbon or with metals. For example, particles coated with carbon or aluminum were phagocytized at a faster rate than particles coated with silver. These differences were seen clearly only when autologous serum was used, suggesting that the differences may have been related to the presence of serum constituents that helped in some way 182 Volume 20, Issue 3 Toxicology to mark the particles for phagocytosis. An interesting obser vation was that clearance rates for these particles were not affected for 8 d following inhalation, in spite of the differences in rates of phagocytosis of these particles in vitro. It is clear from several in vitro and in vivo studies that alveolar macrophages can ingest large particles. However, it is not clear what the maximum size of a transportable particle is. In addition to particle size, particle shape may prohibit its transport. For example, some fibers may be ingested by mac rophages, but the fibers may alter the mobility of the macro phages, making them immobile. Asbestos, for example, may be cleared slowly because of its fibrous shape. An alternate possibility, however, is that asbestos fibers induce biochemical changes that are toxic to phagocytic cells attempting to trans port the fibers.96,97 After an alveolar macrophage has ingested a particle, the particle appears to remain with the macrophage as long as the macrophage is viable. The particles are subjected to dissolution and digestion processes within the host macrophage. If this macrophage dies, its debris is ingested by another macrophage, along with the particle or particles contained within the debris. Macrophages may remain stationary in the lung, may migrate within the lung (and ingest additional particles as they migrate), or may exit from the lung. Particles that move into the interstitium and remain there for long periods of time appear to find their way into subpleural and paraseptal positions, and into perivascular positions around pulmonary arterioles and venules associated with bronchioles. The peribronchiolar sites appear to be prominent, long-term storage sites for particles retained in the lung.62 The pulmonary retention sites and cellular constituents of the pulmonary region involved with long-term retention can be readily seen in animals exposed to large amounts of partic ulate materials. Large aggregates of particle-laden macro phages have been observed in alveoli near terminal bronchioles and within alveolar and peribronchial interstitial tissues in rats exposed repeatedly to diluted diesel exhaust.981" These ag gregates of diesel soot were associated with focal inflamma tion, Type 2 pneumonocyte proliferation, and fibrosis, depending on the diesel soot exposure concentration and length of exposure. Similar responses were seen in rats exposed re peatedly to T i0 2112 and to fly ash ."3 " 4 These observations on particle accumulations in lung tissue were not limited to small animal species. MacFarland et a l." 5 repeatedly exposed cynomolgus monkeys, 23.5 h/d for 18 months, toO. 16 or 0.46 mg fly ash per cubic meter. The authors noted accumulations of the particles in alveolar macrophages, adjacent walls, and peribronchiolar lymphoid follicles. In a later study, MacFarland et a l." 6 exposed rats and monkeys to respirable dusts of raw and processed shale and noted accu mulations of pigment and particle-laden macrophages in lung tissues of both species. The retention sites for particles retained in the pulmonary region appear to be the same for small and large quantities of most types of particles. Accumulations of particle-laden mac rophages can cause damage to epithelial barriers and contribute to progressive lung disease by releasing enzymes and other ch em icals."7 Considerable work is being done to understand the origin, longevity, function, and fate of pulmonary alveolar macro phages. There is a critical need for further work, with emphasis on species similarities and differences because pulmonary mac rophages have such important roles in retention and clearance of inhaled materials. To speculate, differences in longevity and function of pulmonary macrophages may be the basis for most or all of the differences in retention and clearance of particles observed among laboratory animal species and humans. C. Type 1 and Type 2 Cells After inhalation exposures, Type l cells contain foreign particles, presumably for long periods of time. However, it may be that the particles were in transit through the Type l epithelium. Sanders and Adee6' noted that after inhalation of plutonium dioxide by rats plutonium particles were engulfed by alveolar macrophages and Type l alveolar epithelial cells. They did not determine whether or not the Type l cell rep resented a retention site for the particles. In addition, Barnhart et a l.101 noted the presence of diesel exhaust particles in Type 1 cells of rats. Adamson and Bowden84 observed the presence of carbon particles in the cytoplasm of Type 1 pneumonocytes and in cells of the pulmonary interstitium, including interstitial macrophages. Brody et a l." 8 and Sorokin and Brain55 found inhaled particles within pulmonary alveolar macrophages, Type 1 pneumonocytes, and in interstitial locations within a few hours after exposure. Kyono et a l." 9 studied localization patterns for lead par ticles in the lungs of rats after inhalation exposures to a lead fume having an activity median diameter of 1.1 mm. They noted the usual accumulations of lead in alveolar macrophages, but there were also lead particles in Type 1 and Type 2 pneu monocytes. For every type of cell containing lead particles, phagosomes and lysosomes were the main site of retention within the cell. These observations of particles in Type 1 and Type 2 pneu monocytes have all been from studies with rodents. It is not clear if this is a species-related phenomenon or if similar results pertain to other species, including humans. D. Interstitium While phagocytosis and transport of particles in macro phages and other phagocytic cells appear to be the dominant methods of trapping particles in lung tissue, some particles may directly enter the alveolar interstitium by endocytosis, or by passing between cells lining the interstitium. Phagocytosis of particles appears to occur within a few hours of their dep osition in the lung. During the time the particles are uningested. 1989 183 Critical Reviews In / have the potential to penetrate into the interstitium. Whether or not there is penetration may depend upon the integrity of the alveolar lining cells. In addition, there have been several reports indicating that both the ability to penetrate and the probability of penetrating the pulmonary epithelium depends on the amount of material deposited, or the mass load ing 55.120.121 -phe transepithelia! passage of free particles may be related to the number of alveolar macrophages available to ingest the deposited particles; with an increased particle load, a level exceeding the saturation point for increasing macro phage numbers may be reached.67-84-85 122 123In such a situation, the probability of particle penetration may increase. Transepithelial transport of fine particles has been dem onstrated for a variety of materials including carbon, latex, bacteria, silica, and asbestos.85120124125 The higher the number of particles, the greater the possibility that some of them will reach the interstitium where they may be phagocytized by in terstitial macrophages.85 Bowden16 provided a descriptive summary of the role of the macrophage in keeping the lung free of foreign particles and of the consequences, particularly as they would affect the pulmonary interstitium, of perturbing this defense system. Ad amson and Bowden122 irradiated mice to deplete monocytes and reduce the ability of the mice to produce macrophages. When the lung was then intratracheally instilled with carbon tid e s, it was unable to recruit adequate numbers of mac . ^phages to respond to the challenge. This resulted in a de creased capacity for phagocytosis and the number of free carbon particles in the alveoli increased. The likelihood that some of the carbon particles would cross the alveolar epithelium and accumulate in interstitial macrophages increased, and an in creased number of carbon particles translocated to lung-asso ciated lymph nodes, suggesting an association between the accumulation of particles in the interstitium and translocation of particles to the lung-associated lymph nodes. Similar results might be seen if the ability of the lung to provide sufficient numbers of phagocytes to respond to the particle load was overloaded, or if there was an inadequate cellular response to a limited number of particles. Although particles may enter the pulmonary interstitium by direct passage across the cytoplasm of the Type 1 pneu monocyte cell,67 the possibility of macrophage transport of particles from the alveolus to the interstitium exists. One recent study concluded that alveolar macrophages can return to the pulmonary interstitium and transport phagocytized particles to lung-associated lymph nodes.72 This issue deserves additional attention, and resolving it will significantly advance our un derstanding of the mechanisms involved in particle transport within lung tissue and in retention of particles in specific pul monary tissues. Particle Redistribution The initial dispersion of deposited particles in the lung changes with time. After clearance of a portion of the deposited particles, the distribution of the remaining particles becomes less uniform. Changes in retention and distribution patterns for inhaled particles can have important implications for the sub sequent biological effects of inhalation exposures. Particle movement in the lung will cause a changing pattern of cells at risk. Particle clumping could increase the risk of damage to cells proximate to the clumped particles (if the particles are toxic), it could reduce the dissolution-absorption rate, and it could decrease the availability of the particles for physical clearance. In some cases, the long-term retention and clearance patterns of the particles might be markedly influenced by par ticle redistribution and clumping. Thus, the phenomena as sociated with redistribution and clumping need to be addressed to help understand the long-term fate of particles deposited in the pulmonary region, and to determine if the temporal changes are species-dependent. In one study addressing this issue, Diel et al.126 evaluated distribution patterns for monodisperse 238P u02particles inhaled by Syrian hamsters. Particle movement was not measured di rectly, rather it was inferred from the relative distributions of the 238P u 0 2 particles in lung tissue. Temporal distribution of the particles with respect to specific anatomic structures in the lung was measured. These measurements were used to cal culate the distributions of particles relative to their distance from the lung periphery, airways, foci of damage, and other particles in the lung. The inhaled particles deposited prefer entially in the central regions of the lung. With time, the par ticles appeared to migrate toward the periphery and were cleared from all regions of the lung with equal efficiency, unless the lung was damaged as a result of the presence of the radioactive particles. Migration of particles over time resulted in a less uniform distribution in the lung. These results were confirmed in a study of rat lungs after inhalation of 239P u02. 127 In a comparative study using hamsters and rats, Rhoads et a l.128evaluated 239P u 0 2distribution patterns in lung tissue after inhalation exposure. Again, changes in lung distribution of the 239Pu0 2 particles were noted over the course of the study. In both species, the relative concentration of particles in the sub pleural region increased compared with the concentration in the parenchymal region. There was particle clumping in both species, but particles in the lungs of the rats tended to have a greater degree of clumping with time than those in guinea pig lungs. The relative particle concentration in the peribronchial region of the rats was low initially, then increased with time; the pattern showed no temporal trend in hamsters. This dif ference in patterns for clearance and redistribution of particles near small airways represents a species difference in the long term retention patterns for inhaled particles. Although the previous discussion involved radioactive par ticles, particle movement and redistribution in the lung appears to be similar for all types of particles. Both inert and toxic particles move toward the peribronchial and perivascular con 184 Volume 20, Issue 3 Toxicology nective tissues.84 124 125 The process appears to be common to all mammalian species, but that has not been clearly demon strated. Even if the process is common to humans and other mammalian species, differences in rates and degree of particle redistribution and particle clumping could still result in species differences in retention patterns and biological responses to retained particles. V. PULMONARY LYMPHATIC SYSTEM Leak129 provided an excellent review of the pulmonary lymphatic system and its function. While the basic components of the system appear to be common to all mammalian species, there are significant quantitative differences among species that probably influence the functional effectiveness of their lym phatic transport systems. Leak and Jamuar130summarized known similarities and differences in the distribution of lymphatic vessels in humans and several animal species. The only sub stantive differences noted are that humans and large animal species have thick pleura with many interlobular septa that form an extensive network of lymphatic vessels and collecting vessels throughout the connective tissue. The pleura is gen erally thin in small animals and there is a sparse distribution of lymphatic vessels within the pleura. Lymphoid tissue has been recognized as a constituent of lung tissue for over 100 years. Some of the lymphoid tissues are easily seen because they are external to the lung and are generally found in the mediastinum or close to the tracheo bronchial bifurcation. These lymph nodes are generally referred to as tracheobronchial lymph nodes (TBLN) or lung-associated lymph nodes (LALN). The lymph nodes physically separated from lung parenchyma have been studied extensively and are recognized as structures important to lung defenses and sys temic immune responses. Lymphatic tissue integrally associ ated with the lung interstitium has been recognized and described morphologically, but its role in lung function has not been clearly defined. Lymphoid tissue enclosed within the lung is described as " bronchus-associated lymphoid tissue^' (BALT). Bnmdelet131 included a review of experimental results from studies and observations about BALT in his excellent review of the dust clearance mechanisms in the lung. Studies dating from the 1880s included observations and discussions about the possible role of BALT in the lung. Chamberlain et al.132 and Bienenstock et al.133134 extended observations of earlier investigators and summarized contemporary conclusions rel ative to the function of BALT in lung tissue. Chamberlain et al.132described the ultrastructure of BALT in rat lungs as being similar to that of a lymph node, composed predominently of lymphocytes. Cytokinetic studies indicate that both locally de rived, replicating cells and recirculating cells form the pool of thymus-derived lymphocytes that constitute BALT.135 As de scribed by Chamberlain et a l.,132 the following are significant differences between BALT and lymph nodes. (1) BALT is always within the lamina propria; (2) BALT does not have clearly defined borders; (3) BALT does, not have organized capsules; and (4) BALT has no marginal lymphatic sinus. The BALT forms aggregates in the walls of bronchi and bronchi oles, interstitial connective tissue, and pleural connective tis sue. The most prominent aggregates of BALT are associated with bronchi and bronchioles. Aggregates of BALT range from small collections of mononuclear cells limited to the lamina propria of airways, to large nodules that extend through the muscularis and merge into the peribronchial and peribronchio lar connective tissue or the walls of adjacent alveoli. In every case, the nodules appear to be in direct contact with the bron chial or bronchiolar epithelium. All classes of blood vessels can be seen within the BALT; they do not exhibit any constant or specific pattern or show any unusual ultrastructural char acteristics. Postcapillary venules identical to those of lymph nodes are associated with the BALT. The lymphoepithelium overlying BALT shows distinct morphologic differences com pared with the surrounding epithelium. Epithelium covering the BALT in rats has ciliated and nonciliated cells.132In rabbits, the lymphoepithelial cells are flattened and have irregular mi crovilli, but no cilia.136 Bienenstock et a l.133described morphologic characteristics of BALT in bronchial mucosa of rabbits, Syrian hamsters, humans, mice, and rats. The most extensive observations were reported for rabbits. Additional data were presented by Bi enenstock and Johnston137 and by Gregson et al.138 The cu mulative results from these references are presented in Table 3. No observations of BALT in dogs or monkeys have been reported. The amount of BALT present in the lung and its function may depend on the environment in which the animal lives. As summarized by Chamberlain et al.132and Bienenstock et a l.,135 two main functions have been attributed to BALT. The first function is related to retention of particles in the lung, the second to immune responses. Some particles deposited in the lung are incorporated into BALT and may be retained there indefinitely. BALT might also represent a holding site for particles that are eventually translocated to other lymphatic tissues or excreted onto the mucociliary escalator of the bron chiolar epithelium. Fournier et al.139 injected 0.8-p.m latex particles into the tracheas of rats to determine if any of the particles would be incorporated into BALT. Approximately 700,000 particles were used per rat; none of the latex particles were later found to be associated with BALT. In contrast, ferritin molecules injected into the tracheas of rats were found in BALT. The authors concluded that the relatively small num ber of latex particles could have influenced the results and that lymphoepithelial nodules are structures capable of uptake and transport of certain kinds of particles across the basement membrane. In experiments using rats intratracheally instilled with try pan blue and carmin red, Brundelet131 described the migration 1989 185 Critical Reviews In Table 3 Summary of Morphologic Characteristics of Bronchus-Associated Lymphoid Tissue (BALT) for Selected Species Species Observations Rabbit Syrian hamsters Humans Rats Mice Follicles often seen immediately underlying the bronchial epithelium and in deeper structures Blood vessels not observed inside the follicles, but typically seen bordering BALT on side farthest away from airway lumen Majority of BALT cells appear to be lymphocytes No plasma cells in the BALT follicles No germinal centers observed No BALT observed in newborn rabbits No BALT observed in this species Similar to rabbits, but mainly diffuse collections of subepithelial lymphoid tissue in bronchi BALT found in all adult rats examined, but less BALT present in germ-free rats BALT present in fetal and adult lungs From References 133, 137, and 138, pathways of labeled alveolar macrophages into BALT. Some dye-laden macrophages apparently crossed alveolar walls to penetrate directly into the BALT; most of the dye-laden mac rophages appeared to penetrate into the connective tissue of the inter-alveolar walls or of the pulmonary septa. Once in the connective tissue septa, the dye-laden macrophages could mi grate to a peribronchial lymphoid tissue focus and exit the BALT directly into the bronchial lumina. Similar observations by Green140 of the transport of coal dust-laden nodules at the alveolobronchiolar junction area of the lung demonstrated that particle-laden macrophages passed through these lymphoid nodules and out onto the ciliated mucosa present at the start of the terminal bronchioles. From this point, the macrophages could be carried out of the lung via the mucociliary escalator. In his review of alveolobronchiolar transport, Green62 sug gested that BALT may be a key factor in the processes or mechanisms by which liquid and phagocytic cells pass into and out of the pulmonary interstitium. Alveolobronchiolar transport appears to be a means of getting particles from the interstitium to the bronchial epithelium. The BALT was eval uated as a dynamic kind of tissue that could function to absorb or exude liquid and could allow phagocytic cells to pass back and forth between the interstitium and bronchiolar lumen. In essence, the BALT could represent an excretory pathway for particles or phagocytic cells that enter the interstitium via the alveoli and exit onto the bronchial epithelium. Whether or not BALT is involved in particle clearance from the P region is still an unanswered issue that deserves additional attention. BALT could represent an important type of lung tissue with functions relevant to species differences in physical clearance of particles deposited in the P region. The relationship between BALT and systemic immune re sponses is more speculative. These lymphoid nodules along transport pathways may provide a site for intrapulmonary an tigen processing and antigen production.62 Experimental evi dence suggests that both B and T cells comprise at least part of the cell constituents of BALT.133In general, the BALT might be part of a broad mucosal immunologic system, and its pres ence and function in the lung may not be unique.133 VI. CLEARANCE OF PARTICLES FROM THE RESPIRATORY TRACT A. General Clearance is defined for the purposes of this paper as re moval of debris from the respiratory tract. The debris can include foreign particles, dead or living phagocytic cells, bac teria and other microorganisms, and soluble material. Descrip tions of the basic processes and pathways associated with clearance of the respiratory tract are available in the scientific literature. Information from References 19, 20, 24, 30, 31, 56, and 141 through 146 was used to make generalizations about long-term respiratory tract retention and clearance of deposited particles. In addition to understanding the clearance processes that remove materials from the body, it is also im portant to understand processes and mechanisms associated with translocation of particles or dissolved constituents of the particles to other organs because the respiratory tract can be the portal of entry for toxic agents that have effects on other organs of the body. Figure 4 depicts proposed pathways by which materials can physically move within and out of the respiratory tract. Transport processes cause particles to be removed from the respiratory tract and excreted in feces or transported to thoracic lymph nodes. Some particles may be absorbed directly into the circulatory system. Another important process in clearance is absorption of dissolved constituents of particles. In addition to complete dissolution of particles, constituents of particles may leach from the surfaces or interiors of the particles, leaving the core particle in its original state, except that it is devoid of specific constituents. 186 Volume 20, Issue 3 Toxicology oastromtestmju. TRACT FIGURE 4. Biological fate of particles deposited in the respiratory tracts of mammalian species. Discussions about clearance of materials from the respi ratory tract may focus on biological clearance or on effective clearance. Biological clearance refers to the net result of bi ological activities within the respiratory tract that act to remove the deposited material, its dissolved constituents, and its met abolic products from the respiratory tract. Effective clearance applies to radioactive materials, where the inhaled material is physically decaying in addition to being cleared by biological processes. It is important to make the distinction between dissolution and absorption in the respiratory tract. The term dissolution includes the net result of processes in cells and body fluids that cause constituents of particles to dissociate. These pro cesses may involve solubilization of the particles or elution of constituents from the matrix of the particles. In either case, constituents of the particles become available for absorption into the lymphatic or circulatory systems, for metabolism, or for chemical reactions with, or adsorption to, tissue constitu ents. The solubilized constituents of the particles may remain associated with tissue constituents proximate to the particles, may be translocated and deposited elsewhere in the body, or may be excreted into urine or feces. Absorption refers to trans port phenomena that cause a net transfer of material from the respiratory tract to the lymphatic or circulatory systems. The term absorption does not, by itself, imply a mechanism, and the absorbed material may be in ionic, molecular, or particulate form. Factors that affect the solubilization of particles in phys iological fluids and absorption of their constituents are not fully understood. Particle solubility is known to be influenced by the surface-to-volume ratio and other surface properties of par ticles.147 The rates at which dissolution and absorption pro cesses occur are influenced by the chemical composition of the particles and by other factors, such as temperature history. The temperature history of particles may be one of the most important considerations for oxides. Usually, in controlled lab oratory environments, the solubility of oxides decreases when they are produced at higher temperatures. Mercer14analyzed pulmonary clearance of particles on the basis of particle dissolution rate compared with physical trans location rate. Particle deposits in the lung were assumed to have log-normal size distributions and dissolution was a func tion of surface area of the particles. The smaller particles of the distribution would dissolve and leave the lung faster than the larger particles. Mercer concluded that ( 1) for monodisperse particles, if the dissolution rate constant [k] is known for a material, the time required to dissolve half the mass of particles can be calculated; and (2) when the dissolution half-times are much shorter than the half-times associated with the physical translocation of particles, dissolution will dominate clearance characteristics. Therefore, dissolution-absorption can be the dominant factor in clearance, even for materials having inter mediate or long dissolution half-times. This is significant for many so-called insoluble particles potentially inhaled by hu mans. Physical clearance rates for the particles are slow, so even a modest rate of dissolution can make a significant dif ference in effective clearance rates. In many cases, it is possible, on the basis of general chem ical considerations, to make accurate projections about the dissolution rate for a material in question and also to accurately project its clearance rate from the respiratory tract. However, for other materials, especially those containing multivalent cat ions or anions, it may not be possible to project their in vivo dissolution and absorption rates on the basis of their chemical composition. Weathering of particles, or allowing them to interact with chemicals, will change their surface characteristics and influ ence solubility. In an in vitro dissolution study with relatively insoluble particles of P u02, it was possible to markedly alter the dissolution characteristics of the particles by alternately drying and wetting them .149 Each wet-dry cycle resulted in slight changes in the surface properties of the particles that, in turn, resulted in significantly altered dissolution characteristics. While this process has no direct implications for particles de posited in the human lung, the results point out the need to recognize that environmental weathering may alter the surface characteristics of materials that later may be aerosolized. The particles may then dissolve in the lung at unusual rates relative to what might be predicted based simply on a physical de scription of the material. Phagocytic cells, primarily macrophages, clearly play a role in dissolution-absorption of particles retained in the res piratory tract. Dissolution of some types of particles is believed to occur within the phagosomes due to the acidic milieu in those organelles.150131 It seems clear that pH is an important factor in dissolution of particles, particularly for metallic par ticles. However, difficulties associated with evaluating the fac tors that influence dissolution-absorption rates for inhaled particles are compounded by the fact that it is difficult to precisely predict or measure pH in locations where particles are retained. Then too, while particles may be more soluble 1989 187 Critical Reviews In . phagosomes, the dissolved material may not diffuse or be transported away from the dissolution site. Snipes et a l.132 concluded, from simulation modeling, that mice, rats, and dogs all showed the same in vivo dissolution absorption rates for the same-sized, relatively insoluble, fused aluminosilicate particles. The same result was demonstrated for relatively soluble aerosols of cerium oxalate and cerium chloride inhaled by mice, hamsters, rats, and dogs.133A similar result might be expected for other kinds of particles and is an important factor to consider in species comparisons of effective clearance for inhaled materials. Overall, the data suggest that there are no significant differences among laboratory animal species and humans with regard to in vivo dissolution rates for any materials deposited in the respiratory tract. Differences in dissolution-absorption, if they exist, appear likely to be as sociated with the fate of the material after dissolution. In other words, the material may dissolve to the same extent in all species, but differences in local tissue reactions or metabolism may result in retention of the solubilized material or metabolites near the site of deposition or reaction. In one study related to the issue of species differences in dissolution-absorption, Oberdrster et a l.'34 studied clearance of ,09Cd from the lungs of rats and monkeys after inhalation of 109Cd-labeled aerosols of CdCl2 and Cd-oxide. The inhaled ''admium was cleared ten times faster from the lungs of the s than from the lungs of monkeys. The Cd was retained very tenaciously in the lungs of the monkeys as a result of binding with tissue constituents, but the tissue binding did not occur in the rats, or occurred to a lesser extent. Bailey et al.133 are conducting a study that includes an interspecies comparison of the translocation of 37Co from lung to blood after inhalation of 37Co-oxide, the results of which should provide additional information about species similarities and differences with re gard to dissolution and absorption of materials deposited in the lung. There are other reports indicating that presumably soluble constituents of inhaled particles are retained for longer periods of time in the lung than would be predicted on the basis, of their solubility. Examples of such inorganic materials are beryllium136 and americium.137 The authors' interpretation of the prolonged retention of these materials in the lung was that the particles did dissolve, but their constituents adsorbed to, or chemically reacted with, tissue components. Therefore, the deposited materials, or some form of the materials, were re tained near the site where deposition occurred. This same phe nomenon occurs with organic materials. For example, covalent binding of benzo(a)pyrene or metabolites to cellular macro molecules resulted in an increased pulmonary retention time for that compound after inhalation exposures of rats.138Certain chemical dyes139 are also retained in the lung, where they may ssolve and become associated with lipids or react with other wOnstituents of lung tissue. Understanding these phenomena and recognizing species similarities and differences are im portant for evaluating lung retention and clearance processes and interpreting results of inhalation studies. Some chemical compounds deposited in the lung in par ticulate forms are mobilized faster than can be explained by their known chemical properties at the normal lung fluid pH of about 7 .4 .160 In addition to in vivo chemical forces acting to dissolve particles, physical forces may influence the dis solution rate indirectly by altering the form of the particles and the surface area available for dissolution. Raabe et a l.161 re ported a higher in vitro solubility for 238P u02 particles than for 239Pu0 2 particles. Park et a l.162 noted that :38Pu02 particles changed physical characteristics when they were stored in water compared to when they were in biological tissues. The change was quite rapid in water suspension, where radiation exposure damaged the particles and caused physical disruption of the particle matrix; this damage culminated in radiolytic fragmen tation of 238Pu0 2. The result was smaller, more soluble particles of 238Pu0 2. Diel and Mewhinney163 noted the same phenom enon for particles of 238P u02 in lung tissue. B. Determining Clearance Most experiments designed to study clearance actually study retention of a test material. Clearance rate projections are in ferred from measurements of the amounts of material present in the respiratory tract as a function of time after deposition. An example of a noninvasive experiment to study clearance is one in which the subject is exposed to a radioactive test aerosol that allows external counting of the subject by using a whole body counter or a suitable collimated detector. Several mea surements of radioactivity in the subject are made over time. The time-dependent change in the amount of the radioactive test material remaining in the experimental subject is used to determine the clearance rate of the material. It is possible to conduct serial sacrifice experiments with laboratory animals to evaluate retention and clearance as they relate specifically to pulmonary tissue and the LALN. In most studies, the T-B and P regions are not segregated, so the results reflect lung clearance, where lung is defined as the combination of the P region and that portion of the T-B region from the major bronchi to the terminal bronchioles. Referring to lung retention and clearance as retention and clearance of the P region results in only a small error because most of the material retained in the lung after a few days is associated with the P region. Figure 5 shows generalized retention patterns for many respirable-sized particles inhaled by rats and dogs in a single exposure. Both curves originate at 100% on the vertical axis. Immediately after deposition, particles that deposit in the N-P and T-B regions start to clear, and clearance of those two regions is essentially complete within a day or so. Particles retained in the respiratory tract longer than a few days are mostly associated with the P region, with some exceptions discussed later. The shapes of the retention curves presented 188 Volume 20, Issue 3 Toxicology in Figure 5 emphasize the fact that, in the rat, 80 to 90% of the particles that deposit in the respiratory tract are found in itially in the combined N-P and T-B regions; the other 10 to 20% are found in the P region. In the dog, about 50% of the initially deposited particles are found in the combined N-P and T-B regions and about 50% is found in the P region. The pattern demonstrated by the dog is similar to what is observed for nosebreathing humans. FIGURE 5. Respiratory tract retention patterns for inhaled particles. C. Clearance Patterns for the Nasopharyngeal Region While most particles that deposit in the N-P region clear rapidly in all mammalian species, a small percentage of the particles is retained for long time periods. Long-term retention of inhaled particles in the N-P region has been reported by Stuart,168 Snipes et al.,36 152 and Whaley et al.165 Stuart con cluded that particles were retained for relatively long times in the heads of Beagle dogs. He based his conclusion on analyses of longitudinal scans of the dogs that showed long-tenh reten tion of promethium oxide particles. The study by Snipes et a l.152 included mice, rats, and dogs, all exposed to monodis perse or polydisperse l34Cs-labeled fused aluminosilicate par ticles. In all three species, 0.001 to 1% of the initial internally deposited burden of particles was retained in the N-P region and was removed only by dissolution and absorption of the particles. Particles were retained at sites in close proximity to the basement membrane of nasal airway epithelium, as seen in autoradiograms. In the other study by Snipes et al.,36 3-, 9-, and 15-p.m latex microspheres were inhaled by rats and guinea pigs. About 1 and 0.1% of all three sizes of microspheres were retained in the head airways of the rats and guinea pigs, respectively. For rats, the 9- and 15-p.m microspheres cleared with half-times of 23 d; for guinea pigs, the same microspheres cleared with half-times of about 9 d. The 3-p.m microspheres were cleared from the head airways of the rats and guinea pigs with bio logical half-times of 173 and 346 d, respectively. The smaller particles are apparently more likely to penetrate the epithelium and reach long-term retention sites. . Whaley et a l.165 studied retention and clearance of radio labeled, 3-p.m polystyrene latex particles instilled onto the epithelium of the maxillary and ethmoid turbinates of Beagle dogs. Retention of the particles at both sites after 30 d was about 0.1% of the amount initially deposited. Autoradiographs of turbinate tissue indicated that the particles were retained in the epithelial submucosa of both regions. D. Clearance Patterns for the Tracheobronchial Region The reader is referred to a review by Lippmann and Schlesinger29 that provides considerable detail on T-B clear ance in humans and experimental animals. This review em phasizes observations made in recent years relative to long term retention of particles in and beneath the epithelium of the T-B region. While it is accepted that most material that deposits in the T-B region clears within hours or days, in recent years there have been a number of studies demonstrating that small por tions of the materials that either deposit in the T-B region or are cleared through the T-B airways are retained with retention half-times on the order of weeks or months. Patrick and Stirling166 noted that about 1% of barium sulfate particles in stilled intratracheally into rats remained in the bronchial tissue for at least 30 d. In a followup study, Stirling and Patrick167 used autoradiography to demonstrate the temporal retention patterns for the retained 133BaSO,, particles in T-B airways. Particles of barium sulfate were retained within macrophages in the tracheal wall for at least 7 d after intratracheal instillation of 133B aS04. By 2 h after instillation, some of the particles were buried in the tracheal wall. After 24 h, when most of the initial deposition of particles had cleared, 74% of 133BaSO,, particles located on autoradiograms were located in macro phages proximate to the basement membrane. After 7 d, prac tically all of the remaining particles were incorporated into the walls of the airways. The authors did not determine the mech anisms by which the particles were moved into the airway epithelium. However, the rapid phagocytosis of particles de posited on the airway surfaces after inhalation or other means of exposure suggests that macrophages could be involved in particle transport into airway epithelium. It was not clear whether the intratracheal instillation procedures influenced the results of these studies. Gore and Thome168 exposed rats by inhalation to polydis perse aerosols containing 45 or 87 mg UOj/m3. At 2, 4, 7, and 35 d after inhalation of the U 0 2, autoradiography was used to determine the locations of particles retained in the T-B and P regions. The authors did not report seeing particles of U 0 2 1989 189 Critical Reviews In ained in the airways, but did note two phases of clearance, fhe first phase was associated with a clearance half-time of 1.4 d, the second phase with a clearance half-time of about 16 d. The faster clearance was presumably associated with par ticles deposited on the conducting airways during the inhalation exposure; the longer-term clearance was associated with clear ance of U 0 2 particles from the P region. In a separate study, Gore and Patrick1*''1evaluated the distribution of U 0 2 particles in the upper respiratory tracts of rats for up to 14 d after inhalation of aerosols similar to those used by Gore and Thom e.168 Retention of U 0 2 at airway bifurcations was noted, as was retention of particles in the trachea. In another study, Gore and Patrick170 also compared the retention sites of inhaled U 0 2 particles and intratracheally in stilled barium sulphate particles. Both types of particles were found in macrophages at sites near the basement membrane of the airways of the T-B region. The macrophages appeared to have engulfed the particles in the airways, then passed through the airway epithelium and remained in the vicinity of the base ment membrane. About 4% of the U 0 2 in lungs of rats was associated with intrapulmonary airways.171 172 Retention of inhaled or instilled particles in conducting airways has been observed by other investigators. Watson and Brain173 observed similar results with aerosols of gold colloid and iron oxide. Both types of particles were found in bronchial tithelium, but more of the iron oxide was observed, sug gesting a possible particle size effect or a relationship between the process of material uptake and chemical composition of the material. Both types of particles were found in bronchial epithelial cells, but neither gold nor iron oxide particles were seen in interstitial macrophages. In a recent inhalation study, Briant and Sanders174exposed rats to chain-aggregate aerosols of U-Pu. These authors ob served retained particles of U-Pu in the larynx, trachea, carina, and bronchial airways throughout the course of the 84-d study. The amounts retained varied, but were at any time approxi mately 1% of the concurrent lung burden. The lung cleared the U-Pu with a biological half-time of 100 d, and the relative amounts of U-Pu in the airways suggested comparable particle clearance rates from the airways. Particles of U-Pu retained in the airways were located in epithelial cells. These demonstrations of long-term particle retention in the conducting airways of the respiratory tract have important im plications for exposure, dose, and response patterns for inhaled materials. Because long-term retention of particles in respi ratory airways has been observed in several animal species, it is possible that it occurs in all mammalian species. If also true for humans, particle retention in tissue constituents of the T-B region has important implications for human respiratory tract dosimetry of inhaled, relatively insoluble particles. Ex'osure and damage to sensitive cells of the T-B epithelium, msequent to inhalation of toxic particles, could be much higher than previously predicted. There have, in fact, been a few reports indicating that relatively insoluble particles associated with cigarette smoke are retained in the epithelium of the tracheobronchial tree of humans.175 177These reports strongly suggest that the processes associated with particle retention in airway epithelium of the respiratory tract are similar among mammalian species. E. Clearance Patterns for the Pulmonary Region For most inhaled materials, retention time in the P region is sufficiently long that both physical translocation and dis solution-absorption can be important factors in clearance. These two processes are the key elements of long-term particle re tention and clearance for the P region of the respiratory tract. Physical translocation of particles from the P region takes place predominantly through (1) the mucociliary escalator and (2) transport to the LALN. Dissolution makes constituents of the particles available for absorption or for chemical reactions, including metabolism within the tissues of the respiratory tract.178 Because a broad range of particle sizes may be inhaled, it is relevant to know whether or not particle size is an important factor in physical clearance processes. Morrow et a l.179 sug gested that clearance is not particularly dependent on particle size. Most of the retention-clearance data have been difficult to interpret because of uncertainties regarding dissolution and absorption of the test particles and the relationships between particle size and physical clearance processes. In the late 1960s, monodisperse aerosols became available for use in basic studies designed to determine relationships between aerosol particle size and clearance patterns from the pulmonary region. Snipes et a l.152 exposed mice, rats, and dogs to monodisperse, fused aluminosilicate particles radiolabeled with 134Cs. These rela tively inert particles were in the size range of 0.7- to 2.8-p.m aerodynamic diameter and were physically cleared to both the LALN and to the gastrointestinal tract at the same rate within a species. While the study used a relatively narrow size range for inhaled particles, the results demonstrated that particle size, at least within the range commonly encountered in the work place and environment, did not appear to affect physical clear ance of deposited particles. The continuous movement of macrophages out of the lung via the mucociliary escalator provides the opportunity to clear significant amounts of material from the lung each day. LaBelle and Brieger57 demonstrated that phagocytic cells are involved in particle clearance from the lung. They noted that the rates of phagocyte movement out of the lung were almost identical to the rates of elimination of particles from the lung. The rate of macrophage movement out of the lung via this pathway was estimated by Spritzer et al.180 to be 1.24 to 2.47 million per hour for rats. Lehnert and Morrow74 suggested that the number of alveolar macrophages leaving the lung daily was 0.28 mil lion in rats. Brain80estimated that macrophages leave the lungs of cats at the rate of 2.05 million per hour. Rates for macro phages leaving the lung via this pathway have not been reported 190 Volume 20, Issue 3 Toxicology for other species, and it is possible that considerable variability exists within and among species. Significant variability within a species would make it difficult to reach conclusions about the relationship between numbers of macrophages leaving the lung per day and clearance. There is no question that macro phages are important in physical clearance of particles from the lung, but rates for physical clearance appear to be controlled by factors other than numbers of macrophages leaving the lung per day. No one has clearly defined the mechanism(s) by which phagocytic cells travel to the mucociliary escalator. Some phagocytic cells migrate from the pulmonary alveoli to the ciliated airways. However, the limited amount of data available that relates to this issue suggests that access to this clearance pathway can also be gained through the interstitium.181183 As discussed in the section of this review concerning BALT, movement of macrophages onto the mucociliary escalator may be facilitated by the presence of BALT and of pores or openings in the lymphoepithelium that allow what appears to be a one way transport of particles to the mucociliary epithelium starting at the bronchioles. Both retention and clearance, of at least part of an inhaled burden of particles, are associated with the interstitium and interstitial macrophages. Some phagocytic cells that follow interstitial clearance pathways appear to be resident interstitial macrophages that have ingested particles passing through the alveolar epithelium. It is known that a variety of free particles may be transported across the epithelium.8485 120125 184 The likely mechanism for this process is endocytosis by Type 1 pneu mocytes, followed by transfer of the particle into the intersti tium. Interstitial macrophages might remain where they are in the interstitium and degrade phagocytized materials, or they might move within the interstitium. Their movement may take them to a lymphatic channel, where they can further translocate to regional lymph nodes. Or, as discussed earlier, interstitial macrophages can presumably also find their way to the mu cociliary escalator. Some investigators do not agree that an interstitial transport of particles, or macrophages, from the alveoli to the bronchi can occur. Sorokin and Brain55 studied histological sections of mouse lungs obtained at different times after exposure of the mice to an iron oxide aerosol. They reported that both free particles and particles inside macrophages were carried from the alveolar lumen into the bronchiolar lumen and onto the mucociliary escalator. It is generally accepted that particles deposited in the alveoli can reach the mucociliary escalator as free particles or as phagocytized particles. However, particles are rapidly phagocytized after deposition in the P region and most clearance appears to be macrophage mediated. The ques tion as to whether or not interstitial transport of particles from alveoli to bronchi can occur has not been conclusively answered and represents an area where additional research is needed. Understanding how particles are cleared from the lung to LALN is important to a basic understanding of how the lung defense mechanisms function in normal or altered states. There are proponents of a mechanism for free particle transport from the pulmonary region to LALN. For example, Lehnert et al.77 suggested that free particles are transported to the lymph nodes where they may be engulfed by resident mononuclear phago cytes. It has been suggested that bronchoalveolar macrophages migrate daily, in significant numbers, to hilar lymph nodes;185 this would provide the means of transporting particles from portions of the lung to those lymph nodes. It seems clear that at least some, if not most or all, particles translocated from the lung to lymph nodes are in phagocytic cells during the transport process.17-72 Few particles remain in the lung for very long as free particles, and there have been no studies whose results clearly indicated that free particles migrated across the alveolar epithelium and into lymphatic vessels. More effort is needed to clearly define the mechanisms for transport of par ticles from the lung to LALN and to determine the extent to which the mechanisms are the same or dissimilar among all mammalian species. The rates of particle translocation from lung to LALN appear to vary considerably among species. Rats and mice have particle translocation rates from the P region to LALN that are quite different from those of guinea pigs, dogs, and possibly humans. After a few days following a single inhalation ex posure, transport of particles from the P region to LALN ap peared to be negligible in mice and rats,152 but continued at a constant rate in guinea pigs and dogs.152186 No experimental information is available about the rates of translocation of par ticles from the P region to LALN in humans. However, data for amounts of particles accumulated in the lungs of humans exposed repeatedly to dusty environments187190 suggest that relatively insoluble particles accumulate in LALN of humans at rates that may be comparable to those observed for guinea pigs, dogs, and monkeys. The translocation rates of particles from the P region to LALN are not constant for all types and amounts of particles deposited in the lung. Klosterkotter and Biinemann191 noted that increased lung burdens of particles caused increased trans location to LALN in rats. Rates of elimination from lung were maximal during the first month, then became increasingly slower. There were considerable differences in lymphatic trans port for various types of particles and also for individual an imals. Only small amounts of inert and fine-grained dusts of titanium dioxide and gamma-aluminum oxide penetrated into the lymph passages, except when large amounts were depos ited. In contrast, quartz exhibited an affinity for the lymph passages. In some of the tests, over 50% of the amount of silica eliminated from the lung was found in the mediastinal lymph nodes. Klosterkotter and Biinemann191 concluded that the lymphatic clearance route is used mainly for pulmonary clearance of toxic substances (e.g., quartz), while its role is negligible in the case of inert substances such as TiO,. In a 1989 191 Critical Reviews In ;r report, Klosterkotter and Gono192 noted that concurrent exposure to quartz and T i0 2 led to a threefold increase in the quantity of T i0 2 observed in the lymph nodes of exposed rats compared with the amounts observed after exposure to only T i0 2. This indicates that the accelerated lymphatic clearance was not specific for translocation of quartz. Exposures to other types of particles have resulted in in creased translocation rates from lung to LALN. Kilpperet a l.193 noted an increased clearance of tantalum to regional lymph nodes for large lung burdens of insufflated tantalum. Adamson and Bowden122 noted an increased accumulation of carbon par ticles in hilar lymph nodes of mice under conditions where macrophage response to the burden of particles was reduced and interstitial burdens of the particles were elevated. Adamson and Bowden attributed their results to an increased direct pen etration of the carbon particles into the interstitium, followed by transport to the lymph nodes. Vostal et a l.194 reported el evated lymph node burdens of diesel exhaust particles in LALN in rats and guinea pigs that were exposed for long periods of time to relatively high concentrations of diluted diesel exhaust. Ferin123 concluded that low exposure concentrations favor a primary removal pathway for insoluble, particle-laden mac rophages via the mucociliary escalator in rats; with increasing exposure levels, there is an increase in the translocation of free particles to the pulmonary lymphatic system. Even with low tid e burdens, however, particles may be found in the lym phatics soon after exposure. Vincent et al.195 reached a similar conclusion in a more recent study in which rats were chronically exposed to T i0 2 or quartz dust at concentrations ranging from 0.01 to 90 mg/m3. Exposure times ranged up to 222 d. Similar results for accumulations of particles in LALN were observed for both types of mineral dusts. Lymph node burdens were negligible or nonexistent when exposure concentrations of the dusts were low or when lung burdens of the mineral dusts were low. However, for exposures to the higher concentrations of the dusts, or after lung burdens of the dusts accumulated to masses involving several milligrams, measurable amounts of the dust particles were noted in the lymph nodes. The limits of detection for T i0 2 and quartz were estimated to be 12 and 10 |xg, respectively. On the order of 1 mg or more of either type of dust was accumulated in lungs before detectable amounts of the dusts were found in LALN. One interpretation of this result is that both ,,dusts were accumulating in LALN for all exposure conditions, but that the amounts could not be quan tified. Numerous studies with a variety of radioactive particles have demonstrated that particles are transported to LALN under conditions where microgram quantities of the particles were deposited in the lungs. While the results for translocation to LALN may have been subject to detection limitations, these authors clearly demonstrated that T i0 2 and quartz ac- mulated in LALN and that accumulation occurred at faster .es under exposure conditions where substantial lung burdens accumulated. The specific clearance routes and amounts of deposited material cleared from the pulmonary region may also be in fluenced by the mode of exposure. As one example, Ferin123 and Ferin and Feldstein121 exposed rats to T i0 2 by inhalation or intratracheal instillation. By 25 d after exposure, the contents of the lymph nodes accounted for less than 1% of the total lung burden, which ranged between 0.1 and 1 mg. A substantial increase in the TiOz content of the hilar lymph nodes was observed for lung burdens greater than 1 mg. With lung burdens in the range of 10 mg, about 4% of the TiO, was found in the lymph nodes. The rate of translocation of T i0 2 particles from the lung to the hilar lymph nodes was almost directly propor tional to the lung burden of T i0 2. Ferin and Feldstein12' con cluded that, with high lung burdens, macrophages could no longer contain all of the particles and thus the probability that particles would enter the interstitium and translocate to lymph nodes was increased. F ew erT i02particles were found in lymph nodes after inhalation than after intratracheal instillation. The authors concluded that one reason for this might be that the carrier fluid facilitated particle penetration. However, intratra cheal instillation procedures generally cause an influx of phag ocytic cells into the lung, which could also be a cause for the increased translocation of particles. Clearance rates for one material may be influenced by the presence of other materials. LaBelle and Brieger5781 noted a faster pulmonary clearance of intratracheally injected uranium dioxide particles in rats when the uranium dioxide particles were injected together with other particles. The total number of particles was sufficient to increase the number of phagocytic cells that could be lavaged from the lung. In a similar study, Ferin et al.58 found that clearance of S i0 2 particles was faster when macrophages were stimulated by intratracheal injection of trypan blue or by inhalation of T i0 2. Clearance was slower when the rats were pretreated with X-ray irradiation. In this study, the stimulation of macrophages was offset by the ra diation damage that reduced the ability of the lung to recruit additional phagocytic cells. A large number of papers published over the last 3 decades contain information relevant to understanding translocation of materials from the lung to LALN. Whether or not clearance from the P region to the lymphatic system occurs most sig nificantly for cytotoxic particles, the process of lymphatic transport is common to all mammalian species, and all types of particles appear to be cleared from the P region to local lymph nodes. However, the rates of transport and amounts of particles cleared to the lymph nodes depend, to a significant extent, on factors that include the animal species, physico chemical properties of the particles, occurrence of biological reactions to the particles, and the amounts of particles inhaled or injected into the lung. Thomas14119* 197 discussed the implications of particle translocation from the lung to LALN when the particles contain specific radionuclides, but he presented information that is 192 Volume 20, Issue 3 Toxicology relevant to all types of particles. Since the LALN represent a trap for particles cleared from the lung, particles can accu mulate to high concentrations in LALN. Thomas141 196 noted that the ratio of LALN burden to lung burden for relatively insoluble particles clearly increases with time after exposure for dogs and monkeys and may have the same pattern for rats and humans. By about 100 d, the concentrations of particles in LALN and lung are approximately equal; thereafter, the ratio of concentrations for LALN/lung increases and may reach 100 or more, depending on the life span of the individual and retention characteristics of the particles in the lymph nodes and lung. Comparable results were demonstrated for single or re peated exposures. The movement of macrophages from the lung to the LALN affords the opportunity to transport particles out of the lung, but the result is to sequester, or trap, the particles in what is generally perceived to be a dead-end compartment. Some re ports have suggested that particles may appear in post-nodal lymph, from which they enter the blood and are translocated to other sites. Transmigrated particle-laden macrophages, as well as free particles, have been found in various extrapul monary organs.198'203 One interpretation of these results is that particles normally leave the LALN only as a result of disso lution-absorption processes. However, the LALN may have been damaged to the extent that the macrophages, or the par ticles delivered to the LALN, could pass through the nodes and enter the circulatory system. The consequence of particles passing into the bloodstream is that the particles may become trapped in the reticuloendothelial system of organs such as liver, spleen, kidney, and skeleton. LeFevre et a l.198 reported accumulations of particles in the livers and spleens of coal miners. The results may have been related to relatively large lifetime accumulations of particles in the lung that influenced the release of a portion of the particles into the general cir culation. Radioactive particles have been observed in liver199200 and spleen200 of dogs after inhalation exposures. This could have occurred as a result of direct transport of particles. How ever, the particles were large relative to the sizes of p3rticles that might pass directly through pulmonary cell membranes. Another possibility is that translocation of particles from lung to lymph nodes caused accumulations of particles of ^ 'PuOz in the nodes; the accumulations of particles then caused suf ficient radiation damage to the lymph nodes to allow direct passage of the particles into the circulatory system. Gearhart et al. 199 observed particles of plutonium oxide in the livers of only those dogs that had been exposed to relatively large amounts of 238P u 0 2 and not until more than 1350 d after a single in halation exposure. This would have allowed ample time for accumulation of ^P uO z particles in the lymph nodes as a consequence of normal pulmonary clearance processes, radia tion and chemical damage to the nodes, and release of a portion of the lymph node burden to the circulatory system. Macrophages may phagocytize particles, then transport the particles directly into blood vessels.201-204 However, if this pro cess occurs, it is probably a rare phenomenon because particles transported from the lung in phagocytic cells appear to be exclusively transported to the lung-associated, lymphatic tis sues. Entry of macrophages into the lymphatic system is more likely than entry into blood capillaries since lymphatic vessels have looser endothelial cell connections and wider intercellular junctions than capillaries.67 Also, there may be movement of particles directly from the interstitium into the circulatory sys tem. Gross and Westrick205 hypothesized that very small par ticles could pass directly from lung tissue into the circulatory system, but gave no indication about what they considered to be particle size limitations for this process. This hypothesis has been pursued in several studies, and evidence has been presented to support the idea that particles can pass directly from lung parenchyma into blood vessels (and lymphatic ves sels) without having been carried by mobile cells. In one study, Stradling et al.206 noted that a suspension of 238P u 0 2, having particle sizes < 5 p.m, was substantially converted by frag mentation to a suspension of particles in the size range of 0.001 to 0.025 p.m in 32 weeks. Most of the small particles were 0.001 -jim diameter, and the authors suggested that very small particles of P u 0 2 could pass directly from lung tissue into the circulatory system. In a similar study, Raabe et al.207 noted an unusually high apparent in vivo dissolution-absorption rate for highly, insoluble weapons-grade P u02. In agreement with the results of Stradling et al.,206 Raabe et al.207 concluded that the apparent high degree of dissolution-absorption resulted from fragmentation of the P u02 into particles small enough to move readily into the blood rather than being dissolved. Diel and Mewhinney163 had a different interpretation of results from a similar study. They quantitated the enhanced in vivo fragmen tation of 238Pu0 2 particles in canine lung tissue and concluded that the alpha decay of Pu damaged the crystalline lattice of the particles, thereby providing a larger surface area for the particles in lung tissue. The larger surface area of the 238P u02 resulted in a more rapid rate of particle dissolution, which in turn allowed for an increased rate of absorption of the solu bilized Pu. There may be preferential drainage of specific particle sizes via the lymphatic vessels or capillaries. Electron microscopic studies by Lauweryns and Baert208 demonstrated that carbon particles (diameter of 25 nm) were cleared from the lungs of rabbits via the lymphatics, while ferritin molecules (diameter of 10 nm) were absorbed by both blood capillaries and lym phatics. Both types of particles were seen in the interstitium and the lymph capillaries within half an hour after their intra tracheal administration. The quantitative role of blood capil laries and lymphatics in alveolar clearance was also evaluated by Meyer et al. ,209 who studied the removal of labeled albumin from the alveolar spaces in dogs. These authors found that, during a 4-h observation j>eriod, albumin absorption via the blood was about 11 times higher than absorption via the lym- 1989 193 Critical Reviews In itics, emphasizing the importance of regional blood flow in absorption of pulmonary exudates. However, considering the fact that blood flow through the lung is about 400 times higher than lymph flow, these authors estimated that the " efficiency" of particle absorption via the lymphatics was about 37 times that of blood. F. Intra- and Inter-Species Variability Many measurements of pulmonary retention and clearance have been conducted on a variety of laboratory animal species. For some exposure materials, data from more than one labo ratory are available for the same species. In many cases, at least two laboratory animal species were exposed to the same aerosolized material, so direct comparisons among species are possible. Relatively few human inhalation exposures to the same materials as used for the animal studies have occurred, so only a limited number of direct comparisons are possible between laboratory animals and humans. Table 4 contains a summary of selected results for pul monary retention of inhaled materials after single inhalation exposures to small masses of relatively insoluble materials. Studies of less than about 3-months duration were not consid ered. The variability in these results was caused by several factors. In many cases, the reported results did not allow di vision of the lung burden between short- and long-term d ear re. Also, for most studies, solubility and amounts of -osorption of the exposure materials were not known or were not reported. The broad range of particle sizes would have influenced deposition patterns, the ratios of amounts of material cleared rapidly or slowly, and dissolution-absorption rates, but probably not the physical clearance of particles from the pul monary region. The information shown in Table 4 was used as the basis for approximations of biological clearance rates for particles inhaled by the species listed in Table 5. In addition, approx imations are included for the fractions of pulmonary burdens initially deposited in the P regions that were subjected to shortor long-term clearance. These approximations are not arith metic averages; rather, they represent estimates of trends de picted in the data in Table 5. These approximations clearly will not apply to all types of inhaled particles. For example, in some cases, deposition and clearance may be influenced by the physicochemical and/or biological characteristics of the inhaled material. However, the generalizations that led to Table 5 allow comparisons for the consequences of chronic inhalation exposures among these animal species and humans that might not otherwise be possible. Figure 6 demonstrates pulmonary retention curves based on the data in Table 5. These patterns clearly demonstrate similarities and differences in pulmonary retention and clear'ce among these species that might be applicable to a variety inhaled materials. The net retention and clearance patterns for the pulmonary burdens of material were similar for guinea pigs, monkeys, dogs, and humans. For these species, about 20 to 30% of the initial burden of particles cleared with a half time on the order of 1 month; the balance cleared with a half time of several hundred days. Rats and mice cleared about 90% of the deposited material with a half-time of about 1 month and 10% with a half-time greater than 100 d. The relative division of the pulmonary burden between short- and long-term clearance represents a significant difference between most ro dents and larger mammals and has considerable impact on long term patterns for retention of material acutely inhaled, as well as for accumulation patterns for materials inhaled in repeated exposures. VII. MODELING PULMONARY RETENTION AND CLEARANCE OF PARTICLES A. Models for Effective Lung Retention and Clearance A widely used method for expressing effective clearance and retention of lung burdens of materials is to fit the data to a single- or multi-component exponential function. This pro cedure does not differentiate between physical clearance of particles and absorptive processes, but assumes that the effec tive reduction of the lung burden occurs in a way that can be mathematically expressed as a single, first-order process or as the sum of two or more first-order processes. In this procedure, the decrease in the lung burden of particles is assumed to be proportional to the lung burden. The time-dependent decrease in the lung burden is d(L) = - k(L)dt (1) and L(t)/Lo = e x p (-k t) (2) where L(t) represents the lung burden of particles at time t and Lqrepresents the initial lung burden of particles. Because 100% of the initial lung burden is in a single compartment in this simple case, the expression becomes L, = Lo exp( - kt) (3) More generally, the form of the expression is L, = L0 2 A, exp( - k^t) (4) where A* = fraction number i of the lung burden; k* = clear ance rate for fraction number i; and, t = time, usually ex pressed in days after inhaling the particles. For repeated exposures, this simple model can be used to estimate what the equilibrium lung burdens of particles would be for lung deposition of a known mass of material per day. 194 Volume 20, Issue 3 Toxicology Table 4 Comparative Pulmonary Clearance Data for Relatively Insoluble Particles Inhaled by Laboratory Animals and Humans Species matrix Particle size |iin Measure Mouse Rat Guinea pig Dog Monkey Human FAP FAP FAP Ru Oxide Pu Oxide Diesel soot FAP FAP FAP FAP Latex Pu Oxide Pu Oxide UA FAP Diesel soot Latex Coal dust Coal dust Ce Oxide FAP FAP FAP FAP Nb Oxide Pu Oxide Pu Oxide Pu Oxide Pu Oxide Pu Oxide Pu Oxide Pu Oxide Tantalum UA Zr Oxide Pu Oxide Pu Oxide FAP FAP Latex Latex Pu Oxide Graphite and PuO, Pu Oxide Th Oxide Teflon Zr Oxide 0.7 1.5 2.8 0.38 0.2 0.12 1.2 0.7 1.5 2.8 3.0 < 1 .0 2.5 ~1--2 2.0 0.12 3.0 2.4 1.9 0.09-- 1.4 2.1-- 2.3 0.7 1.5 2.8 1.6-- 2.5 1-- 5 4.3 1.1-- 4.9 0.1--0.65 0.72 1.4 2.8 4.0 0.3 2.0 2.06 1.6 1 4 3.6 5 0.3 6 <4--5 <4--5 4.1 2.0 AMAD AMAD AMAD CMD CMD MMAD CMD AMAD AMAD AMAD CMD CMD AMAD CMD AMAD MMAD CMD MMAD MMAD MMD AMAD AMAD AMAD AMAD AMAD CMD MMD MMAD CMD AMAD AMAD AMAD AMAD CMD AMAD CMAD AMAD CMD CMD CMD CMD MMD AMAD CMD CMD CMD AMAD Pulmonary burden* P, T, Pr Tr Study (days) 0.93 34 0.93 35 0.93 36 0.88 28 0.86 20 0.37 6 0.62 20 0.91 34 0.91 35 0.91 36 0.39 18 0.20 20 0.75 30 0.67 20 0.22 29 0.09 13 0.15 20 0.15 21 0.15 21 ~1 0.10 200 0.10 3.9 0.32 87 0.22 32 0.40 1.9 0.47 4.5 0.14 40 0.27 50 0.27 30 0.42 0.5 0.30 4.5-- 45 0 07 0.07 0.07 0.12 0.14 0.63 0.38 0.09 0.09 0.09 0 61 0.80 0.25 0.33 0.78 1.00 1.00 1.00 LOO 1.00 0.91 0.85 0.85 0.85 1.00 1.00 1.00 0.90 0.90 0.68 0.78 0.60 0.53 LO 1.0 1.0 0.86 0.73 0.73 0.58 1.00 LOO 1.00 1.00 0.60 1.00 146 17) 201 230 460 80 180 173 210 258 63 180 250 500 385 >2000 83 1000 --700 >570 440 257 341 485 >300 1500 300 400 1000 680 1400 1800 860 120 340 500--900 770-- 1100 350 670 2% 150-- 300 240 240--290 1000 300-- 400 200-- 2500 224 850 850 850 490 525 330 492 850 850 850 190 350 800 768 1100 432 190 160 301-- 392 140 181 850 850 850 128 280 300 468 -4000 730 730 730 155 127 128 200 990 372-- 533 372-- 533 -480 160 300 566 427 427 300 261 Ref. 152 152 152 237 237 238 239 152 152 152 36 240 241 242 243 238 36 244 245 246 247 152 152 152 248 237 249 250 251 252 252 252 253 254 255 256 257 258 258 259 260 261 262 263 263 264 255 Note: FAP = fused aluminosilicate particles; AMAD = activity median aerodynamic diameter; MMAD = mass median aerodynamic diameter; CMD = count median diameter; MMD = mass median diameter. Some aerosols were monodisperse, most were polydisperse, with geometric standard deviation in the range of 1.5 to 4 Clearance half-times are approximations for biological clearance, the net result of dissolution-absorption processes and physical clearance processes. In some examples, the original data were subjected to a computer curve-fit procedure to derive the values for P and T. ' Pulmonary burden = P |e"""J>1/Tl + P2e """i "/T2, where P, and P2 equal fractions of the initial pulmonary deposition, T, and T ; equal retention half-times in days, and t equals days after exposure 1989 195 Critical Reviews In Table 5 Average Pulmonary Retention Parameters* for Insoluble Particles Inhaled by Selected Animal Species, Including Humans Species Pulmonary retention parameters P, T, P> Tj Mouse 0.9 30 0.1 240 Rat 0.9 25 0.1 210 Guinea pig 0.2 29 0.8 570 Dog 0.3 30 0.7 700 Monkeyl> 0.3 30 0.7 700 Human 0.3 30 0.7 7006 Pulmonary retention (fraction of initial deposition) = P, e x p f(- In 2)t/T,] + P2 exp [ ( - In 2)t/T2], where P, and P2 = fractions of pulmonary burden in fast- and slow-clearing components, respectively; T, and T2 = clearance half-times (days) for P, and P;. respectively; and t = time in days. 6 Assumed the same as for dogs. A ,[l - exp( --k ,g ] LB - { k, + A JI - exp( --k2t)] X (mass/day) (6) B. Basic Components of a Materials Balance Model 1. Anatomical Compartments Models are useful in deposition, retention, and clearance studies to describe the fate of inhaled materials. To allow this, the model must be designed to approximate the physiological or anatomic compartments in the system being studied. The compartments generally correspond to the three major regions of the respiratory tract (N-P, T-B, and P), LALN, blood, and one or more additional internal organs such as liver, skeleton, and kidney, as well as to excreta. Provisions are made in the models for appropriate entry of the material into the system, physical removal or translocation of the material among regions or compartments of the system, and removal of the material from the system (exhaled air, urine, feces, physical decay, or chemical alteration). An important fust step in modeling, therefore, is to define the model. The model must be based on anatomical and func tional organization and must produce results that can be used to mathematically describe the time-dependent fate of inhaled material. The model must also describe the time-course of movement of the inhaled material among the model compart ments and provide a mathematical means of estimating ex posure or dose to each compartment. Figure 7 presents an example of a simple model that can be used for relatively insoluble inhaled materials. Note that a dissolution-absorption factor is included because even relatively insoluble materials have dissolution and absorption rates that influence the reten tion and clearance of the inhaled material EXPOSURE ATMOSPHERE FIGURE 6. Comparative pulmonary retention o f particles inhaled by se lected animal species, including humans. The procedure is to integrate the expression to infinite time. The resulting mathematical expressions can be solved to de termine the equilibrium lung burdens. Where lung retention and clearance can be represented by a single- or two-component exponential expression, respectively, . _ [1 - exp( - kt >] LB = A --------- ---------- - X (mass/day) md (5) FECES ORGANS FIGURE 7. Model for simulating particle retention and clearance patterns in laboratory animals and humans. 196 Volume 20, Issue 3 Toxicology The transport rates for moving the inhaled material between compartments and out of the system can be represented by either constants or complex mathematical expressions. The form of the expression is secondary to its utility in describing the time-dependent fate of the inhaled material. The dissolu tion-absorption function may be the same or different for the respiratory and gastrointestinal tracts. This can be determined experimentally and should be used in cases where unusual degrees of dissolution-absorption in the gastrointestinal tract would influence interpretation of the modeling results. An ex ample of where this could happen is in studies conducted with a moderately soluble material that deposits in the skeleton or liver after absorption from the lung or gastrointestinal tract. In these cases, it would be important to compare the relative exposures of tissues in the respiratory tract with exposures of liver or skeleton. 2. Experimental Organ and Tissue Data Determination of tissue and organ content of the test ma terial as a function of time after a single exposure is critical to modeling the fate of inhaled materials. The data set has to agree with the compartments defined in the model. Typically, these would include, as a minimum, the major regions of the respiratory tract, LALN, and carcass. Blood and other organs would be defined as compartments, if needed, to clarify the fate and tissue exposure patterns produced by the inhaled ma terial. The tissue and organ data for various times throughout the study, with the first samples collected immediately after the exposure, are evaluated and expressed in terms of percent of the initial inhaled burden. Because changes in both the model system and the body will usually occur fastest at early times after exposure, the sampling is traditionally weighted to yield more data early in the study, with sampling times becoming less frequent with increasing time after exposure. 3. Excreta Data Solubilized and absorbed material can be redeposited in internal organs such as liver or skeleton, can be ex c ite d in urine, or can be excreted across the gastrointestinal tract into feces by a pathway termed endogenous fecal excretion. Phys ical clearance of particles from all three regions of the respi ratory tract results in feces being a major route for clearance of most inhaled materials. 4. Solubility-Absorbability o f Test Material To accurately assess retention and clearance patterns for single inhalation exposures and make projections for repeated exposures, it is crucial to know the solubility-absorbability of the test material. It is not possible to accurately project the magnitudes of lung burdens of materials that would accumulate under repeated exposure conditions without having an approx imation for the extent to which the material would dissolve in physiological fluids and be absorbed from the respiratory tract. Unfortunately, adequate information about in vivo solubility is a major unknown in most inhalation studies and for most ac cidents involving inhalation of hazardous materials. The advantage of using an in vivo procedure is that it avoids the criticisms associated with using in vitro test systems; the material of interest is tested under conditions where it can be dissolved in biological fluids and absorbed across biological membranes. Morrow et al.210injected radiolabeled metal oxide particles into muscle of rabbits and rats, measured the retention of the test materials at the injection sites, and related retention of the intramuscularly injected material to lung retention of the ma terial in dogs. Clearance kinetics were similar for the lung and muscle burdens of radiolabeled test materials. In a similar study, Morrow et al.2" injected neutron-activated coal dust suspensions into muscle of rats and then monitored the injection sites. The intramuscular injection provided a clearance rate for the neutron-activated coal dust that was comparable to that observed for the inhalation studies. It appeared that removal of the radioactive constituents of the coal dust occurred as a consequence of dissolution-absorption of the coal dust particles rather than as a preferential leaching of radionuclides from the matrices of the particles. Thomas et al.212 used a similar intramuscular injection pro cedure with rats to determine the in vivo absorption of four chemical forms of barium. After the injection procedure, the rats were measured periodically to determine the amounts of m Ba remaining at the sites of injection. The most insoluble form of barium was barium incorporated in fused alumino silicate particles. This form of barium cleared slowly, presum ably by dissolution of the particles and absorption of the ra diolabel, with a half-time of 1400 d. Using the same approach, Ferin213 injected a suspension of T i0 2 particles into muscle of rats. Absorption of the T i0 2 was negligible over a 118-d test period. Assuming that the solubility of T i0 2 is the same in lung and muscle, results from this intramuscular injection study affirmed the author's obser vations that the T i0 2 was relatively insoluble in the lung and helped confirm that clearance of T i0 2 particles from the lungs of rats was predominantly by physical clearance processes. While the intramuscular injection procedure provides data for the approximate dissolution-absorption characteristics of a material in vivo, intratracheal instillation of a test material can yield data about absorption of the material directly from the lung. It is important to precisely determine the amount of material placed into the lung and account for its clearance. After rapid clearance of the small portion of the test material typically deposited on tracheobronchial airways during the in tratracheal instillation procedure, clearance of the lung burden occurs by physical clearance and dissolution-absorption. Cuddihy et al.133 used another type of approach to estimate dissolution-absorption of materials from tissue and the excreta content of the material. In their approach, the rate at which 1989 197 Critical Reviews In adionuclides appeared in bone, liver, and remaining tissues or were excreted into urine was controlled by the solubilization and absorption of the materials retained in the respiratory tract. The data for tissue and urine content of the radionuclides as a function of time were used to approximate the dissolution absorption rates directly. In vitro test systems have been used in some instances to approximate the dissolution properties of test materials. Mor row et al.210-214 studied pulmonary retention and clearance of several metal oxides inhaled by dogs. They simultaneously approximated the solubility of the metal oxides by using an ultrafiltration procedure, with several buffered and unbuffered biological solutions, including human or bovine serum. Results provided a ranking of ultrafiltrability for the metal oxides that corresponded to the ranking for clearance from the lungs of the dogs; the greater the ultrafiltrability, the more rapid the clearance. This provided a ranking useful for evaluating the results of in vivo determinations of clearance by absorption of the pulmonary burdens of the metal oxides. Moss and Kanapilly215 presented an overview of in vitro techniques used to measure the dissolution rates of selected materials. The authors made the point that in vitro dissolution tests are relatively simple compared with in vivo procedures. Although true, results from in vitro test systems are difficult to relate directly to in vivo results. Particles being dissolved i vivo are subject to unknown conditions relative to pH and enzyme action, both of which may not be constant over time and are difficult, at best, to simulate in vitro. The in vitro test systems serve as useful screening procedures and ways to ob tain relative dissolution rates for a variety of materials, but they may not produce results directly applicable to in vivo dissolution-absorption rates. This results from the inability to precisely simulate in vivo dissolution conditions and tissue reactions with substances dissolved from particles or leached from their surfaces. VIII. REPEATED INHALATION EXPOSURES A. General Respiratory tract clearance processes begin to decrease the burden of particles deposited during a single inhalation ex posure. Given sufficient time, the deposited particles would be almost completely removed by these clearance processes. However, single inhalation exposures may be the exception rather than the rule. Repeated or chronic exposures produce lung burdens of the inhaled material that generally increase with time until the rate of deposition is balanced by the rate of clearance. This is defined as the " equilibrium lung burden" . The accumulation patterns are unique to each animal species and are possibly unique to the inhaled material, especially if ie material alters deposition and/or clearance patterns. It is important to evaluate these accumulation patterns because they dictate what the equilibrium lung burdens of material will be for repeated exposures to a specified exposure atmosphere. Alternatively, these patterns indicate the relative concentrations of exposure atmospheres that should be used to produce target lung burdens of the inhaled test substance. In studies where, more than one species is exposed to a test material, the ac cumulation patterns can be used to predict " equivalent con centrations" of airborne materials. Equivalent concentrations are defined as species-dependent concentrations of airborne material that, when repeatedly inhaled, produce equal lung deposits of the inhaled material per gram of lung tissue during a specified exposure period. B. Lung Overloading with Inhaled Materials When small amounts of particles are inhaled and deposited in the lung, the lung's clearance processes are able to remove the particles at a rate sufficient to keep the lung relatively free from accumulations of the particles. However, when the air concentrations of the particles are high, or exposures long, the lung cannot clear the particles fast enough, and it tends to accumulate particles until the equilibrium lung burden is reached. If lung clearance processes are altered by the amounts of ma terial inhaled during the exposure, or by the accumulated masses of particles, the lung burden increases beyond the equilibrium lung burden. This process will be referred to as lung overloading. Literature on the topic of lung overloading in laboratory animals has grown markedly in recent years. Most of the ex perimental work has been conducted with rats exposed chron ically to diesel exhaust.99 l03',05 n0111-216'2'9 Exposures covered a broad spectrum of conditions and exposure times, but all exposures produced significant lung burdens of diesel soot. Impairment of particle clearance from the lung has been a common consequence of exposures of rats to large amounts of diesel exhaust. This means that lung burdens of diesel soot particles accumulated to the extent that lung clearance rates decreased relative to normal rates. The consequence was a shift in the relationship between deposition and clearance, such that the lung accumulated particles at a faster rate than occurred during the early phase of the exposure. The change was quan titated in a few studies. For example, in studies with rats exposed repeatedly to diesel exhaust, Vostal et a l.104 and Chan et al.220 reported dose-related slowing of clearance of l4Clabeled diesel particles following periods of 20-h/d exposures to concentrations of diesel exhaust up to 6 mg soot per cubic meter. Chan et al.220 noted that the rate of alveolar clearance of diesel particles from lungs of rats was dependent upon the total diesel particle burden. Normal clearance rates occurred when lung burdens were less than 0.8 mg of diesel soot, but slowed when lung burdens were 6.5 mg of soot. This report was one of the first in a series of reports that confirmed the phenomenon now widely recognized as altered lung clearance resulting from lung overloading with relatively insoluble, par ticulate material. Griffis et al.218 reported an approximate dou bling of lung clearance half-times in rats following 18 weeks 198 Volume 20, Issue 3 Toxicology of exposure, 7 h/d, to diesel exhaust at concentrations of 4.1 mg soot per cubic meter. Results of more recent studies have been summarized by Wolff et al.1" Rats were exposed for 24 months to 0, 0.35, 3.5, or 7.0 mg diesel soot per cubic meter. Lung burdens as high as 20.5 mg soot per gram lung were produced. None of the exposures resulted in altered tracheal mucociliary clearance, but long-term pulmonary clearance was decreased by a factor of about 4 after 24 months of exposures to either 3.5 or 7.0 mg diesel soot per cubic meter. Heinrich et al.217 exposed hamsters and rats to 4 mg of diesel exhaust per cubic meter for 19 h/d, 5 d/week. The ex posure did not alter alveolar clearance of a test aerosol of 59Fe20 3 inhaled by hamsters after 12 months of exposure to the diesel exhaust. However, clearance of this same test aerosol inhaled by rats was slowed by a factor of 2.5 after only 3 months of exposure. These results confirm previous findings that inhalation of diesel exhaust can alter pulmonary clearance, but also emphasizes the fact that considerable species varia bility may exist in terms of lung accumulations of diesel soot and biological responses associated with exposure to the same air concentration of diesel soot. Lung burdens of diesel soot were not reported, so it is difficult to judge the extent to which results were influenced by the amounts of diesel soot accu mulated in the lungs of the rats and hamsters. Progressive accumulations of particles in rodent lungs with long-term exposures have not been unique to diesel exhaust. Bolton et al.221 and Vincent et al.222 described an " overload" phenomenon in rats, with respect to accumulation of asbestos, when it exceeded about 1.5 mg per lung. Impairments in lung clearance have also been observed following inhalation of pul verized coal combustion dust at 38 mg/m3 and fluidized bed combustion fly ash at 37 mg/m3 for 4 weeks, resulting in lung burdens of about 3 mg per rat.219 Rats exposed to coal dust appear to produce accumulations of particles in lung that have the same magnitude and pathological appearance as those in rats exposed to diesel exhaust when similar exposure conditions are used. The lung overload effect seems to be nonspecific and may apply to all types of particles that are not very soluble and therefore are persistently retained in the lungs. The effect does not appear to involve deposition, rather it is a consequence of abnormal pulmonary clearance. The reduced clearance is ac companied by large accumulations of particle-containing mac rophages in pulmonary air spaces. This phenomenon has been observed a number of times in rats, but there are no definitive data available for other species. Collectively, studies on par ticle overloading indicate that the phenomenon occurs when at least 1 to 3 mg of particles per gram of lung has been deposited. Particle overloading of the lung appears to be uniquely asso ciated with specific accumulation patterns of particle-laden macrophages and cumulative lung burdens of particles.88223 There are morphologic changes in the lung consistent with inhibited phagocytic clearance of particles. These changes are manifest in macrophage accumulations, epithelial cell prolif eration, inflammatory reactions, and increased amounts of par ticles in the pulmonary interstitium and LALN. The pulmonary macrophage is significantly involved with the lung overload phenomenon and may, in fact, help trigger the process; toxi- cological alterations in macrophage function due to the inhaled materials may be an important aspect of the immediate- and long-term lung responses to excessive lung burdens of parti cles.117 Alternatively, the numbers of particles deposited in the lung simply overwhelm the functional abilities of macrophages to deal with them normally, and they tend to accumulate and initiate the sequelae typically noted in lung tissue as a con sequence of lung overloading with particles. It is not clear whether the mass of retained particles or the volume occupied by the particles is more important in initiating and perpetuating the lung overloading process. Morrow88 sug gested that the volume of particles available for phagocytosis is the significant factor in the lung overloading process and that the inability of particle-laden alveolar macrophages to translocate to the mucociliary escalator is correlated to an av erage composite particle volume per alveolar macrophage in the lung. When the particulate volume in a macrophage exceeds approximately 60 p.m3 per macrophage, with a uniform dis tribution of particles over the pulmonary pool of alveolar mac rophages (--2.5 x 107 macrophages in the Fischer 344 rat), the lung overload effect appears to be initiated. When lung burdens of particles have accumulated to the extent that normal lung clearance patterns are altered, the as sumption is made that lung clearance, in general, is altered. This conclusion is justified for lung burdens in the range of tens of milligrams of test particles per gram of lung, at least in rats. It is not clear, however, that lung burdens in the mil ligram-per-gram lung range can produce this effect. When any quantity of test particles is inhaled, the particles are sequestered to some extent in the lung. When large quantities of particles are inhaled under repeated exposure conditions, the particles accumulate in a nonuniform pattern, mainly in accumulations of macrophages within alveoli. The accumulations of macro phages laden with particles become less available for clearance with time as a consequence of their burial in the large amounts of material being repeatedly inhaled. These large daily deposits of particles overwhelm the capacity of the lung defense mech anisms. However, prior to incorporation of the inhaled material into the sequestered lung burden, which makes it less available for physical clearance and possibly for dissolution-absorption, the deposited material may be cleared at a normal rate. This means that the lung may have the ability to clear newly de posited materials at a normal rate, in spite of the presence of a substantial lung burden of sequestered particles, if the re peated or chronic exposure is terminated. The accumulated lung burden that triggers impaired clear ance has not been precisely determined. Additional research is needed to determine the magnitudes of lung burdens of diesel 1989 199 Critical Reviews In jt or other kinds of particles that initiate or result in altered clearance. The results should also demonstrate whether or not a relationship exists between rate of accumulation of lung bur dens of particles and the extent of any changes in clearance processes or rates. In addition, it is important to evaluate the degree to which lung function can be returned to normal after the overloading phenomenon. It is common practice to evaluate the extent to which lung clearance has been altered by using a radiolabeled test aerosol inhaled after the lung-loading process. Different opinions have been expressed as to the kind of aerosol most appropriate for this test. Using diesel exhaust as an example, one argument is that if a study is designed to determine the amount of inhaled diesel exhaust that alters clearance, then a test aerosol inhaled after the lung overloading with diesel exhaust particles should be as physically similar as possible to diesel exhaust. For other kinds of exposure materials, the test aerosol should be selected to match the physical characteristics of those test materials as nearly as possible. It is argued that using an aerosol different from the test material would yield invalid results because the two aerosols might not deposit or clear the same way. A number of studies have determined the deposition and clearance of other types of aerosols inhaled after the lung overloading process. The results of these studies have impor tant implications relative to the reversibility or irreversibility altered lung clearance resulting from the lung burden of uiesel soot. Studies have been done in which lung overloading with diesel soot was followed by inhalation of a test aerosol of Cs-labeled, fused aluminosilicate particles111 or 14C-labeled diesel soot220 to determine the effects of the lung burdens of diesel soot on the rate of lung clearance. Heinrich et al.217 measured clearance of iron oxide particles after 18-months exposure to 3.9 mg diesel soot per cubic meter. Results of ail of these studies indicated that, at some point in the lung over loading process, the lung demonstrated a diminished ability to clear particles. Thereafter, the rate of lung overloading accel erated with continued exposures. There are no data on lung burdens of diesel particle^ in humans. However, data are available for other inhaled mate rials. One example is fumes from arc welders. Kalliomaki et al.224 reported retained lung burdens of arc welding particles of about 0.2 g after 5- to 30-years exposure of shipyard arc welders. Another example is coal dust. Lung accumulations of coal dust on the order of 10 to 40 g have been reported for British coal miners.223'228 Stober et a l.187 reported lung burdens of as much as 25 g in Saar coal miners and of 50 g in Ruhr coal miners. Dobreva et al.229 and Verma et al.230 reported comparable amounts of particulates in lungs of hard rock min ers. These lung burdens in miners represented the net lungretained burdens after many years of repeated exposure to mine mospheres. A lung burden of 10 to 20 g in humans coneponds to 10- to20-mg particles per gram lung, which is similar to the lung burden of diesel soot in rats after 24 months of exposure to 3.5 or 7.0 mg soot per cubic meter. Results from humans, as they become available, will represent important data for comparing results among species. A complicating factor with human exposures is the defi nition of the exposure history. A more difficult problem is relating industrial and environmental exposures to. other fac tors. For example, the extent to which personal habits, age, smoking history, and other exposures contribute to the lung burdens of specific materials inhaled by humans in work en vironments is not known. McClellan231 made the point that cigarette smoking is the dominant cause of air pollution-in duced disease and, as such, limits our potential for developing an understanding about the human health effects of inhaled materials through human studies. The alternative is to use hu man data, but to supplement it with information from studies ranging from molecular to whole animal studies. Considerable work needs to be done to compare and contrast results from laboratory animal studies with the expanding human data base. C. Comparative Lung (and LALN) Burdens from Repeated Exposures of Man and Animals The following discussion presents some basic considera tions and an approach to making projections for lung burdens in laboratory animals and humans exposed to the same airborne material or to concentrations of the airborne material that pro duce similar lung burdens. It is important to recognize these species differences when designing studies or evaluating re sponses to inhalation exposures. The approach used in this example was simulation mod eling. The desired result from all models is a mathematical way to evaluate, compare, and predict lung accumulation pat terns for inhaled materials. The simulation approach is rela tively simple and was based on the GASP-IV language.232 The model (Figure 7) included only the pulmonary region and LALN to simplify the presentation. Particles deposited in the P region during inhalation exposure were assumed to clear by (1) phys ical translocation of particles from the pulmonary region [MP(t)] to the gastrointestinal tract, (2) physical translocation of par ticles to the LALN [ML(t)J, and (3) dissolution-absorption of particles [A(t)J. The transfer rates represent the fraction of the material in each respective compartment that is removed per day. The mechanical clearance rates were all assumed to be independent of each other. The dissolution-absorption rate (per cent of the total mass of particles absorbed per day) was as sumed to be constant among species and to be the same for particles in the P region and in the LALN. An important assumption was that deposition, retention, and clearance of the chronically inhaled particles were the same as for a small mass of particles inhaled during a single acute inhalation exposure. Stated another way, each increment of the repeatedly inhaled aerosols was assumed to deposit and clear the respiratory tract as if it were the only material inhaled and its deposition and clearance were not influenced by either its mass or the mass of particles already in the respiratory tract. Transfer rates for simulation modeling of the fate of par- 200 Volume 20, Issue 3 Toxicology tides inhaled by mice, rats, guinea pigs, monkeys, dogs, and humans are summarized in Table 6. These represent transfer rates that yielded curves giving close approximations of the equations presented in Table 5. A dissolution-absorption rate of 0.000693 per day for the retained particles was inherent in the simulations for each species. Respiratory minute volumes and lung weights (Table 2) appropriate for each species were used for these simulations. The aerosol concentration was arbitrarily set at 0.1 -mg particles per cubic meter, with an aerodynamic diameter of 1.0 p.m. The deposition fractions were established based on the infor mation in Figures 8 to 11, which represent approximations based on data from Lippmann,23 Raabe et a l.,26 and Schlesinger.233 The simulated exposures were set at 8 h/d, 5 d/week, for 2 years. A summary of the exposure information input for the simulation model is presented in Table 7. The modeling projections for accumulation of the particles in lungs and LALN of these six species are presented in Figures 12 and 13. These simulations demonstrate the patterns for accumulating pulmonary and LALN burdens of inhaled ma terial and also make the point that the amount of material accumulated per gram of lung (specific lung burden) is different for the different species exposed to the same aerosol. Rats and mice have a high daily relative deposition for the inhaled par ticles. Their accumulation patterns show a rapid initial accu mulation of particles. However, clearance is relatively rapid for both rats and mice, which dictates that equilibrium pul monary burdens for this exposure material will be reached during the 2-year exposure. The other four curves reflect sim ilar particle accumulation patterns in the other species; those patterns are dictated by smaller relative daily deposition rates and slower clearance rates for the particles inhaled by these species. According to these projections, guinea pigs, monkeys, dogs, and humans would not reach an equilibrium pulmonary burden of this exposure material during the 2-year repeated exposure. The projected pulmonary burdens among these spe cies after 2 years of simulated repeated exposures differed by a factor of almost 12. These results represent reasonable projections using cur rently available information about deposition, retention, and clearance for these laboratory species and humans. It has to' be emphasized that the patterns demonstrated in Figures 12 and 13 are influenced by the physical clearance parameters and dissolution-absorption characteristics of the inhaled material. In addition, if inhalation of an aerosol influences respiratory patterns, the inhaled material will affect the rates and patterns by which the aerosolized material accumulates in the lung. Likewise, translocation patterns from the P region to LALN appear to be influenced by the chemical composition of inhaled material, as well as by the mass of material. Therefore, while the patterns demonstrated in these figures may be representative for inhalation exposures to relatively low air concentrations of relatively inert materials, the patterns cannot be assumed to be definitive for all materials inhaled in any quantities by these six species. There are only limited data available to compare with these kinds of projections for repeated exposures. In a 5-year study using uranium oxide, daily inhalation of 5 mg U 0 2 dust per cubic meter caused a rapid build-up of uranium in the lungs and tracheobronchial lymph nodes of dog and monkeys, but not in rats.234-235 Concentrations of uranium in the lungs of rats reached an equilibrium level that was nearly an order of mag nitude lower than the uranium concentrations found in lungs of dogs or monkeys. Concentrations of uranium in the TBLN were also much lower for rats than for dogs or monkeys, for which the values were quite similar. These patterns observed experimentally for uranium oxide agree reasonably weli with the simulated projections presented in Figures 12 and 13 for repeated inhalation of relatively insoluble particles. In another study, reported by Wolff et a l.,m F344 rats were exposed to diesel exhaust for 2 years. Exposures were 7 h/d, 5 d/week to 0.35 mg diesel soot per cubic meter. The data Table 6 Transfer Factors for Modeling Pulmonary Retention and Clearance of Particles* Inhaled by Selected Animal Species, Including Humans Species Mechanical particle clearance from the pulmonary region, MP(t) Clearance to lymph nodes, M(L) Mouseb Rat6 Guinea pig* Dog*, monkey*1, human" 0.023 exp( -0.008t) + 0.0013 0.028 exp ( --O.Olt) + 0.0018 0.007 exp (- 0 .0 3 0 + 0.0004 0.008 ex p (-0.0221) + 0.0001 0.0007 exp( - 0.5t) 0.0007 exp( - 0.50 0.00004 0.0002 Dissolution-absorption half-time assumed 1000 d. * Adapted from Reference 152. ' Adapted from Reference 187. " Assumed the same as for dogs. I 1989 201 Critical Reviews In FIGURE 8. Pulmonary deposition of particles inhaled by rats and mice (fraction of the total amount inhaled). PARTICLE MASS MEDIAN AERODYNAMIC DIAMETER (pm) FIGURE 9. Pulmonary deposition of particles inhaled by guinea pigs (frac tion of the total amount inhaled). PARTICLE MASS MEDIAN AERODYNAMIC DIAMETER (pm) FIGURE 10. Pulmonary deposition of particles inhaled by dogs and monkeys (fraction of the total amount inhaled). FIGURE 11. Pulmonary deposition of particles inhaled by humans (fraction of the total amount inhaled). Table 7 Summary of Exposure Information Used for Simulating Pulmonary and Lung-Associated Lymph Node Burdens of Particles in Mice, Rats, Guinea Pigs, Dogs, Monkeys, and Humans Common Parameters Values Exposure atmosphere Mass median aerodynamic diameter Particle dissolution-absorption Exposure pattern Duration of repeated exposure 0.1 mg particles per cubic meter 1 pm 1000 d half-time 8 h/d; 5 d/week 2 years Specific Parameters Species Respiratory minute volume (1) Pulmonary deposition fraction Mouse Rat Guinea pig Monkey Dog Human (mouthbreathing) 0.04 0.20 0.46 0.70 3.6 20 0.10 0.10 0.20 0.30 0.30 0.30 Particles (pg) deposited)day/g lung 0.96 0.64 1.10 0.46 0.47 0.29 for lung content of the diesel soot and a simulation model152 projection for the data are presented in Figure 14. The model was modified by (1) defining the diesel soot dissolution-ab sorption rate as 0.000693 per day, (2) using a value of 0.16 as the pulmonary deposition fraction, and (3) using 0.2 l/min as the respiratory minute volume for these rats. The model used the assumption that deposition, retention, and clearance 202 Volume 20, Issue 3 Toxicology FIGURE 12. Simulation model projections for accumulation of panicles in tbe pulmonary region of selected mammalian species, including humans, with chronic exposures 8 h/d, 5 d/week to 0.1 mg particles per cubic meter; panicle dissolution-absorption half-time assumed to 1000 d. Results are presented as micrograms of particles per gram lung. FIGURE 13. Simulation model projection for accumulation of panicles in the lung-associated lymph nodes (LALN) of selected mammalian species and humans with chronic exposures 8 h/d, 5 d/week to 0 . 1 mg panicles per cubic meter, panicle dissolution-absorption half-time assumed 1000 d. Results are presented as micrograms of panicles in LALN per gram of lung. FIGURE 14. Mean values for diesel soot content of lungs ( S E ) and sim ulation model results for diesel soot content of rat lungs for rats exposed 7 h/d, 5 d/week to 0.35 mg diesel soot per cubic meter. of every daily intake of diesel exhaust soot were the same from the first exposure to the last exposure, 2 years later. The sim ulation fit the data well enough to conclude that the model was reasonable for this repeated exposure to diesel exhaust. The fact that the simulation accounted for the observed patterns of lung accumulation of diesel particles strongly suggests that the assumptions were valid for this exposure concentration and accumulated lung burden of diesel soot. However, it is clear that the assumptions are not valid for comparable exposures of rats to 3.5 or 7 mg diesel soot per cubic meter111 where lung overloading with diesel soot occurred. The agreement between observed and simulated patterns for lung and LALN accumulations of uranium and diesel soot particles lends a degree of confidence to the assumption that appropriate models can be used to make accurate projections, relative to lung and LALN burdens that would result from repeated exposures. D. Equivalent Concentrations of Exposure Material Another way to model pulmonary accumulations of inhaled particles is to select the endpoint lung burden. As an example, 75-p.g particles per gram lung was chosen because it was the specific lung burden projected for humans in Figure 12. Next, the aerosol concentrations needed to reach that burden after 2 years of repeated exposure, 8 h/d, 5 d/week were computed for the other species. Figure 15 presents the results of simu lations for all six species, using the equivalent air concentra tions. This figure indicates (1) the relative air concentrations of this aerosol projected to produce the same pulmonary con centration of particles in the six species, and (2) the patterns that would be projected for accumulations of particles in the P region. It emphasizes the fact that different air concentrations of the test material are needed for different species to produce 1989 203 Critical Reviews In FIGURE 15. Simulation model results for pulmonary accumulation of par ticles in selected species exposed to achieve 75 pg particles per gram lung during a 2-year exposure 8 h/d, 5 d/week to an aerosol containing I-pm mass 'edian aerodynamic particles with a dissolution-absorption half-time of 1000 Results include relative air concentrations for these species exposed to the defined aerosol. the same endpoint lung burden during a repeated exposure. The patterns for accumulation of particles in lung were identical for guinea pigs, monkeys, dogs, and humans because of the assumptions used in the simulations. The actual accumulation patterns may be influenced by air concentrations of exposure materials and by the chemical composition of the exposure material. These simulation projections used the assumption that dep osition and clearance of inhaled materials would be independ ent of the amount of material inhaled, as well as of the amount of material accumulated in the lung. Also, the potential changes in lung accumulation patterns resulting from irritation or toxic effects of the test material were not considered. Results from the literature generally indicate that these assumptions are valid, at least for nuisance dusts or materials with low toxicity, until relatively large quantities (milligrams per gram of lung tissue) of the test material have accumulated in the lung. These examples were for a hypothetical test aerosol and commonly used laboratory animal species. The same process could be used for evaluating and making projections for a variety of test aerosols and additional animal species, including humans. The most important information necessary for these -ejections is ( 1) the deposition and clearance parameters for me species, and (2) the dissolution-absorption characteristics of the test material. These are the factors that dictate the ac cumulation patterns for the inhaled material. IX. SUMMARY This review summarizes contemporary scientific knowl edge relative to the fate of particles inhaled by mammalian species. Processes involved in deposition, retention, and clear ance are similar among species. There are important differences among species in amounts of materials deposited in the various regions of the respiratory tract; the bases for these differences are reasonably well understood. The bases for species differ ences in retention and clearance are not understood. Under standing retention and clearance patterns and the biological mechanisms associated with them is essential to a complete understanding of physiological processes in a given species and for making extrapolations among species. The available data base for laboratory animals contains a wealth of information on single, acute inhalation exposures. The complementary data base for repeated exposures is grow ing, but is still relatively small. It is important that we expand and begin to understand the data base for repeated or chronic inhalation exposures since they are the most likely types of industrial and environmental exposures. Having a means of accurately relating laboratory animal data to the sparse amount of human data now available and likely to be available in the future is vital to inhalation toxi cologists and organizations that establish exposure standards. A desired outcome of this kind of review is a statement as to which animal species is the best surrogate for humans. One aspect of the answer to this question is that there is no standard human. Considerable variability exists as a consequence of race, sex, age, size, health status, and other factors. The same considerations apply to all species. In addition, each species has its unique deposition, retention, and clearance patterns for inhaled materials, as well as potential species-dependent re sponses to inhaled materials or the injury patterns they produce. While no one species can adequately serve as a human sur rogate, the composite data from animal studies can supplement human data to allow reasonable projections about human in halation exposures. ACKNOWLEDGMENTS Portions of the research reviewed in this article were spon sored by the U.S. Department of Energy, Office of Health and Environmental Research under Contract Number DE-AC0476EV01013. The author is indebted to colleagues at the Love lace Inhalation Toxicology Research Institute and associates in the scientific community who produced information used in this review. 204 Volume 20, Issue 3 Toxicology REFERENCES 1. Schreider, J. P. and Ranbe, O. G ., Anatomy of the nasal-pharyngeal airway of experimental animals. Anal. Rec.. 200, 195, 1981. 2. Phalen, R. F., Inhalation Studies, Foundations and Techniques. CRC Press, Inc., Boca Raton, FL, 1984. 3. McLaughlin, R. F., Jr., Tyler, W. S ., and Canada, R. O., Subgross pulmonary anatomy in various mammals and man, JAMA. 175, 148, 1961. 4. McLaughlin, R. F ., Tyler, W. S ., and Canada, R. O ., A study of the subgross pulmonary anatomy in various mammals, Am. J. A nal., 108, 149, 1961. 5. Jeffery, P. K., Morphologic features of airway surface epithelial cells and glands. Am. Rev. Respir. Dis., 128, S14, 1983. 6 Tyler, W. S ., Small airways and terminal units: comparative subgross anatomy of lungs, Am. Rev. Respir. Dis., 128, S32, 1983. 7. Bowden, D. H., Cell turnover in the lung, Am. Rev. Respir. Dis., 128, S46, 1983. 8. Shorter, R. G ., Titus, J. L., and Divertie, M. B., Cell turnover in the respiratory tract, Dis. Chest, 46, 138, 1964. 9. Shorter, R. G ., Titus, J. L., and Divertie, M. B., Cytodynamics in the respiratory tract of the rat. Thorax, 21, 32. 1966. 10. Shorter, R. G ., Cell kinetics o f respiratory tissues, both normal and stimulated, in Morphology o f Experimental Respiratory Carcinogen esis, Netlesheim, P., Hanna, M. G ., Jr., and Deatherage, J. W ., Jr.. Eds., U.S. Atomic Energy Commission, Division of Technical Informaiton, Washington, D.C., 1970, 45. 11. Godleski, J. J. and Brain, J. D., The origin of alveolar macrophages in mouse radiation chimeras. J. Exp. Med., 136, 630, 1972. 12. Coggie, J. E. and Tarling, J. D., Cell kinetics o f pulmonary alveolar macrophages in the mouse, Cell Tissue Kinet., 15, 139, 1982. 13. van Furth, R., Diesselhoff-den Duik, M. M. C ., Sluiter, W ., and van Dissel, J. T., New perspectives on the kinetics of mononuclear phagocytes, in Mononuclear Phagocytes: Characteristics, Physiology and Function, van Furth, R., Ed., Martinus Nijhoff, Dordrecht, The Netherlands, 1985, 201. 14. Bowden, D. H. and Adamson, I. Y. R., Role of monocytes and interstitial cells in the generation of alveolar macrophages. 1. Kinetic studies of normal mice, Lab. Invest., 42, 511, 1980. 15. Adamson, I. Y. R. and Bowden, D. H., Role of monocytes and interstitial cells in the generation of alveolar macrophages. U. Kinetic studies after carbon loading, Lab. Invest.. 42, 518, 1980. 16. Bowden, D. H., Macrophages, dust, and pulmonary diseases, Exp. Lung Res., 12, 89, 1987. 17. Hannsen, A. G., Mason, M. J., Muggenburg, B .'A ., Gillett, N. A., Jarpe, M . A ., and Bice, D. E ., Migration of neutrophils from lung to tracheobronchial lymph node, J. Leukocyte Biol., 4 1 ,9 5 , 1987. 18. Yamada, Y., Cheng, Y.-S., Yeh, H. C., and Swift, D. L., Inspi ratory and expiratory deposition of ultrafine particles in a human nasal cast, Inhal. Toxicol., Premier Issue, I, 1988. 19. Morrow, P. E ., Bates, D. V., Fish, B. R., Hatch, T. F., and Mercer, T. T., Deposition and retention models for internal dosimetry of the human respiratory tract. Health Phys., 12, 173, 1966. 20. Hatch, T. E. and Gross, P., Pulmonary Deposition and Retention o f Inhaled Aerosols, Academic Press, New York, 1964. 21. Heyder, J., Armbruster, L., Gebhart, J., Grein, E., and Stahlbofen, W., Total deposition of aerosol particles in the human respi ratory tract for nose and mouth breathing, J. Aerosol Sci., 6, 311, 1975. 22. Stahlhofen, W., Gebhart, J., and Hyder, J., Experimental deter mination of the regional deposition of aerosol panicles in the human respiratory tract. Am. Ind. Hyg. Assoc. J., 41, 385, 1980. 23 Lippmann, M., Regional deposition of panicles in the human respi ratory tract, in Handbook o f Physiology. Section 9: Reactions to En vironmental Agents, Lee, D. H. K., Falk, H. L., Murphy, S. D., and Geiger, S. R ., Eds., American Physiological Society, Bethesda. MD. 1977, 213. 24. Morrow, P. E., Clearance kinetics o f inhaled panicles, in Respiratory Defense Mechanisms (Pan II), Brain, J. D., Proctor, D. F., and Reid, L. M ., Eds., Marcel Dekker, New York. 1977, 491. 25. Phalen, R., Kenoyer, J., and Davis, J., Deposition and clearance o f inhaled panicles. Comparison of mammalian species, in Proc. Annu Conf. onEnviron. Toxicol., Vol. 7, AMRL-TR-76-125. NTIS,Springfield, VA, 1977, 159. 26. Raabe, O. G., Yeh, H. C., Newton, G. J., Phalen, R. F., and Velasquez, D. J., Deposition of inhaled monodisperse aerosols in small rodents, in Inhaled Particles TV (Part 1), Walton, W. H., Ed.. Per gamon Press, Oxford, UK, 1977, 3. 27. Brain, J. D. and Valberg, P. A., Deposition of aerosol in the res piratory tract, Am. Rev. Respir. Dis., 120, 1325, 1979. 28. Yeh, H. C ., Respiratory Tract Deposition Models, Final Report, LF72, Lovelace Inhalation Toxicology Research Institute. Albuquerque, NM, 1980. 29. Lippmann, M. and Schlesinger, R. B., Interspecies comparisons of panicle deposition and mucociliary clearance in tracheobronchial air ways, J. Toxicol. Environ. Health, 13, 441, 1984 30. Raabe, O . G ., Deposition and clearance of inhaled particles, in Oc cupational Lung Disease, Gee, J. B. L., Morgan, W. K. C., and Brooks, S. M ., Eds., Raven Press, New York, 1984, 1 31. Stuart, B. O ., Deposition and clearance of inhaled particles, Environ. Health Perspect., 55, 369, 1984. 32. Schlesinger, R. B., Comparative deposition of inhaled aerosols in experimental animals and humans: a review, J Toxicol. Environ. Health. 15, 197, 1985, 33. McMahon, T. A ., Brain, J. D., and LeMott, S., Species differences in aerosol deposition, in Inhaled Particles TV (Part 1), Walton, W. H ., Ed., Pergamon Press, Oxford, UK, 1977, 23. 34. Leith, D. E ., Mass transport in mammalian lungs: comparative phys iology, J. Toxicol. Environ. Health. 13, 251, 1984. 35. Svartengren, M., Falk, R., Linn m an, L., Philipson, K., and Camner, P., Deposition of large particles in human lung, Exp. Lung R es., 12, 75, 1987. 36. Snipes, M. B., Olson, T. R., and Yeh, H. C ., Deposition and re tention patterns for 3-, 9-, and 15-p.m latex microspheres inhaled by rats and guinea pigs, Exp. Lung Res.. 14, 37, 1988. 37. Sweeney, T. D., Brain, J. D., and LeMott, S., Anesthesia alters the pattern of aerosol retention in hamsters, J. Appl. Physiol., 54, 37, 1983. 38. Newton, P. E. and Pfledderer, C ., Measurement of the deposition and clearance of inhaled radiolabeled particles from rat lungs, J. Appl Toxicol.. 6, 113, 1986. 39. Lippmann, M., Recent advances in respiratory tract particle deposi tion. in Advances in M odem Environmental Toxicology, Vol. 8, Oc cupational and Industrial Hygiene: Concepts and Methods, Esmen. N. A. and Mehlman, M. A.. Eds., Princeton Scientific Publishers. Princeton, NJ, 1984, 75. 40. Aiarie, Y., Irritating properties of airborne materials to the upper respiratory tract, Arch. Environ. Health, 13, 433, 1966. 41. Bruce, M. C ., Bruce, E. N., Leith, D. E ., and Murphy, S. D., Diethyl maleate and/or ozone (10 ppm) reduce ventilation by 60-- 80% in awake mice (Abstr ), Physiologist, 22, 16, 1979. 42. Silver, E. H., Leith, D. E ., and Murphy, S. D., Potentiation by triorthotolyl phosphate of acrylate ester-induced alterations in respi ration. Toxicology, 22, 193. 1981. 43. Chang, J. C. F ., Steinhagen, W. H ., and B arrow , C. S., Effect of single or repeated formaldehyde exposure on minute volume of B6C3F, mice and F344 rats, Toxicol. Appl. Pharmacol.. 61, 451. 1981. 44. Medinsky, M. A., Dutcher, J. S,, Bond, J. A., Henderson, R. F., 1989 205 Critical Reviews In M auderly, J . L ., Snipes, M . B ., Mewhinney, J . A., Cheng, Y. S., and B irnbaum , L. S ., Uptake and excretion of [l4C]methyl bromide as influenced by exposure concentration, Toxicol. Appl. Pharmacol.. 78. 215, 1985. 45. L ippm ann, M. and A lbert, R. E ., The effect of particle size on the regional deposition of inhaled aerosols in the human respiratory tract. Am. Ind. Hyg. Assoc. J.. 30, 257, 1969. 46. Foord, N., Black, A., and Walsh, M ., Regional deposition of 2.5-- 7.5 M-mdiameter inhaled particles in healthy male non-smokers, J. Aerosol Sri.. 9, 343, 1978. 47. Stahlbofen, W ,, G ebbart, J ., and Heyder, J . , Biological variability of regional deposition of aerosol panicles in the human respiratory tract. Am. Ind. Hyg. Assoc. J.. 42, 348, 1981. 48. C han, T . L. and L ippm ann, M ., Experimental measurements and empirical modelling of the regional deposition of inhaled panicles in humans, Am. Ind. Hyg. Assoc. J.. 41, 399, 1980, 49. Em m ett, P. C ., Aitken, R. J . , and H annan, W. J ., Measurements of the total and regional deposition of inhaled panicles in the human respiratory tract, J. Aerosol Sci., 13, 549, 1982. 50. H eyder, J ., G ebbart, J . , Stahlbofen, W ., and Stuck, B., Biological variability of panicle deposition in the human respiratory tract during controlled and spontaneous mouth-breathing, Ann. Occup. Hyg.. 26, 137, 1982. 51. Cuddthy, R. G ., M cClellan, R. O ., and GrllTItb, W. C ., Variability in target organ deposition among individuals exposed to toxic sub stances, Toxicol. Appl. Pharmacol.. 49, 179, 1979. 52. L undgren, D. L ,, H ahn, F. F ., and M cClellan, R. O ., Effects of single and repeated inhalation exposure of Syrian hamsters to aerosols of '-" CeOj, Radial. Res.. 90, 374, 1982. 53. H okna, B., Scanning electron microscopic observation of particles deposited in the lung, Arch. Environ. Health. 18, 330, 1969. 54. Evans, J . C ., Evans, R. J ., Holmes, A., Hounam, R. F ., Jones, D. M ., M organ, A., and W alsh, M ., Studies on the deposition of inhaled fibrous material in the respiratory tract of the rat and its sub sequent clearance using radioactive tracer techniques. I. UICC crocidolite asbestos, Environ. Res., 6, 180, 1973. 55. Sorokin, S. P. and B rain, J . D ., Pathways of clearance in mouse lungs exposed to iron oxide aerosols, Anal. Rec.. 181. 581, 1975. 56. Brody, A. R. and Roe, M . W ., Deposition pattern of inorganic par ticles at the alveolar level in the lungs of rats and mice. Am. Rev. Respir. Dis., 128, 724, 1983. 57. LaBelie, C. W. and B rieger, H ., Patterns and mechanisms in the elimination of dust from the lung, in Inhaled Particles and Vapours. Davies, C. N., Ed., Pergamon Press, Oxford, UK, 1961, 356. 58. Ferin, J ., U rbankova, G ., and Vlckova, A ., Pulmonary clearance and the function of macrophages. Arch. Environ. Health. 10?*790. 1965. 59. F erin, J . , The mechanism of elimination of deposited particles from the lungs, Ann. Occup. Hyg.. 10, 207, 1967. 60. Sanders, C. L. and Adee, R. R., Phagocytosis of inhaled plutonium oxide-OTPu particles by pulmonary macrophages, Science. 162, 918, 1968. 61. Sanders, C. L. and Adee, R. R ., Ultrastructural localization of in haled OTP u 0 2 particles in alveolar epithelium and macrophages, Health Phys.. 18, 293, 1970. 62. G reen, G. M ., Alveolobronchiolar transport mechanisms. Arch. In tern. Med., 131, 109, 1973. 63. C am ner, P ., H elistroni, P ., and L undborg, M ., Coating 5 p. particles with carbon and metals for lung clearance studies. Arch. Environ. Health, 27, 331. 1973. 64. C am ner, P ., L undborg, M ., and H elistroni, P ., Alveolar macro phages and 5 pm particles coated with different metals. Arch. Environ. Health. 29, 211, 1974. 65. Hibbs, J. B., J r ., T aintor, R. R., Chapm an, H. A., J r., and Weinberg, J. B., Macrophage tumor killing: influence of the local environment. Science. 197, 279, 1977. 66. Gee, J. B. L. and Khandwala, A. S., Motility, transport, and en- docytosis in lung defense cells, in Respiratory Defense Mechanisms (Part II), Brain, J. D., Proctor, D. F., and Reid. L. M .. Eds., Marcel Dekker, New York, 1977, 927. - 67. Lauweryns, J. M. and Baert, J. H., Alveolar clearance and the role of the pulmonary lymphatics. Am. Rev. Respir. Dis.. 115, 625, 1977. 68. Sorokin, S. P., Phagocytes in the lungs: incidence, general behavior, and phylogeny, in Respiratory Defense Mechanisms (Part II), Brain, J. D., Proctor, D. F., and Reid, L. M., Eds., Marcel Dekker, New York, 1977, 711. 69. Berry, J. P., Henoc, P., and Galle, P., Phagocytosis by cells of the pulmonary alveoli. Am. J. Pathol.. 93, 27, 1978. 70. Hocking, W. G. and Golde, D. W., The pulmonary-alveolar mac rophage (first of two parts), N. Engl. J. Med., 301, 580, 1979. 71. Brain, J. D., Macrophages in the respiratory tract, in Handbook o f Physiology. Section J: The Respiratory System. Fishman, A. P .. Fisher, A. B., and Geiger, S. R., Eds., American Physiological Society, Belhesda. MD. 1985, 447. 72. Harmsen, A. G., Muggenburg, B. A., Snipes, M. B., and Bice, D. E., The role of macrophages in particle translocation from lungs to lymph nodes. Science. 230, 1277, 1985. 73. Lehnert, B. E. and Morrow, P. E., Association of 5,iron with alveolar macrophages during alveolar clearance, Exp. Lung. Res.. 9, 1. 1985. 74. Lehnert, B. E. and Morrow, P. E,, Characteristics of alveolar mac rophages following the deposition of a low burden of iron oxide in the lung, J. Toxicol. Environ. Health, 16, 855. 1985. 75. Lehnert, B. E., Valdez, Y. E ., and Bomaiaski, S. H., Lung and pleural "free-cell responses" to the intrapulmonary deposition of par ticles in the rat, J. Toxicol. Environ. Health. 16, 823, 1985 76. Lehnert, B. E., Valdez, Y. E., and Holland, L. M., Pulmonary macrophages: alveolar and interstitial populations. Exp. Lung Res.. 9. 177. 1985. 77. Lehnert, B. E., Valdez, Y. E., and Stewart, C. C ., Translocation of particles to the tracheobronchial lymph nodes after lung deposition: kinetics and particle-cell relationships, Exp. Lung Res.. 10. 245. 1986. 78. Blussc van Oud Albius, A. and van Furth, R., Origin, kinetics, and characteristics of pulmonary macrophages in the normal steady state, J. Exp. Med.. 149, 1504, 1979. 79. Gehr, P., Lung morphometry, in Lung Modelling fo r Inhalation o f Radioactive Materials, Smith, H. and Gerber. G.. Eds.. EUR9384EN. Proceedings of a meeting Jointly organized by the Commission of European Communities and the National Radiological Protection Board. Luxembourg, Office for the Official Publications of the European Com munities. 1984, I. 80. Brain, J. D., Free cells in the lungs: some aspects of their role, quantitation, and regulation. Arch Intern. Med.. 126. 477. 1970. 81. LaBelle, C. W. and Brieger, H., The fate of inhaled particles in the early postexposure period II. The role of pulmonary phagocytosis. Arch. Environ. Health. 1. 423, I960. 82. Gross, P., de TreviUe, R. T. P., Tolker, E. B., Kaschak. M., and Babyak, M. A., The pulmonary macrophage response to irritants: an attempt at quantitation. Arch. Environ. Health. 18. 174, 1969. 83. Bingham, E,, PfUzer, E. A,, Barkley, W., and Radford, E. P., Alveolar macrophages: reduced number in rat after prolonged inhalation of lead sesquioxide. Science. 162. 1297. 1968. 84. Adamson, I. Y. R. and Bowden, D. H., Adaptive response of the pulmonary macrophagic system to carbon. II. Morphologic studies. Lab. Invest.. 38. 430, 1978. 85 Adamson, I. Y. R. and Bowden, D. H.. Dose response of the pul monary macrophagic system to various particulates and its relationship to transepithelial passage of free panicles. Exp. Lung Res . 2. 165. 1981 206 Volume 20, Issue 3 Toxicology 86. Bowden, D. H. and Adamson, I. Y. R., Adaptive responses of the pulmonary macrophagic system to carbon. I. Kinetic studies, Lab Invest., 38, 422, 1978. 87. Brain, J. D., The effects of increased particles on the number of alveolar macrophages, in Inhaled Particles III, Vol. 1, Walton, W. H., Ed., Unwin Brothers Limited, Old Woking, Surrey, UK, 1971, 209. 88. Morrow, P. E ., Possible mechanisms to explain dust overloading of the lungs, Fundam. Appl. Toxicol., 10, 369, 1988. 89. Bingham, E ,, Barkley, W ., Murphy, R., and Vassalk>, ., Inves tigation of alveolar macrophages from rats exposed to coal dust, in Inhaled Particles TV (Part 2), Walton, W. H., Ed., Pergamon Press, Oxford, UK, 1977, 543. 90. Castranova, V., Bowman, L ., Reasor, M. J., Lewis, T ., Tucker, J., and Miles, P. R., The response of rat alveolar macrophages to chronic inhalation of coal dust and/or diesel exhaust, Environ. Res.. 36, 405, 1985. 91. Holt, P. F ., Translocation of asbestos dust through the bronchiolar wall. Environ. Res., 27 , 255, 1982. 92. Hahn, F. F,, Newton, G . J., and Bryant, P. L., In vitro phagocytosis of respirable-sizes monodispersc particles by alveolar macrophages, in Pulmonary Macrophage and Epithelial Cells, Sanders, C. L.. Schnei der, R. P., Dagle, G. E., and Ragan, H. A., Eds., ERDA Series 43. U.S. Department of Commerce. Springfield, VA, 1977, 424. 93. Hohna, B., Lung clearance of mono- and di-disperse aerosols deter mined by profile scanning and whole-body counting: a study on normal and SOj-exposed rabbits, Acta Med. Scand. (Suppl), 474, 1, 1967. 94. Lundborg, M. and Hohna, B., In vitro phagocytosis of fungal spores by rabbit lung macrophages, Sabouraudia, 10, 152, 1972. 95. Camner, P. and Lundborg, M., Alveolar macrophages and Teflon particles coated with carbon and metals, in Pulmonary Macrophage and Epithelial Cells, Sanders, C. L., Schneider, R. P., Dagle, G E., and Ragan, H. A., Eds., Energy Research and Development Admin istration Technical Information Center, Springfield. VA, 1977. 405. 96. Misra, V., Rahman, Q ., and Viswanathan, P. N ., Biochemical changes in guinea pig lungs due to amosite asbestos. Environ. Res.. 16, 55, 1978. 97. Goodglick, L. A. and Kane, A. B., Role of reactive oxygen metab olites in crocidolite asbestos toxicity to mouse macrophages, Cancer Res., 46, 5558, 1986. 98. Wiester, M. J., Dtis, R., and Moore, W., Altered function and histology in guinea pigs after inhalation of diesel exhaust. Environ. Health, 22, 285, 1980. 99. Karagianes, M. T., Palmer, R. F., and Busch, R. H., Effects of inhaled diesel emissions and coal dust in rats. Am. Ind. H%g. Assoc. J., 42, 382, 1981. 100. White, H. G. and Garg, B. D., Early pulmonary response of the rat lung to inhalation of high concentration of diesel particles, J. Appl. Physiol., 1, 104, 1981. 101. Barnhart, M. I., Salley, S. O., Chen, S. -T., and Puro, H., Mor phometric ultrastrucrural analysis of alveolar lungs of guinea pigs chronically exposed by inhalation to diesel exhaust (DE), in Toxico logical Effects o f Emissions from Diesel Engines, Lewtas, J., Ed., Elsevier, New York, 1982, 183. 102. Kaplan, H. L., MacKenzie, W. F., Springer, K. J,, Schreck, R. M., and Vostal, J. J., A subchronic study of the effects of exposure of three species of rodents to diesel exhaust, in Toxicological Effects o f Emissions from Diesel Engines. Lewtas. J., Ed., Elsevier, New York, 1982, 161. 103. Pepelko, W. E ., EPA studies on the toxicological effects of inhaled diesel engine emissions, in Toxicological Effects o f Emissions from Diesel Engines, Lewtas, J., Ed., Elsevier, New York, 1982, 121. 104. Vostal, J. J., Schreck, R. M., Lee, P. S., Chan, T. L., and Soderholm, S. C., Deposition and clearance of diesel particles from the lung, in Toxicological Effects o f Emissions from Diesel Engines, Lew tas, J., Ed., Elsevier, New York, 1982, 143. 105. Vostal, J. J., White, H. J., Strom, K. A., Siak, J. -S., Chen, K. -C ., and Dziedzk, D., Response of the pulmonary defense system to diesel particulate exposure, in Toxicological .Effects o f Emissions from Diesel Engines. Lewtas, J., Ed., Elsevier, New York, 1982,201. 106. Green, F. H. Y., Boyd, R. L., Danner-Rabovsky, J., Fisher, M. J., Moorman, W. J., Ong, T. -M., Tucker, J., Vallyathan, V . , W hong, W.-Z., Zoldak, J., and Lewis, T ., Inhalation studies of diesel exhaust and coal dust in rats, Scand. J. Work Environ. Health, 9, 181, 1983. 107. Strom, K. A., Response of pulmonary cellular defenses to the inhal ation of high concentrations of diesel exhaust, J. Toxicol. Environ. Health. 13. 919, 1984. 108. Mauderly, J. L., Gillett, N. A., Henderson, R. F., Jones, R. K., and McClellan, R. O ., Relationships of lung structural and functional changes to accumulation of diesel exhaust particles. Proc. 6th Int. Symp. on Inhaled Particles, Cambridge, England, September 2 to 9, 1985, in press. 109. Strom, K. A. and Garg, B. D., Retention and clearance of diesel paniculate in the lungs of rats (Abstr ), Toxicologist, 5, 179, 1985. 110. Wolff, R. K., Henderson, R. F., Snipes, M. B., Sun, J. D., Bond, J. A., Mitchell, C. E., Mauderly, J. L., and McClellan, R. O., Lung retention of diesel soot and associated organic compounds, in Carcinogenic and Mutagenic Effects o f Diesel Engine Exhaust, Ishinishi, N., Koizumi, A., McClellan, R. O., and Stber. W ., Eds.. Elsevier, New York, 1986, 199. 111. Wolir, R. K., Henderson, R. F., Snipes, M. B., Griffith, W. C., Mauderly, J. L., Cuddihy, R. G., and McClellan, R. O., Alterations in panicle accumulation and clearance in lungs of rats chronically exposed to diesel exhaust. Fundam. Appl. Toxicol., 9, 154, 1987. 112. Lee, K. P., Henry, N. W., Ill, Trochimowicz, H. J., and Rein hardt, C. F., Pulmonary response to impaired lung clearance in rats following excessive TiO, dust deposition, Environ. Res., 41, 144, 1986. 113. Raabe, O. G., Tyler, W. S., Last, J. A., Schwartz, L. W., Lollini, L. O., Fisher, G. L., Wilson, F. D., and Dungworth, D. L., Studies of the chronic inhalation of coal fly ash by rats. Ann. Occup. Hyg., 26, 189, 1982. 114. Shami, S. G., Silbaugh, S. A., Hahn, F. F., Griffith, W. C., and Hobbs, C. H., Cytokinetic and morphological changes in the lungs and lung-associated lymph nodes of rats after inhalation of fly ash, Environ. Res., 35, 373, 1984. 115. MacFarland, H. N., lllrich, C. E., Martin, A., Krumm, A., Busey, W. M., and Alaric, Y., Chronic exposure of Cynamolgus monkeys to fly ash, in Inhaled Particles HI, Vol. I, Walton, W. H., Ed., Unwin Brothers Limited, Old Woking, Surrey, UK. 1971, 313. 116. MacFarland, H. N., Coate, W. B., Disbennett, D. B., and Ack erman, L. J., Long-term inhalation studies with raw and processed shale dusts, Ann. Occup Hyg.. 26, 213, 1982. 117. Brain, J. D., Toxicological aspects of alterations of pulmonary mac rophage function, Annu. Rev. Pharmacol. Toxicol., 26, 547, 1986. 118. Brody, A. R., Roe, M. W., Evans, J. N., and Davis, G. S-, Dep osition and translocation of inhaled silica in rats: quantitation of particle distribution, macrophage participation, and function. Lab. Invest.. 47, 533. 1982. 119. Kyono, H., Homma, K., Nagatani, T., Watanabe, T., and Kawai, K-, Localization of inhaled lead particles in the rat lung. Ind. Health. 12, 49, 1974. 120. Brody, A. R., Hill, L. H., Adkins, B., Jr., and O'Connor, R. W., Chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages, Am. Rev Respir. Dis.. 123, 670, 1981. 121. Ferin, J. and Feldstein, M. L., Pulmonary clearance and hilar lymph 1989 207 Critical Reviews In node content in rats after particle exposure, Environ. Res.. 16, 342, 1978. 122. Adamson, I. Y. R. and Bowden, D. H., Effects of irradiation on macrophagic response and transport of particles across the alveolar epithelium. Am. J. Pathol.. 106, 40, 1982. 123. Ferin, J . , Effect of particle content of lung on clearance pathways, in Pulmonary Macrophage and Epithelial Cells, Sanders, C. L., Schnei der, R. P., Dagle, G. E., and Ragan, H. A., Eds., Energy Research and Development Adm inistration Technical Information Center, Springfield, VA, 1977, 414. 124. Bowden, D. H. and Adamson, I. Y. R., Bronchiolar and alveolar lesions in the pathogenesis of crocidolite-induced pulmonary fibrosis in mice, J. Pathol., 147, 257, 1985. 125. Bowden, D. H. and Adamson, I. Y. R., Pathways of cellular efflux and particulate clearance after carbon instillation to the lung, J. Pathol , 143, 117, 1984. 126. Did, J. H., Mewhinney, J. A., and Snipes, M. B., Distribution of inhaled " PuO, particles in Syrian hamster lungs. Radial. Res.. 88, 299, 1981. 127 Lundgren, D. L., Damon, E. G ., Did, J. H., and Hahn, F, F., The deposition, distribution and retention of inhaled " ThiO, in the lungs of rats with pulmonary emphysema. Health Phys. .4 0 ,2 3 1 , 1981. 128. Rhoads, K., Mahaffey, J. A., and Sanders, C . L., Distribution of inhaled " PuO, in rat and hamster lung, Health Phys., 42, 645, 1982. 129. Leak, L. V., Pulmonary lymphatics and their role in the removal of interstitial fluids and particulate matter, in Respiratory Defense Mech anisms (Part U), Brain, J. D ., Proctor, D. F.. and Reid, L. M ., Eds., Marcel Dekker, New York, 1977, 631. 130. Leak, L. V. and Jamuar, M. P., Ultrastructure of pulmonary lym phatic vessels. Am. Rev. Respir. Dis.. 128. S59, 1983. 131. Brandeiet, P. J ., Experimental study of the dust-clearance mechanism of the lung. I. Histological study in rats of the intra-pulmonary bronchial route of elimination, Acta Pathol. Microbiol. Scand., Suppl. 175, 1, 1965. 132. Chamberlain, D. W., Nopajarooasri, C., and Simon, G. T., Ul trastructure of the pulmonary lymphoid tissue. Am. Rev. Respir. D is., 108, 621, 1973. 133. Bienenstock, J., Johnston, N ,, and Perey, D. Y. E ., Bronchial lymphoid tissue. 1. Morphologic characteristics. Lab. Invest., 28, 686, 1973. 134. Bienenstock, J ., Johnston, N., and Perey, D. Y. E., Bronchial lymphoid tissue. U. Functional characteristics. Lab. Invest., 28, 693, 1973. 135. Bienenstock, J,, Clancy, R. L,, and Perey, D. Y. E., Bronchus associated lymphoid tissue (BALT): its relationship to m ucosaljm munity, in Immunologies and Infectious Reactions in the Lung, Kirk patrick, C. H. and Reynolds, H. Y., Eds., Marcel Dekker, New York, 1976, 29. 136. Racz, P,, Tenner-Racz, K., Myrvik, Q. N,, and Fainter, L. K., Functional architecture of bronchial associated lymphoid tissue and lymphoepithelium in pulmonary cell-mediated reactions in the rabbit, J. Reticuloendoth. Soc., 22, 59, 1977. 137. Bienenstock, J. and Johnston, N., A morphologic study of rabbit bronchial lymphoid aggregates and lymphoepithelium, Lab. Invest.. 35, 343, 1976. 138. Grcgson, R. L., Davey, M. J., and Prentice, D. E., Bronchusassociated lymphoid tissue (BALT) in the laboratory-bred and wild rat, Rattus norvegicus. Lab. Anim.. 13, 239, 1979. 139. Fournier, M., Vai, F., Derenne, J. P., and Pariente, R., Bronchial lymphoepitheliaJ nodules in the rat. Am. Rev. Respir. Dis.. 116, 685, 1977. <40. G reen, G . M ., Alveolobronchiolar transport; observations and hy pothesis of a pathway, Chest, 59 (Suppl.), IS, 1971. 141. Thomas, R. G ., Tracheobronchial lymph node involvement following inhalation of alpha emitters, in Radiobiology o f Plutonium, Stover, B. J. and Jee, W. S. S., Eds., J. W. Press, Salt Lake City, UT. 1972, 231. 142. Kilburn, K. H., Clearance mechanisms in the respiratory tract, in Handbook o f Physiology, Section 9: Reactions to Environmental Agents, Lee, D. H. K., Falk, H. L., Murphy, S. D., and Geiger, S. R., Eds.,American Physiological Society, Bethesda, MD, 1977, 243. 143. Camner, P., Alveolar clearance, Eur. J. Respir. Dis. Suppl.. 61, 59, 1980. 144. Lippmann, M ., Yeates, D. B., and Albert, R. E., Deposition, re tention, and clearance of inhaled particles, Br. J. Ind. Med., 37, 337, 1980. 145. Jones, J. G ., Clearance of inhaled particles from the alveoli, in Aer osols and the Lung: Clinical and Experimental Aspects. Clarke, S. W. and Pavia, D., Eds., Butterworths, London, 1984, 170. 146. Schlesinger, R. B., Clearance from the respiratory tract, Fundam. Appl. Toxicol., 5, 435, 1985. 147. Morrow, P. E., Alveolar clearance of aerosols. Arch. Intern. Med.. 131. 101, 1973. 148. Mercer, T . T ., On the role of panicle size in the dissolution of lung burdens, Health Phys., 13, 1211, 1967. 149. Mewhinney, J. A., Eidson, A. F., and Wong, V. A., Effect of wet and dry cycles on dissolution of relatively insoluble panicles containing Pu, Health Phys., 53, 377, 1987. 150. Lundborg, M., Lind, B., and Camner, P., Ability of rabbit alveolar macrophages to dissolve metals. Exp. Lung Res. . 1 , 11. 1984. 151. Lundborg, M., Eltliind, A., Lind, B., and Camner, P., Dissolution of metals by human and rabbit alveolar macrophages, Br. J. Ind. M ed., 42. 642, 1985. 152. Snipes, M. B., Boecker, B. B., and McClellan, R. O ., Retention of monodisperse or polydisperse aluminosilicate particles inhaled by dogs, rats, and mice, Toxicol. Appl. Pharmacol., 69, 345, 1983. 153. Cuddihy, R. G., Boecker, B. B., and Griffith, W. C-, Modelling the deposition and clearance of inhaled radionuclides, in Biological Implications o f Radionuclides Released from Nuclear Industries, Vol. 2, 1AEA-SM-237/40, International Atomic Energy Agency, Vienna, Austria, 1979, 77. 154. Oberd6rster, G-, Cox, C ., and Baggs, R., Long term lung clearance and cellular retention of cadmium in rats and monkeys. J. Aerosol Sci., 18, 745, 1987. 155. Bailey, M. R., Kreyling, W. G., Andre, S., Batchelor, A., Black, A., Collier, C. C-> Drossehneyer, E., Ferron, G. A., Foster, P., Haider, B., Hodgson, A., Masse, R., Metivier, H., Moores, S. R., Morgan, A., Muller, H. L., Patrick, G., Pickering, S., Ramsden, D., Stirling, C ., and Talbot, R. J., An interspecies comparison of the translocation of material from lung to blood, Ann. Occup Hyg.. 32. 975, 1988. 156. Reeves, A. L. and Vorwald, A. J., Beryllium carcinogenesis. II. Pulmonary deposition and clearance of inhaled beryllium sulfate in the rat. Cancer Res., 21, 446, 1967. 157. Mewhinney, J. A. and Griffith, W. C ., Models of Am metabolism in Beagles and humans. Health Phys.. 42, 629, 1982 158. Medinsky, M. A. and Kampcik, S. J., Pulmonary retention of (l4C]benzo(a]pyrene in rats as influenced by the amount instilled. Tox icology, 35, 327, 1985. 159. Medinsky, M. A., Cheng, Y. S., Kampcik, S. J., Henderson, R. F., and Dutcber, J. S., Disposition and metabolism of '"C-solvent yellow and solvent green aerosols after inhalation. Fundam Appl Toxicol., 7, 170, 1986. 160. Kanapilly, G . M., Alveolar microenvironment and its relationship to the retention and transport into blood of aerosols deposited in the alveoli, Health Phys.. 32, 89, 1977. 161. Raabe, O. G., Kanapilly, G . M., and Boyd, H. A., Studies of the in vitro solubility of respirable particles of 2MPu and 2WPu oxides and 208 Volume 20, Issue 3 Toxicology an accidentally released aerosol containing " 'Pit, in Inhalation Toxi cology Research Institute Annual Report LF-46, Lovelace Biomedical and Environmental Research Institute, Albuquerque, NM, 1973, 24. 162. Park, J. F., Catt, D. L., Craig, D. K., Olson, R. J., and Smith, V. H., Solubility changes of u *Pu oxide in water suspension and effect on biological behavior after inhalation by Beagle dogs, in Proc. 3rd Int. Cong. Ini. Radiat. Protect. Assoc., Snyder, W. S., Ed., CONF930907-P1, U. S. Atomic Energy Commission, Technical Information Center, Oak Ridge, TN, 1974 , 719. 163. Diet, J. H. and Mewhinney, J. A ., Fragmentation of inhaled " *Pu02 particles in lung. Health Phys., 44, 135, 1983. 164. Stuart, B. O., Promethium oxide inhalation studies, in Pacific North west Laboratory Annual Report fo r 1965 in the Biological Sciences, BNWL-280tn. Battelle-Northwest, Richland, WA. 1966, 56. 165. Whaley, S. L., WotfT, R. K., Muggenburg, B. A., and Snipes, M. B., Mucociliary clearance and particle retention in the maxillary and ethmoid turbinate regions of Beagle dogs, J. Toxicol. Environ. Health. 19, 569, 1986. 166. Patrick, G . and Stirling, C ., The retention of particles in large airways of the respiratory tract, Proc. R. Soc. Land. B. Ser. V, 198, 455, 1977. 167. Stirling, C. and Patrick, G ., The localisation of particles retained in the trachea of the rat, J. Pathol., 131, 309, 1980. 168. Gere, D. J. and Thorne, M. C ., The distribution and clearance of inhaled uranium dioxide particles in the respiratory tract of the rat. in Inhaled Particles TV (Part 1), Walton, W. H., Ed., Pergamon Press, Oxford, UK, 1977, 275. 169. Gore, D. J. and Patrick, G ., The distribution and clearance of inhaled UO; particles on the first bifurcation and trachea of rats, Phys. Med. Biol., 23, 730, 1978. 170. Gore, D. J. and Patrick, G ., A quantitative study of the penetration of insoluble panicles into the tissue of the conducting airways, Ann. Occup. Hyg., 26, 149, 1982. 171. Gore, D. J., The spatial and temporal distribution of inhaled UO; particles in the respiratory tract o f the rat. II. The relative concentration of UO; between the intrapulmonary airways and the pulmonary tissue, Radiat. Res., 93, 276, 1983. 172. Patrick, G ., The retention of various types and sizes of particles in the large airways of the rat: implications for assessing the risk of lung cancer, in Current Concepts in Lung Dosimetry. Fisher, D. R., Ed., PN L-11049, National Technical Information Service, Springfield, VA, 1983, 66. 173. Watson, A. Y. and Brain, J. D., Uptake of iron oxide particles by mouse airway epithelium, Lab. Invest., 40, 450, 1979. 174. Briant, J. K. and Sanders, C. L ., Inhalation deposition and retention patterns of a U-Pu chain aggregate aerosol. Health Phys., 53, 365, 1987. 175. Little, J. B,, Radford, E. P., Jr., McCombs, H. L,, and Hunt, V. R., Distribution of polonium-210 in pulmonary tissues of cigarette smokers, N. Engl. J. Med., 273, 1343, 1965. 176. Radford, E. P. and Martel!, E. A ., Polonium-210:lead-210 ratios as an index of residence times of insoluble particles from cigarette smoke in bronchial epithelium, in Inhaled Particles TV (Part 2), Walton, W. H., Ed., Pergamon Press, Oxford, UK, 1977, 567. 177. Cohen, B. S., Harley, N. H., Schlesinger, R. B., and Lippmann, M., Nonuniform particle deposition on tracheobronchial airways: im plications for lung dosimetry, Ann. Occup. Hyg.. 32, 1045, 1988. 178. Dahl, A. R., Bond, J. A., Petridou-Fischer, J., Sabourin, P. J., and Whaley, S. J., Effects of the respiratory tract on inhaled materials, Toxicol. Appl. Pharmacol.. 93, 484, 1988. 179. Morrow, P. E ., Gibb, F. R., and Gazioglu, K., The clearance of dust from the lower respiratory tract of man. An experimental study, in Inhaled Particles and Vapours 1. Davies, C. N., Ed., Pergamon Press, Oxford, UK, 1967. 351. 180. Spritzer, A. A., Watson, J. A., Auid, J. A., and Guetthoff, M . A ., Pulmonary macrophage clearance: the hourly rates of transfer of pulmonary macrophages to the oropharynx of the rat, Arch. Environ. Health, 17, 726, 1968. 181. Brundetet, P. J., An intra-pulmonary route o f dust elimination in rats, in Inhaled Particles and Vapours II, Davies, C. N., Ed., Pergamon Press, Oxford. UK, 1967, 49. 182. Tucker, A. D., Wyatt, J. H., and Undery, D., Clearance of inhaled particles from alveoli by normal interstitial drainage pathways, J. Appl. Physiol., 35, 719, 1973. 183. Kilburn, K. H-, Clearance zones in the distal lung, Ann. N.Y. Acad. Sei., 221,276,1974. 184. Barry, B. E. and Crapo, J. D., Macrophages are the major cell to increase in the pulmonary interstitium after 3 months exposure to crysotile asbestos, Fed. Proc., 40, 828, 1981. 185. Corry, D., Kuikarni, P., and Lipscomb, M. F ., The migration of bronchoalveolar macrophages into hilar lymph nodes, Am. J. Pathol.. 115. 321. 1984. 186. Snipes, M. B. and McClellan, R. O ., Model for deposition and long term disposition of ,J4Cs-labeled fused aluminosilicate particles inhaled by guinea pigs, in Inhalation Toxicology Research Institute Report, LM F -II5, Muggenburg, B. A. and Sun, J. D., Eds., Albuquerque, NM, National Technical Information Service, Springfield, VA, 1986, 91. 187. Stber, W., Einbrodt, H. J., and Klosterktter, W., Quantitative studies of dust retention in animal and human lungs after chronic inhalation, in Inhaled Particles and Vapours II, Davies, C. N., Ed., Pergamon Press, Oxford, UK. 1967, 409. 188. Carlberg, J. R., Crable, J. V,, LUntiaca, L. P., Norris, H. B., Holtz, J. L., Mauer, P., and Wolowicz, F. R., Total dust, coal, free silica, and trace metal concentrations in bituminous coal miner's lungs. Am. Hid. Hyg. Assoc. J., 32, 432, 1971. 189. Mclnroy, J. F., Stewart, M. W., and Moss, W. D., Studies of plutonium in human tracheobronchial lymph nodes, in Radiation and the Lymphatic System, Ballou, J. E., Ed.. CONF-740930, Energy Research and Development Administration Technical Information Cen ter, Washington, D C ., 1976, 54. 190. Cottier, H., Meister, F., Zlmmennann, A., Kraft, R., Burkhardt, A., Gehr, P., and Poretti, G., Accumulation of anthracotic particles along lymphatics of the human lung: relevance to " hot spot" formation after inhalation of poorly soluble radionuclides. Radiat. Environ. Biophys., 26, 275, 1987. 191. Klosterkttcr, W. and Bnemann, G ., Animal experiments on the elimination of inhaled dust, in Inhaled Particles and Vapours, Davies, C. N., Ed., Pergamon Press, Oxford, UK, 1961, 327. 192. Klosterkttcr, W. and Gooo, F,, Long-term storage, migration and elimination of dust in the lungs of animals, with special respect to the influence of polyvinyl-pyridine-n-oxide, in Inhaled Particles III. Vol. 1. Walton, W. H., Ed., Unwin Brothers Limited, Old Woking, Surrey, UK, 1971, 273. 193. Kpper, R. W., Bianco, A., Gibb, F. R ., Landman, S., and Mor row, P. E,, Uptake and retention of insufflated tantalum by lymph nodes, in Radiation and the Lymphatic System, Ballou, J. E., Ed., CONF-740930, Energy Research and Development Administration Technical Information Center, Washington, D C., 1976, 46. 194. Vostal, J . J ., Chan, T . L ., Garg, B. D., Lee, P. S., and Strom, K . A ., Lymphatic transport of inhaled diesel particles in the lungs of rats and guinea pigs exposed to diluted diesel exhaust. Environ. In ternat., 5, 339, 1981. 195. Vincent, J. H., Jones, A. D., Johnston, A. M., McMillan, C ., Boiton, R. E ., and Cowie, H., Accumulation of inhaled mineral dust in the lung and associated lymph nodes: implications for exposure and dose in occupational lung disease. Ann. Occup. Hyg.. 31, 375. 1987. 196. Thomas, R. G ., Transport of relatively insoluble materials from lung to lymph nodes. Health Phys., 14, 111, 1968. 1989 209 Critical Reviews In 197. Thomas, R. G ., An interspecies model for retention of inhaled par ticles, in Assessment o f Airborne Particles, Fundamentals, Applica tions, and Implications to Inhalation Toxicity, Mercer, T. T., Morrow, P. E., and Stber, W ., Eds., Charles CThomas, Springfield, LL, 1972, 405. 198. LeFevre, M. E., Green, F. H. Y., Joel, D. D., and Laqueur, W., Frequency of black pigment in livers and spleens of coal workers: correlation with pulmonary pathology and occupational information. Hum. Pathol., 13, 1121, 1982. 199. Gearhart. J. M., Did, J. H., and McClellan, R. O ., Intiahepatic distribution of plutonium in beagles. Radial. Res., 84, 343, 1980. 200. Guilmette, R. A., Mggenburg, B. A., Hahn, F. F., Mewhinney, J. A., Seiler, F. A., Boecker, B. B., and McClellan, R. O., Do simetry of " "PuOj in dogs that inhaled monodisperse aerosols of O9P u 0 2, Radial. Res., 110, 199, 1987. 201. Lee, K. P,, Bassas, C. E,, G riffith, F. D,, and Waritz, R. S., Pulmonary response and transmigration of organic fibers by inhalation exposure, Am. J. Pathol., 102, 314, 1981. 202. Hoorihane, D. O'B., A biopsy series of mesotheliomata, and attempts to identify asbestos with some of the tumors, Ann. N.Y. Acad. Sei., 132, 647, 1965. 203. Pooley, F ., Locating fibers in the bowel wall. Environ. Health Perspect.. 9, 235, 1974. 204. Holt, P. F., Transport of inhaled dust to extrapulmonary sites. J. Pathol., 133, 123, 1981. 205. Gross, P. and W estrick, M., The permeability of lung parenchyma to particulate matter. Am. J. Pathol.. 30. 195, 1954. 206. StradUng, G. N., Ham, G. J., Smith, H., Cooper, J., and Breadmore, S. E ., Factors affecting the mobility of plutonium-238 dioxide in the rat, Int. J. Radial. Biol., 34, 37, 1978. 207. Raabe, O . G ., Teague, S. V ., Richardson, N. L ., and Nelson, L. S., Aerodynamic and dissolution behavior of fume aerosols pro duced during the combustion of laser-ignited plutonium droplets in air, Health Phys.. 35, 663, 1978. 208. Lanweryns, J. M. and Baert, J. H., The role of the pulmonary lymphatics in the defenses of the distal lung: morphological and ex perimental studies of the transport mechanisms of intratracheally in stilled particles, Ann. N.Y. Acad. Sei., 221, 244, 1974. 209. Meyer, E. C ., Dominguez, E. A. M., and Bensch, K. G ., Pulmonary lymphatic and blood absorption of albumin from alveoli, Lab. Invest.. 20, 1, 1969. 210. Morrow, P. E., Gibb, F. R., Davies, H., and Fisher, M., Dust removal from the lung parenchyma: an investigation of clearance stim ulants, Toxicol. Appl. Pharmacol., 12, 372, 1968. 211. Morrow, P. E., Gibb, F. R., Beiter, H., and Kpper, R. W., Pulmonary retention of neutron-activated coal dust, Arclr Environ. Health, 34, 178. 1979. 212. Thomas, R. G., Ewing, W. C., Catron, D. L., and McClellan, R. O ., In vivo solubility of four forms of barium determined by scan ning techniques. Am. Ind. Hyg. Assoc. J., 34, 350, 1973. 213. Ferin, J., Lung clearance of particles, in Air Pollution and the Lung. Aharonson, E. F., Ben-David, A.. Klingberg, M. A., and Kaye, M.. Eds., John Wiley & Sons, New York, 1976, 64. 214. Morrow, P. E., Gibb, F. R., and Johnson, L., Clearance of insoluble dust from the lower respiratory tract. Health Phys.. 10, 543, 1964. 215. Moss, O . R. and KanapUly, G. M., Dissolution of inhaled aerosols, in Generation o f Aerosols, Willeke, K., Ed., Ann Arbor Science Pub lishers, Ann Arbor, MI, 1980, 105. 216. Rudd, C. J. and Strom , K. A ., A spectrophotometnc method for the quantitation of diesel exhaust particles in guinea pig lung. J. Appl. Toxicol.. I, 83, 1981. 217. Heinrich, II., Muhle, H., Takenaka, S., Ernst, H., Fuhst, R., Mohr, U., Pott, F., and Stber, W., Chronic effects on the respiratory tract of hamsters, mice and rats after long-term inhalation of high concentrations of filtered and unfiltered diesel engine emissions, J. Appl. Toxicol., 6, 383, 1986. 218. Griffis, L. C., Wolff, R. K., Henderson, R. F., Griffith, W. C ,, Mokler, B. V., and McClellan, R. O., Clearance of diesel soot particles from rat lung after a subchronic diesel exhaust exposure, Fundam. Appl. Toxicol.. 3, 99, 1983. 219. Wotff, R. K., Hanson, R. L., Henderson, R. F., Benson, J. "M., Mauderly, J. L., Hahn, F. F., and Hobbs, C . H., Inhaled panicle accumulation and clearance after exposure to potentially toxic panicles. Am. Rev. Respir. Dis. (Abstr ), 131, A211, 1985. 220. Chan, T . L ., Lee, P . S ., and H ering, W. E ., Pulmonary retention of inhaled diesel particles after prolonged exposures to diesel exhaust, Fundam. Appl. Toxicol., 4, 624, 1984. 221. Bolton, R. E., Vincent, J. H., Jones, A. D., Addison, J., and Beckett, S. T., An overload hypothesis for pulmonary clearance of UICC amosite Fibres inhaled by rats, Br. J. Ind. M ed.. 40, 264, 1983. 222. Vincent, J. H,, Johnston, A. M., Jones, A. D,, Bolton, R. E., and Addison, J., Kinetics of deposition and clearance of inhaled mineral dusts during chronic exposure, Br. J. Ind. Med., 42, 707, 1985. 223. Morrow, P. E ., The setting of particulate exposure levels for chronic inhalation toxicity studies, J. Am. Coll. Toxicol., 6, 533, 1986. 224. Kalliomaki, P. L., Korhonen, O., Vaaranen, V., KalUomaki, K., and Koponen, M ., Lung, retention and clearance of shipyard arc welders, Int. Arch. Occup. Environ. Health, 42, 83, 1978. 225. Rossiter, C. E., Rivers, D., Bergm an, I., Cassweil, C., and Na geischmidt, G ., Dust content, radiology and pathology, in simple pneumoconiosis of coal workers, in Inhaled Panicles and Vapours II. Davies, C. N., Ed., Pergamon Press, Oxford, UK, 1967, 419. 226. Davis, J. M. G., Chapman, J., Codings, P., Douglas, A. N., Fernie, J., Lamb, D., and Ruckley, V. A., Variations in the histological patterns of the lesions of coal workers' pneumoconiosis in Britain and their relationship to lung dust content. Am. Rev. Respir Dis.. 128, 118, 1983. 227. Ruckley, V. A., Gauld, S. J., Chapman, J. S., Davis, J. M. G., Douglas, A. N., Fernie, J. M., Jacobsen, M., and Lamb, D., Emphysema and dust exposure in a group of coal workers. Am. Rev. Respir. Dis.. 129, 528, 1984. 228. Soutar, C. A. and Hurley, J. F ., Relation between dust exposure and lung function in miners and ex-miners, Br. J. Ind. Med.. 43, 307, 1986. 229. Dobreva, M., Burilkov, T., Kolev, K., and Lavoia, P., Character istics of lung dusts and their relation to dust exposure and pathological findings in the lungs, in Inhaled Panicles TV (Part 2), Walton, W. H ., Ed., Pergamon Press, Oxford, UK, 1977, 717. 230. Venna, D. K., Muir, D. C. F,, Stewart, M. L., Julian, J. A., and Ritchie, A. C., The dust content of the lungs of hard-rock miners and its relationship to occupational exposure, pathological and radiological findings. Ann Occup. Hyg., 26, 401, 1982. 2 3 1. McClellan, R. O ., Role of inhalation studies with animals in defining human health risks for vehicle and power plant emissions. Environ. Health Perspect., 47, 283, 1983. 232. Pritsker, A. A. B., The Gasp TV Simulation Language. Wiley, New York, 1974. 233. Schleslnger, R. B., Deposition and clearance of inhaled particles, in Concepts in Inhalation Toxicology. McClellan, R. O. and Henderson, R. F., Eds., Hemisphere Publishing, New York, 1989, 163, 234 Leach, L. J,, Maynard, E. A., Hodge, H. C ,, Scott, J. K,, Yuile, C . L., Sylvester, G. E., and Wilson, H. B., A five-year inhalanon study with natural uranium dioxide (U 0 2) dust. 1. Retention and biol ogic effect in the monkey, dog and rat. Health Phys., 18. 599. 1970. 235. Leach, L. J., Yuile, C. L., Hodge, H. C ., Sylvester, G. E .. and Wilson, H. G -, A five-year inhalation study with natural uraniumdioxide (UOj) dust. II. Postexposure retention and biologic effects in the monkey, dog and rat. Health Phys., 25. 239, 1973 210 Volume 20, Issue 3 Toxicology 236. Snyder, W. S., Cook, M. J., Karhausen, L. R-, Naset, E. S., Howells, G. P., and Tipton, I. H., International Commission on Radiological Protection No. 23, Report o fthe Task Group on Reference Man, Pergamon Press, Oxford, UK, 1975. 237. Bair, W . J ., Deposition, retention, translocation and excretion of radioactive particles, in Inhaled Particles and Vapours, Davies, C. N., Ed., Pergamon Press, Oxford, UK, 1961, 192. 238. Lee, P. S., Chan, T. L., and Hering, W, E ., Long-term clearance of inhaled diesel exhaust particles in rodents, J. Toxicol. Environ. Health, 12, 801, 1983. 239 Bailey, M. R., Hodgson, A., and Smith, H., Respiratory tract re tention of relatively insoluble particles in rodents. J. Aerosol Sci.. 16, 279, 1985. 240. Langham, W. H., Determination of internally deposited radioactive isotopes from excretion analyses. Am. Ind. Hyg. Assoc. Q , 17, 305, 1956. 241. Sanders, C. L., Dagle, G. E., Cannon, W. C., Craig, D. K., Powers, G. J., and Meier, D. M., Inhalation carcinogenesis of high- fired 2" Pu0 2in rats. Radial. Res.. 68, 349, 1976. 242. Galibin, G. P. and Parfenov, Y. D., Inhalation study on metabolism of insoluble uranium compounds, in Inhaled Particles III, Vol. 1, Walton, W. H., Ed., Unwin Brothers Limited, Old Woking. Surry, UK, 1971, 201. 243. Snipes, M. B., Boecker, B. B., and McClellan, R. O ., Respiratory tract clearance of inhaled particles in laboratory animals, in Lung Mo delling fo r Inhalation o f Radioactive Materials, Smith. H. and Gerber, G . , Eds., EUR9384EN, Proceedings of a meeting jointly organized by the Commission of European Communities and the National Radiol ogical Protection Board, Luxembourg, Office for Official Publications of the European Communities, 1984 , 63. 244. Gibb, F. R., Beiter, H. B., and Morrow, P. E., Studies o f Coal Dust Retention in the Lungs Utiliting Neutron-activated Coal. UR- 3490-679, National Technical Information Service, Springfield, VA, 1975. 245. Morrow, P, E. and Yuile, C. L., The disposition of coal dusts in the lungs and tracheobronchial lymph nodes of dogs, Fundam. Appl Toxicol., 2, 300, 1982. 246. Stuart, B. O., Casey, H. W., and Bair, W. J., Acute and chronic effects of inhaled l44C e 0 2 in dogs. Health Phys., 10, 1203, 1964. 247. Boecker, B. B. and McClellan, R. O., The effects of solubility on the bioassay for inhaled radionuclides, in Diagnosis and Treatment o f Deposited Radionuclides, Komberg, H. A. and Norwood. W. D., Eds., Excerpta Medica, New York, 1968, 234 248. Cuddihy, R. G ., Deposition and retention of inhaled niobium in beagle dogs. Health Phys., 34, 167, 1978. ' 249 Bair, W. J,, Willard, D. H., Herring, J. P,, and George, L. A., H, Retention, translocation and excretion of inhaled " 'PuOj, Health Phys., 8, 639, 1962. 250. Morrow, P. E., Gibb, F. R ., Davies, H., Mitola, J., Wood, D., Wraight, N., and Campbell, H. S., The retention and fate of inhaled plutonium dioxide in dogs, Health Phys., 13, 113, 1967. 251. Park, J. F., Bair, W. J., and Busch, R. H., Progress in beagle dog studies with transuranium elements at Battelle-Northwest, Health Phys., 22, 803, 1972. 252. Guilmette, R. A ., Diel, J. H., Muggenburg, B. A., Mewhinney, J. A ., Boecker, B. B., and McClellan, R. O ., Biokinetics of inhaled 2MPu0 2in the Beagle dog: effect of aerosol panicle size, Int. J. Radiat. Biol., 45, 563, 1984. 253. Bianco, A., Gibb, F. R ., Kilpper, R. W., Landman, S., and Mor row, P. E ., Studies of tantalum dust in the lungs. Radiology, 112, 549, 1974. 254. Fish, B. R., Inhalation of uranium aerosols by mouse, rat. dog and man, in Inhaled Particles and Vapours, Davies, C. N., Ed., Pergamon Press, Oxford, UK, 1961, 151. 255. Waligora, S. J . , J r . , Pulmonary retention of zirconium oxide (,5Nb) in man and Beagle dogs. Health Phys., 20, 89, 1971. 256. Nolibe, D., Metivier, H., Masse, R., and LaFuma, J., Therapeutic effect of pulmonary lavage in vivo after inhalation of insoluble radio- active particles, in Inhaled Panicles TV (Part 2), Walton, W. H. and McGovern, B., Eds.. Pergamon Press, Oxford, UK, 1977, 597. 257. LaBauve, R. J ., Brooks, A. L ., M auderly, J . L ., Hahn, F. F., Redman, H. C., Macken, C., Slauson, D, O., Mewhinney, J. A., and McClellan, R. O ., Cytogenic and other biological effects of TM Pu02 inhaled by the Rhesus monkey, Radiat. Res.. 82, 310, 1980. 258. Bailey, M. R., Fry, F. A., and James, A. C ., Long-term retention of particles in the human respiratory tract, J. Aerosol Sci., 16, 295, 1985. 259. Bohning, D. E., Atkins, H. L., and Cohn, S. H., Long-term particle clearance in man: normal and impaired, Ann. Occup. Hyg., 26, 259, 1982. 260. Booker, D. V., Chamberlain, A. C., Rundo, J., Muir, D. C. F., and Thomson, M. L., Elimination of 5 p particles from the human lung. Nature, 215, 30, 1967. 261. Johnson, L. J., Dean, P. N., and Ide, H. M., In vivo determination of the late-phase lung clearance of u ,Pu following accidental exposure, Health Phys., 22, 410, 1972. 262. Ramsden, D., Bains, M. E. D., and Fraser, D. C-, In vivo and bioassay results from two contrasting cases of plutonium-239 inhala tion, Health Phys.. 19, 9. 1970. 263. Newton, D., A case of accidental inhalation of protactinium-231 and actinium-227, Health Phys., 15, 11, 1968. 264. Philipson, K., Falk, R., and Camner, P., Long-term lung clearance in humans studied with Teflon particles labeled with chromium-51. Exp. Lung Res., 9, 31, 1985. 1989 211