# Chapter 4. Dosimetry

4.1. Introduction

Particle dosimetry refers to the characterizati on of deposition, translocation, clearance, and retention of particles and their constituents within the respiratory tract and extrapulmonary tissues. This chapter summarizes basic concepts presented in dosimetry chapters of the 1996 and 2004 PM AQCDs (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ), and updates the state of the science based upon new literature appearing since pub lication of these PM AQCDS. Although the basic understanding of the mechanisms governing depositi on and clearance of inhaled particles has not changed, there is significant additional informati on on the role of certain biological determinants such as gender, age and lung disease on deposition and clearance. Additionally, new studies have further characterized the retention and translocat ion of ultrafine particles (UFPs; also commonly referred to as nanoparticles) following deposition in the respiratory tract. The dose from inhaled particles deposited and retained in the respiratory tract is governed by a number of factors. These include exposure conc entration and duration, activity and ventilatory parameters, and particle properties (e.g., particle si ze, hygroscopicity, and solubility in airway fluids and cellular components). The basic characteristics of particles as they relate to deposition and retention, as well as anatomical and physiologi cal factors influencing particle deposition and retention, were discussed in depth in Chapter 10 of 1996 PM AQCD and updated in Chapter 6 of the 2004 PM AQCD. Species differences between huma ns and rats in particle exposures, deposition patterns, and pulmonary retention were also reviewed in Brown et al. (2005, 089308 ). The current review of PM dosimetry focuses mainly on issues that may affect the susceptibility of an individual to adverse effects as well as issues that affect our ability to extrapolate findings between studies (e.g., in vitro to in vivo) and between species. Other than a brief overview in this introductory section, the disposition (i.e., depositi on, absorption, distribution, me tabolism, and elimination) of fibers and unique nano-objects (viz., dots, hollow sphe res, rods, fibers, tubes) is not reviewed herein. Substantial exposures to fibers and unique nano-obj ects generally occur in the occupational settings rather than the ambient environment. The deposition by interception of micro-sized fibers was briefly discussed in the 1996 and 2004 PM AQCD, but fiber retention in the respirato ry tract was not addressed. Airborne fibers (length/diameter ratio ≥ 3), can exceed 150 μ m in length and appear to be relatively stable in air. This is because their aerodynamic size is determ ined predominantly by their diameter, not their length. Fibers longer than 10 μ m can deposit by interception and when aligned with the direction of airflow may penetrate deep into the respiratory tract. Once deposited, macrophage mediated clearance is the primary mechanism of removing micro-sized particles from the pulmonary region. The length of fibers can, however, affect their phagocytosis and clearance. For example, fibers of >17 μm in length are too long to be fully engulfe d by rat alveolar macrophages and can protrude from macrophages (i.e., macrophage frustra tion) (Zeidler-Erdely et al., 2006, 190967 ). Further discussion of the fiber disposition in the respiratory tract is beyond the scope of this chapter. The term “ultrafine particle” has traditionally been used by the aerosol research and occupational and environmental health communities to describe airborne particles or other laboratory generated aerosols used in toxicological studies that are <100 nm in size (based on physical size, diffusivity, or electrical mobility). Generally consistent with the definition of an UFP, the International Organization for Standardization (ISO ) recently defined a nanoparticle as an object with all 3 external dimensions in the nanoscale, i.e., from approximately 1 and 100 nm (ISO, 2008, 190066 ). The ISO also defined a nano-object as a material with one or more external dimensions in

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and

Environmental Research Online) at http://epa.gov/hero . HERO is a database of scientific literature used by U.S. EPA in the process of developing science assessments such as the Integrated Science Assessments (ISA) and the Integrated Risk Information System (IRIS)

.

December 2009 4-1the nanoscale. The terms, nanoparticle and UFP, ha ve been used rather synonymously in the recent literature. However, the terms nanoparticle a nd nano-object are more commonly associated with engineered materials that are created for consum er products and industrial applications. With the current interest in nanotechnologies, many nano-obj ects have been created by manipulating materials at the atomic or molecular scale for the purpose of forming new materials, structures, and devices that exploit the unique physical and chemical properties associated with their nanoscale. Toxicological studies are becoming available that evaluate in vivo translocation and heath effects unique of nano-objects (viz., dots, hollow spheres, rods, tubes). The in vivo disposition of these unique nano-objects is not, however, necessarily releva nt to the behavior of UF aerosols in the urban environment that are created by combustion source s and photochemical formation of secondary organic aerosols. Therefore, the disposition of uni que nano-objects (viz., dots, hollow spheres, rods, fibers, tubes) is not considered in this chapter.



## 4.1.1. Size Characterization of Inhaled Particles

Particle size is a major determinant of the fraction of inhaled particles depositing in and cleared from various regions of the respiratory tract. The distribution of particle sizes in an aerosol is typically described by the lognormal distribution (i.e., the situation in which the logarithms of particle diameter are distributed normally). The ge ometric mean is the median of the distribution, and the variability around the median is the geometric standard deviation (GSD or σ g ) and is given by:

%16 %50

%50 %84 

## d d

d d GSD

g 

## === σ

d i , : Equation 4-1

where: d 16% , d 50% , d 84% are the particle diameters associated with the 16th, 50th (i.e., the median), and the 84th percentiles from the cumulative frequen cy distribution of particle sizes. By definition, GSD must be greater than one. The particle size a ssociated with any percentile of the distribution, is given by



### ( ) Pz

g i 

## dd σ %50 

## =

Equation 4-2

where: z(P) is the normal standard deviate for a given probability. In most cases, the aerosols to which people are naturally exposed are polydisperse. By contrast, most experimental studies of particle deposition and clearance in the lung use monodisperse particles (GSD <1.15). Ambient aerosols may also be composed of multiple size mo des, each mode should be described by its specific median diameter and GSD. Aerosol size distributions may be measured and described in various ways. When a distribution is described by counting particles, the median is called the count median diameter (CMD). On the other hand, the median of a distri bution based on particle mass in an aerosol is the mass median diameter (MMD). Impaction and sedime ntation of particles in the respiratory tract depend on a particle’s aerodynamic diameter (d

ae ), which is the size of a sphere of unit density that has the same terminal settling velocity as the partic le of interest. The size distribution is frequently described in terms of d

ae as the mass median aerodynamic diameter (MMAD), which is the median of the distribution of mass with respect to aerodyn amic equivalent diameter. Alternative descriptions should be used for particles with actual physical sizes below ≈ 0.5 μm because, for these sized particles, aerodynamic properties become less important and diffusion becomes ever more important. For these smaller particles, their physical diameter or CMD are typically used since diffusivity is not a function of particle density. For small irregular sh aped particles and aggregates, the diameter of a spherical particle that has the same diffusion coef ficient in air as the particle in question is appropriate.

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## 4.1.2. Structure of the Respiratory Tract

The basic structure of the human respiratory tract is illustrated in Figure 4-1 . In the literature, the terms extrathoracic (ET) region and upper airways are used synonymously. The term lower airways is used to refer to the intrathoracic airways, i.e., the combination of the tracheobronchial (TB) region which is the conducting airways and the alveolar region which is the functional part or parenchyma of the lung. A recent review of intersp ecies similarities and differences in the structure and function of the respiratory tract is provided by Phalen et al. (2008, 156865 ). Although the structure varies, the illustrated anatomic regi ons are common to all mammalian species with the exception of the respiratory bronchioles. Respiratory bronchioles, the transition region between ciliated and fully alveolated airways, are foun d in humans, dogs, ferrets, cats, and monkeys. Respiratory bronchioles are absent in rats and mice and abbreviated in hamsters, guinea pigs, oxen, sheep, and pigs. The branching structure of the cili ated bronchi and bronchioles also differs between species from being a rather symmetric and dichotomous branching network of airways in humans to a more monopodial branching network in other mammals.

Source: Based on ICRP (1995, 006988 ).



### Figure 4-1. Diagrammatic representation of respiratory tract regions in humans. Structures are anterior nasal passages, ET 1 

### ; oral airway and posterior nasal passages, ET

T 2 

### ; bronchial airways, BB; bronchioles, bb; and alveolar interstitial, AI.

Another species difference relevant to par ticle dosimetry is the route of breathing. For instance, rodents are obligate nose breathers, whereas most humans are oronasal breathers who breathe through the nose when at rest and increasingly through the mouth with increasing activity

December 2009 4-3level. There is inter-individual variability in th e route by which people breathe. Most people, 87% (26 of 30) in the Niinimaa et al. (1981, 071758 ) study, breathed through their nose until an activity level was reached where they switc hed to oronasal breathing. Thirteen percent (4 of 30) of the subjects, however, were oronasal breathers even at rest. These two subject groups are commonly referred to in the literature (e.g. ICRP, 1995, 006988 ) as “normal augmenters” and “mouth breathers,” respectively. In contrast to healthy subjects, Chadha et al. (1987, 037365 ) found that the majority (11 of 12) of patients with asthma or allergic rhinitis breathe oronasally even at rest.

Source: Panel (a) reprinted with permissi on from McGraw Hill (Fishman and Elias, 1980, 156436 )



### Figure 4-2. Structure of lower airways with progression from the large airways to the alveolus. Panel (a) illustrates basic airway anatomy. Structures are epithelial cells, EP; basement membrane, BM; smooth muscle cells, SM; and fibrocartilaginous coat, FC. Panel (b) illustrates the relative amounts of liquid, tissue, and blood with distal progression.

December 2009 4-4The site of particle deposition within the respi ratory tract has implications related to lung retention and surface dose of particles as well as po tential systemic distribution of particles or their constituents. Figure 4-2 illustrates the progressive change in airway anatomy with distal progression into the lower respiratory tract. In the bronchi ther e is a thick liquid lining and mucociliary clearance rapidly moves deposited particles toward the mouth. In general, in the bronchi, only highly soluble materials moving from the air into the liquid laye r will have systemic access via the blood. With distal progression, the protective liquid lini ng diminishes and clearance rates slow. Soluble compounds and some poorly soluble UFPs may cross the air-liquid interface to enter the tissues and the blood especially in the alveolar region.



# 4.2. Particle Deposition

Inhaled particles may be either exhaled or de posited in the ET, TB, or alveolar region. A particle becomes deposited when it moves from the airway lumen to the wall of an airway. The deposition of particles in the respiratory tract depe nds primarily on inhaled particle size, route of breathing (nasal or oronasal), tidal volume (V T ), breathing frequency ( f ), and respiratory tract morphology. The distinction between air passing through the nose versus the mouth is important since the nasal passages more effectively remove i nhaled particles than the oral passage. Respiratory tract morphology, which affects particle transport and deposition, varies between species, the size of an animal or human, and health status. The fraction of inhaled aerosol becoming deposited in the human respiratory tract has been measured experimentally. Studies, using light scatte ring or particle counting techniques to quantify the amount of aerosol in inspired and expired breaths, have characterized total particle deposition for varied breathing conditions and particle sizes. The vast majority of in vivo data on the regional particle deposition has been obtained by scintigraphic methods where external monitors are used to measure gamma emissions from radiolabeled partic les. These scintigraphic data have shown highly variable regional deposition with sites of highly lo calized deposition or “hot spots” in the obstructed lung relative to the healthy lung. Even in the hea lthy lung, “hot spots” occur in the region of airway bifurcations. Mathematical models aid in predicti ng the mixed effects of particle size, breathing conditions, and lung volume on total and regional deposition. Experimentally, however, there is considerable inter-individual variability in total an d regional deposition even when inhaled particle size and breathing conditions are strictly controlled. Section 4.2.4 on Biological Factors Modulating Deposition provides more detailed information on f actors affecting deposition among individuals. In order to potentially become deposited in the re spiratory tract, particles must first be inhaled. The inspirable particulate mass fraction of an aerosol is that fraction of the ambient airborne particles that can enter the uppermost respiratory tract compartment, i.e., the head (Soderholm, 1985, 156992 ). The American Conference of Government al Industrial Hygienists (ACGIH) and the International Commission on Radiological Protection (ICRP) have established inhalability criteria for humans (ACGIH, 2005, 156188 ; ICRP, 1995, 006988 ). These criteria are indifferent to route of breathing and assume random orientation with re spect to wind direction. They are based on experimental inhalability data for d

ae ≤ 100 μ m at wind speeds of between 1 and 8 m/s. For the ACGIH criterion, inhalability is 97% for an d

ae = 1 μ m, 87% for an d

ae = 5 μ m, 77% for an d

ae = 10 μ m, and plateaus at 50% for d

ae above ~40 μ m. The ICRP criterion, which also plateaus at 50% for very large d

ae , does not become of real importance until an d

ae = 5 μ m where inhalability is 97%. Dai et al. (2006, 156377 ) reported slightly lower nasal particle inhalability in humans during moderate exercise than rest (e.g., 89.2 versus 98.1% for 13 μ m particles, respectively). Nasal particle inhalability is similar between an adult and 7-year-old child (Hsu and Swift, 1999, 155855 ). Inhalability into the mouth from calm air in humans also becomes important for d

ae >10 μ m (Anthony and Flynn, 2006, 155659 ; Brown, 2005, 156299 ). Unlike the inhalability from high wind speeds which plateaus at 50% for d

ae greater than ~40 μ m, particle inhalability from calm air continues to decrease toward zero with increasing d

ae . Inhalability data in laboratory animals, suc h as rats, are only available for breathing from relatively calm air (velocity ≤ 0.3 m/s). For nasal breathing, i nhalability becomes an important consideration for d

ae of above 1 μ m in rodents and 10 μ m in humans (Ménache et al., 1995, 006533 ). The inhalability of particles having d

ae of 2.5, 5, and 10 μ m is 80, 65, and 44% in rats, respectively, whereas it only decreases to 96% for an d

ae of 10 μ m in humans during nasal breathing (Ménache et

December 2009 4-5al., 1995, 006533 ). Asgharian et al. (2003, 153068 ) suggested that an even more rapid decrease in inhalability with increasing d

ae may occur in rats. Inhalability is a particularly important consideration for rodent exposures. Section 4.2.3 provides additional discussion of interspecies patterns of particle deposition.



## 4.2.1. Mechanisms of Deposition

Particle deposition in the lung is predominantly governed by diffusion, impaction, and sedimentation. Most discussion herein focuses on these three dominant mechanisms of deposition. Simple interception, which is an important mechanism of fiber deposition, is not discussed in this chapter. Electrostatic and thermophoretic forces as mechanisms of deposition have not been thoroughly evaluated and receive limited discussion. Some generalizations with regard to deposition by these mechanisms follows, but should not be viewed as absolute rules. Both experimental studies and mathematical models have demonstrated that breathing patterns can dramatically alter regional and total deposition for all sized particles. The co mbined processes of aerodynamic and diffusive (or thermodynamic) deposition are important for particles in the range of 0.1 μ m to 1 μ m. Aerodynamic processes predominate above and thermodynamic processes predominate below this range. Diffusive deposition, by the process of Brownian diffusion, is the primary mechanism of deposition for particles having physical diameters of less than 0.1 μ m. For particles having physical diameters of roughly between 0.05 and 0.1 μ m, diffusive deposition occurs mainly in the small distal bronchioles and the pulmonary region of the lung. However, with further decreases in particle diameter below ~0.05 μ m, increases in particle diffusivity shift more deposition proximally to the bronchi and ET regions. Governed by inertial or aerodynamic propertie s, impaction and sedimentation increase with d

ae . When a particle has sufficient inertia, it is una ble to follow changes in flow direction and strikes a surface thus depositing by the process of impa ction. Impaction occurs predominantly at bifurcations in the proximal airways, where linear velocities and secondary eddies are at their highest. Sedimentation, caused by the gravitational se ttling of a particle, is most important in the distal airways and pulmonary region of the lung. In these regions, residence time is the greatest and the distances that a particle must travel to reach the wall of an airway are minimal. The electrical charge on some particles may resu lt in an enhanced deposition over what would be expected based on size alone. With an estimated charge of 10-50 negative ions per 0.5 μ m particle, Scheuch et al. (1990, 006948 ) found deposition in humans (V T = 500 mL, f = 15 min -1 ) to increase from 13.4% (no charge) to 17.8% (charged). This increase in deposition is thought to result from image charges induced on the surface of the airway by charged particles. Yu (1985, 006963 ) estimated a charge threshold level above which deposition fractions would be increased of about 12, 30, and 54% for 0.3, 0.6, and 1.0 μ m diameter particles, respectiv ely. Electrostatic deposition is generally considered negligible for particles below 0.01 μ m because so few of these particles carry a charge at Boltzmann equilibrium. This mechanis m is also thought to be a minor contributor to overall particle deposition, but it may be important in some laboratory studies due to specific aerosol generation techniques such as nebulization. Laborat ory methods such as passage of aerosols through a Kr-85 charge neutralizer prior to inhalation are commonly used to mitigate this effect. The National Radiological Protection Board (N RPB) recently evaluated the potential for corona discharges from high voltage power lines to charge particles and enhance particulate doses (NRPB, 2004, 156815 ). They concluded that electrostatic effects would be the most important for particles in the size range from about 0.1-1 μ m, where deposition may theoretically increase by a factor of three to ten. However, given that onl y a small fraction of ambient particles would pass through the corona to become charged, the small range of relevant particle sizes (0.1-1 μ m), and the subsequent required transport of ch arged particles to expose indivi duals; the NRPB concluded that effects, if any, of electric fields on particle deposi tion in the human respiratory tract would likely be minimal. Thermophoretic forces on particles occur due to temperature differences between respired air and respiratory tract surfaces. Temperature gradients of around 20°C are thought to produce sufficient thermophoretic force to oppose diffusive and electrostatic deposition during inspiration and to perhaps augment deposition by these mechanisms during expiration (Jeffers, 2005, 156608 ). Thermophoresis is only relevant in the extrathoraci c and large bronchi airways and reduces to zero as the temperature gradient decreases deeper in the lung. Theoretical analysis of thermophoresis has been done for smooth walled tubes and is important over distances that are several orders of

December 2009 4-6magnitude smaller than the diameter of the trachea. The alteration of the flow patterns by airway surface features such as cartilaginous rings may af fect particle transport and deposition over far greater distances than thermophoretic force.



## 4.2.2. Deposition Patterns

Knowledge of sites where particle s of different sizes deposit in the respiratory tract and the amount of deposition therein is necessary for und erstanding and interpreting the health effects associated with exposure to partic les. Particles deposited in the various respiratory tract regions are subjected to large differences in clearance mechan isms and pathways and, consequently, retention times. Deposition patterns in the human respiratory tract were described in considerable detail in dosimetry chapters of prior PM AQCD (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ); as such, they are only briefly described here. Predicted total and regional deposition for an adult male during rest and light exercise are illustrated in Figure 4-3 and Figure 4-4 , respectively. Note that a large proportion of inhaled coarse particles in the 3-6 μ m (d

ae ) range can reach and deposit in the lo wer respiratory tract, particularly the TB airways. Although these figures were provi ded in Chapter 6 of the 2004 PM AQCD, they are reproduced here to illustrate changes in deposi tion as a function of particle size and breathing conditions. The predictions were based on two publicly available particle deposition models, the ICRP (1995, 006988 ) and the Multi-Path Particle Dosime try model (MPPD; Version 1.0, ©2002). The ICRP (1995, 006988 ) model was implemented by Lung Dose Evaluation Program (LUDEP; Version 2.07, June 2000). The MPPD 1 model was developed by the CIIT Centers for Health Research with support from the Dutch National Instit ute of Public Health and the Environment.



### Figure 4-3. Comparison of total and regional deposition results from the ICRP and MPPD models for a resting breathing pattern (V T 

### = 625 mL, f = 12 min -1 

### ) and corrected for particle inhalability. Regions are extrathoracic, ET; tracheobronchial, TB; and alveolar, A. Panels a-b are for nose breat hing; panels c-d are for mouth breathing.

1 For more information about this model, the reader is referred to: http://www.ara.com/products/mppd_capabilities.htm

.

December 2009 4-7

### Figure 4-4. Comparison of total and regional deposition results from the ICRP and MPPD models for a light exercise breathing pattern (V T 

### = 1250 mL, f = 20 min -1 

### ) and corrected for particle inhalability. Regions are extrathoracic, ET; tracheobronchial, TB; and alveolar, A. Panels a-b are for nose breathing; panels c-d are for mouth breathing.



## 4.2. 2.1. Tota l Respiratory Tract Deposition

The efficiency of deposition in the respirat ory tract may generally be described as a “U-shaped” curve on a plot of deposition efficien cy versus the log of particle diameter. Total deposition shows a minimum for particle diameters in the range of 0.1 to 1.0 μ m, where particles are small enough to have minimal sedimentation or impaction and sufficiently large so as to have minimal diffusive deposition. Total deposition does not decrease to zero for any sized particle, in part, because of mixing between particle laden tid al air and residual lung air. The particles mixed into residual air remain in the lung following a br eath and are removed on subsequent breaths or gradually deposited. Total deposition approach es 100% for particles of roughly 0.01 μ m (physical diameter) due to diffusive deposition and for particles of around 10 μ m (d

ae ) due to the efficiency of sedimentation and impaction. Total human lung deposition, as a function of particle size, is depicted in Figure 4-5 . These experimental data were obtained by using monodispe rse spherical test particles in healthy adults during controlled breathing on a mouthpiece. Despite the control of inhaled particle size and breathing conditions, this figure illustrates variab ility in deposition efficiencies due to interindividual differences in lung size and anatomical variability in airway dimensions and branching patterns.

December 2009 4-8Source: Data from Kim and Hu (1998, 086066 ) and Kim and Jaques (2000, 012811 ).



### Figure 4-5. Total lung deposition measured in healthy adults (UF, 11 M, 11 F, 31 ± 4 yr; fine and coarse, 11 M, 11 F, 25 ± 4 yr) during controlled breathing on a mouthpiece. Deposition calculated from aerosol bolus measurements between 50 and 500 mL into a breath with 50 mL increments. Illustrated data are means and standard errors. Asterisk indicates significantly greater total deposition in females versus males.



## 4.2. 2.2. Extr athoracic Region

The first line of defense for protecting the lowe r respiratory tract from i nhaled particles is the nose and mouth. Particle deposition in the ET regi on, especially the nasal passages, reduces the amount available for deposition in the TB and alveolar regions. Recent data have become available, but are largely derived from computational fl uid dynamics (CFD) modeling and experimental measurements in casts. As most of these studies do not substantially improve our understanding of deposition in the ET region they are not reviewed here. For particles >1 μ m d

ae , deposition efficiency in the oral and nasal passages has been generally described as a function of an impaction parameter (Stokes number) with the addition of a flow regime parameter (Reynolds number) for th e oral passages (Finlay and Martin, 2008, 155776 ; Grgic et al., 2004, 155810 ; Kelly et al., 2005, 155894 ; Schroeter et al., 2006, 156076 ). For an adult male, the CFD simulations of Schroeter et al. (2006, 156076 ) predicted nasal deposition of 10 μ m d

ae particles was 90%, and 100% for a V E of 7.5 L/min (rest) and 15 L/min (light activity), respectively. Thus, relatively few large coarse particles will pa ss through the nasal passages into the lungs. Since the nasal passages are more efficient at removing inhaled particles than the oral passage, an individual’s mode of breathing (i.e., oral versus nasa l) influences the quantity of particles penetrating to the lung. In limited studies, it has been shown that childre n tend to have more oral breathing both at rest and during exercise and also displayed more vari ability than adults (Becquemin et al., 1999, 155679 ; Bennett et al., 2008, 156269 ; James et al., 1997, 042422 ). In contrast to adults, there is little data on the uptake of particles for oral or nasal breathing in children. Theoretical calculations by Xu and Yu (1986, 072697 ) predict enhanced deposition of particles (>2 μ m) in the head region for children when compared to adults. Studies of fine particle deposition in physical models of the nose, scaled to adult versus children sizes, predict that deposition efficiency in the nose is a function of pressure drop across the nose (Phalen et al., 1989, 156023 ). Consequently, these model analyses suggest that, when properly scaled physiological flows are used in the calculation of nasal deposition, children, who have higher nasal resistance than adults, should have higher nasal deposition compared to adults. Surprisingly, the few studies reporting meas ures of nasal deposition in children, found lower nasal deposition efficiencies for fine particles (1-3 μ m d

ae ) as compared to adults, despite their higher nasal resistances (Becquemin et al., 1991, 009187 ; Bennett et al., 2008, 156269 ). These findings of lesser nasal versus oral breathing and less efficient nasal deposition suggest that children’s lower respiratory tract (i.e., the TB an d alveolar regions) may receive a higher dose of

December 2009 4-9ambient PM compared to adults. Normalized to lung surface area, the dose rate to the lower airways of children versus adults is increased further becau se children breathe at higher minute ventilations relative to their lung volumes (see Section osition).



## 4.2. 4.2.4.2 on age as a factor modulating dep



## 2.3. Trac heobronchial and Alveolar Region

Inhaled particles passing the ET region enter a nd may become deposited in the lungs. For any given particle size, the pattern of particle deposition influences clearance by partitioning deposited material among lung regions. Deposition in the tr acheobronchial airways and alveolar region cannot be directly measured in vivo. Much of the available deposition data for the TB and alveolar regions have been obtained from experiments with radioact ively labeled, poorly soluble particles (U.S. EPA, 1996, 079380 ) or by use of aerosol bolus techniques (U.S. EPA, 2004, 056905 ). In general, the ability of these experimental data to define specific sites of particle deposition is limited to anatomically large regions of the respiratory tract such as the head, larynx, bronchi, bronchioles, and alveolar region. Mathematical modeling can provide more refined predictions of deposition sites. Comparisons of the modeling results obtained with two publicly available models were provided in Figure 4-3 and Figure 4-4 . Highly localized sites of deposition within the bronchi are described in Section 4.2.2.4 . Both experimental and modeling techniques are based on many assumptions that may be relatively good for the healthy lung but not for the diseased lung. For discussion of these issues, the reader is referred to Sections 4.2.4.4 and 4.2.4.5

.



## 4.2. 2.4. Loca lized Deposition Sites

From a toxicological perspective, it is important to realize that not all epithelial cells in an airway will receive the same dose of deposited particles. Localized deposition in the vicinity of airway bifurcations has been analyzed using experi mental and mathematical modeling techniques. In the 1996 PM AQCD, experimental data were availa ble illustrating the peak deposition of coarse particles (3, 5, and 7 μ m d

ae ) in daughter airways during inspiration and the parent airway during expiration, but always near the carinal ridge (Kim and Iglesias, 1989, 078539 ; Kim et al., 1989, 078538 ). In the 2004 PM AQCD, mathematical models predicted distinct “hot spots” of deposition in the vicinity of the carinal ridge for both coarse (10 μ m) and UF (0.01 μ m) particles (Heistracher and Hofmann, 1997, 047514 ; Hofmann et al., 1996, 047515 ). In a model of lung generations 4-5 during inspiration, hot spots occurred at the carinal ridge for 10 μ m d

ae particles due to inertial impaction and for 0.01 μ m particles due to secondary flow patte rns formed at the bifurcation. During expiration, preferential sites of deposition for bot h particle sizes occurred 1) approaching the juncture of daughter airways on the walls formin g and across the lumen from the carinal ridge; and 2) the top and bottom (visualizing the Y-shaped geometry laying horizontal) of the parent airway downstream of the bifurcation. Recent studies further support these findings (Balashazy et al., 2003, 155671 ; Farkas and Balásházy, 2008, 157358 ; Farkas et al., 2006, 155771 ; Isaacs et al., 2006, 155861 ). Most of these studies quantified localized deposition in terms of an enhancement factor. Typically, the enhancement factor is the ratio of the deposition in a pre-specified surface area (e.g., 100 × 100 μ m which corresponds to ~10 × 10 epithelial cells) to the average deposition density for the whole airway geometry. These enhancement factors are ve ry sensitive to the size of the surface considered (Balashazy et al., 1999, 043201 ). The studies by Farkas et al. (2006, 155771 ) and Farkas and Balásházy (2008, 157358 ) investigated the phenomena of localized deposition down to 0.001 μ m particles. The deposition of 0.001 μ m was rather uniform, however, the deposition pattern became increasingly less uniform with increasing particle size. These studies indicate that, for particles greater than ~0.01 μ m, some cells located near the carinal ridge of bronchial bifurcations may receive hundreds to thousands times the average dose (particles per unit surface area) of the parent and daughter airways. Furthermore, the inertial impaction of particles ≥ 1 μ m d

ae at the carinal ridge of large bronchi will increase with increasing inspiratory flows. In a comparison of constricted versus healthy airways, Farkas et al. (2006, 155771 ) also reported that the overall deposition efficiency of 10 μ m d

ae particles at bifurcations downstream of a constriction may be increased by 18 times. Given these considerations, Phalen and Oldham (2006, 156024 ) noted that substantial doses of particles ( ≥ 1 μ m d

ae ) may be justified for in vitro st udies using tracheobronchial epithelial cell cultures.

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## 4.2.3. Interspecies Patterns of Deposition

The primary purpose of this document is to assess the health effects of particles in humans. As such, human dosimetry studies have been stressed in this chapter. Such studies avoid the uncertainties associated with the extrapolation of dosi metric data from laboratory animals to humans. However, animal models have been and continue to be used in evaluating PM health effects because of ethical considerations regarding the types of st udies that can be performed with human subjects. Thus, there is a considerable need to understand dosimetry in animals and dosimetric differences between animals and humans. Limited new data are becoming available. Similar deposition efficiencies have been reported in nasal casts of human and rhesus monkey for 1-10 μ m d

ae for inspiratory flows mimicking resting breathing patterns (Kelly et al., 2005, 155894 ). Oldham and Robinson (2007, 156003 ) recently provided morphological data and predicted particle deposition in an asthma mouse model. Interspecies similarities and differences in deposi tion were described in detail in the last two PM AQCDs (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ). It was concluded that the general pattern of total particle deposition efficiency wa s similar between laboratory animals and humans: deposition increases on both sides of a minimum that occurs for particles of 0.2-1 μ m. There are, however, marked interspecies differences in uptake into the respiratory tract and regional deposition. For instance, the nasal inhalability of 10 μ m d

ae particles is predicted to be 96% in humans, whereas it is only 44% in rats (Ménache et al., 1995, 006533 ). In most laboratory animal species (rat, mouse, hamster, guinea pig, and dogs), deposition in the ET region is near 100% for particles >5 μ m d

ae (Raabe et al., 1988, 001439 ), indicating greater efficiency than that seen in humans. Detailed presentation of dosimetric difference between rats and humans are available elsewhere (Brown et al., 2005, 089308 ; Jarabek et al., 2005, 056756 ). Brown et al. (2005, 089308 ) conducted a thorough evaluation of extrapolations between rats and humans in relation to PM exposures. One of many factors they considered was the choice of a dose metric appropriate for comparison between sp ecies. For example, deposited mass may be an appropriate PM indicator for health effects assoc iated with soluble PM constituents. For health effects associated with insoluble PM, the particle number, surface area, or mass may be appropriate indicators. Given interspecies differences in deposi tion patterns and clearance rates, the question of retained versus deposited dose was also discussed. It was concluded that for acute effects, the incremental dose may be the appropriate type of do se metric. For chronic effects, long-term burden may be more appropriate. For various dose metrics, estimates of particle concentration and exposure duration required for a rat to receive the same dose as received by a human were obtained with consideration of activity levels, mode of breathing, and particle size distributions. It was noted that high PM exposures over the period of months can lead to particle overload in rats (see Section 4.3.4.4 ). Exposure regimes were derived as a functi on of particle size and exposure duration that should avoid overwhelming macrophage mediated cl earance achieving particle overload in rats (see Table 12 in Brown et al., 2005, 089308 ). The dosimetric calculations indicated that to achieve nominally similar acute doses per surface area in rats, relative to humans undergoing moderate to high exertion, PM exposure concentrations for rats would need to be somewhat higher than for humans. Since particle clearance from the lungs of rats is faster than humans, much higher exposure concentrations are required for the rat to simulate retained burdens of humans. Illustrating the complexity of such analyses, in some cases, rats were found to require lower exposures than humans to have comparable doses (generally when c onsidering a scenario of humans at rest).



## 4.2.4. Biological Factors Modulating Deposition

Evaluation of factors affecting particle depositi on is important to help understand potentially susceptible subpopulations. Differences in biological response following pollutant exposure may be caused by dosimetry differences as well as by diff erences in innate sensitivity. The effects of different biological factors on deposition were discussed in the 2004 PM AQCD (U.S. EPA, 2004, 056905 ) and are summarized briefly here.

December 2009 4-11

## 4.2. 4.1. Phys ical Activity

The activity level of an individual is well re cognized to affect their minute ventilation and route of breathing. Changes in minute ventilat ion during exercise are accomplished by increasing both V T and f ( Table 4-1 ). Humans are oronasal breathers tending to breathe through the nose when at rest and increasingly through the mouth with incr easing activity level. There is considerable interindividual variability in both the route by which people breathe and the way breathing pattern changes occur.



### Table 4-1. Breathing patterns with activity level in adult human male.

Activity Awake Rest a Slow Walk a Light Exertion a Moderate Exertion a Heavy Exertion b

Breaths/min

Tidal volume, mL

Minute ventilation, L/min 12

625

7.5 16

813

13 19

1000

19 28

1429

40 26

1923

50

Sources: a Winter-Sorkina and Cassee (2002, 043670 ); b ICRP (1995, 006988 )

Individuals typically breathe through their nose wh ile at rest, switching to oronasal breathing as ventilation increases (Bennett et al., 2003, 191977 ; Niinimaa et al., 1981, 071758 ). The role of the nose in filtering particles is diminished as airflow is diverted from the nose to the mouth during exercise, bringing more particles to the lower respir atory tract. A recent study in adults (Bennett et al., 2003, 191977 ) found that nasal ventilation during exercise varied as a function of both race and gender. African-Americans possessed a greater nasal contribution to breathing during exercise than Caucasians. At similar exercise efforts (i.e., normalized to a % maximum work capacity) the females also had a greater nasal contribution to br eathing during exercise than males. In addition, when individuals increase their ve ntilation with activity the total number of particles inhaled per unit time (i.e., exposure rate) increases, but the fracti onal deposition of particles in each breath also changes with breathing pattern. Figure 4-3 and Figure 4-4 illustrate predicted deposition fractions in the respiratory tract during rest versus light exercise, respectively. During exercise, both V T and f increase. Fractional deposition for a ll particles increases with increased V T

. Increasing the f, however, decreases the fractional deposition of fine and UFPs due to decreased time for gravitational and diffusive deposition. For particles larger than a d

ae of roughly 3 μ m, increasing f can increase the deposition fraction due to increased impaction in the extrathoracic and TB airways. Thus, it should be expected that the change in deposition fracti on with activity will vary among individuals depending on the relative influences of these two variables (i.e., V T and f ) in a given subject and the particle size to which they are e xposed. Experimentally, the lung deposition fractions of fine particles during moderate exercise a nd mouth breathing are unchanged between rest and exercise (Bennett et al., 1985, 190034 ; Morgan et al., 1984, 190035 ). Kim (2000, 013112 ) evaluated differences in deposition of 1, 3, and 5 μ m (MMAD) particles under varying breathing patterns (simulating breathing conditions of sleep, resting, and mild exercise). Total lung deposition increased with increasing V T at a given flow rate and with increasi ng flow rate at a given breathing period. These experimental studies suggest that the total deposited dose rate (i.e., deposition per unit time) of particles will generally increase in direct proportion to the increase in minute ventilation associated with exercise. The changes in ventilation, i.e., breathing patte rn and flow rate, may also alter the regional deposition of particles. Coarse particle deposition in creases in the TB and ET regions during exercise due to the increased flow rates and associated impaction. A rapid-shallow breathing pattern during exercise may result in more bronchial airway ver sus alveolar deposition, while a slow-deep pattern will shift deposition to deeper lung regions (Valberg et al., 1982, 190019 ). Bennett et al. (1985, 190034 ) showed for 2.6 μ m particles that moderate exercise shifted deposition from the lung periphery towards ET and larger, bronchial airways. Similarly, Morgan et al. (1984, 190035 ) showed that even for fine particles (0.7 μ m) TB deposition was enhanced with exercise. This shift in deposition toward the bronchial airways results in a much greater dose per unit surface area of tissue in those regions. Morgan et al. (1984, 190035 ) also found that the apical-to-basal distribution of fine

December 2009 4-12particles increased with exercise, i.e., a shift to wards increased deposition in the lung apices. This shift may be less likely for larger particles, however, whose deposition in large airway bifurcations may preclude their transport to these more apical regions (Bennett et al., 1985, 190034 ).



## 4.2. 4.2. Age

Airway structure and respiratory conditions vary with age, and these variations may alter the amount and site of particle deposition in the re spiratory tract. It was concluded in the 2004 PM AQCD (U.S. EPA, 2004, 056905 ) that significant differences between adults and children had been predicted by mathematical models and observed in experimental studies. Studies generally indicated that ET and TB deposition was greater in child ren and that children received greater doses of particles per lung surface area than adults. Deposition studies in the elderly are still quite limited. A few studies have attempted to measure oronasal breathing in children as compared to adults (Becquemin et al., 1999, 155679 ; Bennett et al., 2008, 156269 ; James et al., 1997, 042422 ). This is important since particles deposit with greater efficiency in the nose relative to the mouth, thereby affecting exposure of the lower respiratory tract. James et al. (1997, 042422 ) found that children (age 7-16 yr, n = 10) displayed more variability than adults with respect to their oronasal pattern of breathing with exercise. However, it was not possible to predict the pattern of the partitioning of ventilation during exercise based on age, gender, or nasal airway resistance. Further, in a limited number of children (age 8

− 16 yr, n = 10), Becquemin et al. (1999, 155679 ) found that the children tended to display more oral breathing both at rest and during exercise than the adults. The highest oral fractions were also found in the youngest childre n. None of these studies, however, was able to show a relationship between nasal resistance and th e relative contribution of nasal breathing in children. Bennett et al. (2008, 156269 ) made preliminary measurements of the relative contributions of oral versus nasal breathing at rest and duri ng incrementally graded submaximal exercise on the cycle ergometer for children (age 6-10 yr, n = 12) and adults (age 18-27 yr, n = 11). There was a trend for children to have a lesser nasal contributio n to breathing at rest and during exercise, but the differences from adults were not statistically significant. Breathing patterns are well recognized to change with increasing age, i.e., V T increase and respiratory rates decrease (Tabachnik et al., 1981, 157036 ; Tobin et al., 1983, 156122 ). Bennett and Zeman (1998, 076182 ) measured the deposition fraction of inhaled, fine particles (2 μ m d

ae ) in children (age 7-14 yr, n=16) and adults (age 19-35 yr, n=12) as they breathed the aerosol with their natural, resting breathing pattern. Among the childre n, variation in deposition fractions, measured by photometry at the mouth, was highly dependent on intersubject variation in V T . On the other hand, they found no difference in deposition fractions between children versus adults for these fine particles. This finding and the modeling predictions (Hofmann et al., 1989, 006922 ) are explained in part by the smaller V T and faster breathing rate of children relative to adults for natural breathing conditions. Bennett et al. (2008, 156269 ) also recently reported measures of fine particle (1 and 2 μ m d

ae ) deposition at ventilation rates typical of light exer cise in children (age 6-10 yr, n=12) and adults (age 18-27 yr, n=11) and showed that, like w ith resting breathing, deposition fractions were predicted by breathing pattern and did not differ or tended to be less in children compared to adults. On the other hand, because children breathe at higher minute ventilations relative to their lung volumes, the rate of deposition of fine particles normalized to lung surface area may be greater in children versus adults (Bennett and Zeman, 1998, 076182 ). Bennett and Zeman (2004, 155686 ) expanded their measures of fine particle deposition during resting breathing to a larger group of healthy children (6-13 yr; 20 boys, 16 girls) and found again that the variation in total deposition was best predicted by V T (r = 0.79, p < 0.001). But both V T and resting minute ventilation increased with both he ight and body mass index (BMI) of the children. Interestingly, these data suggest that for a give n height and age, children with higher BMI have larger minute ventilations and V T at rest than those with lower BMI. These differences in breathing patterns as a function of BMI translated into incr eased deposition of fine particles in the heaviest children. The rate of deposition (i.e., particles depositing per unit time) in the overweight children was 2.8 times that of the leanest children (p < 0.02). Among all children, the rate of deposition was significantly correlated with BMI (r = 0.46, p < 0.004). Some of the increase in deposition fractions of heavier children may be due to their elevated V T , which was well correlated with BMI (r = 0.72, p < 0.001). In 62 healthy adults with normal lung function aged 18-80 yr, Bennett et al. (1996, 083284 ) showed there was no effect of age on the whole lung deposition fractions of 2- μ m particles under

December 2009 4-13natural breathing conditions. Across all subjects, the deposition fractions were found to be independent of age, depending on breathing period (r = 0.58, p < 0.001) and airway resistance (r = 0.46, p < 0.001). In the same adults breathing with a fixed pattern (360 mL V T , 3.4 sec breathing period), there was a mild decrease in deposition with increasing age, which could be attributed to increased peripheral airspace dimensions in the elderly.



## 4.2. 4.3. Gend er

Males and females differ in body size, conductiv e airway size, and ventilatory parameters; therefore, gender differences in deposition might be expected. In some of the controlled studies, however, the men and women were const rained to breathe at the same V T and f. Since women are generally smaller than men, the increased minute ventilation of women compared to their normal ventilation could affect deposition patterns. This may help explain why gender related effects on deposition have been observed in some studies. Kim and Hu (1998, 086066 ) assessed the regional deposition patterns of 1-, 3-, and 5- μ m MMAD particles in healthy adult males and females using controlled breathing. The total fractional deposition in the lungs was similar for both genders with the 1- μ m particle size, but was greater in women for the 3- and 5- μ m particles. Deposition also appeared to be more localized in the lungs of females compared to those of males. Kim and Jaques (2000, 012811 ) measured deposition in healthy adults using sizes in the UF mode (0.04-0.1 μ m). Total fractional lung deposition was greater in females than in males for 0.04- and 0.06- μ m particles. The region of peak fractional deposition was shifted closer to the mouth and peak height was slightly greater for women than for men for all exposure conditions. The total lung deposition da ta from these studies are illustrated in Figure 4-5

. These differences were generally attributed to the smaller size of the upper airways, particularly of the laryngeal structure, in females. In another study (Bennett et al., 1996, 083284 ), the total respiratory tract deposition of 2- μ m particles was examined in adult males and females aged 18-80 yr who breathed with a normal resting pattern. There was a tendency for greater depositi on fractions in females compared to males. However, since males had greater minute ventila tion, the deposition rate (i.e., deposition per unit time) was greater in males than in females. More recently, Bennett and Zeman (2004, 155686 ) found no difference in the deposition of 2- μ m particles in boys versus girls aged 6-13 yr (n = 36).



## 4.2. 4.4. Anat omical Variability

Anatomical variability, even in the absence of respiratory disease, can affect deposition throughout the respiratory tract. The ET region is the first exposed to inhaled particles and, therefore, deposition within this region would reduce the amount of particles available for deposition in the lungs. Variations in relative deposition within the ET region will, therefore, propagate through the rest of the respiratory tract, creating diff erences in calculated doses among individuals. The influence of variations in nasal airway geometry on particle deposition has been investigated. Cheng et al. (1996, 047520 ) examined nasal airway deposition in healthy adults using particles ranging in size from 0.004 to 0.15 μ m and at 2 constant inspiratory flow rates, 167 and 333 mL/s. Inter-individual variability in deposition wa s correlated with the wide variation of nasal dimensions; in that, greater surface area, smaller cr oss-sectional area, and increasing complexity of airway shape were all associated with e nhanced deposition. Bennett and Zeman (2005, 155687 ) have also shown that nasal anatomy influences the efficiency of particle uptake in the noses of adults. For light exercise breathing conditions in adults, their study demonstrated that nasal deposition efficiencies for both 1- and 2- μ m monodisperse particles were significantly less in African Americans versus Caucasians. The lesser nasal ef ficiencies in African-Americans were associated with both lower nasal resistance and less elliptical nostrils compared to Caucasians. Within the lungs, the branching structure of th e airways may also differ between individuals. Zhao et al. (2009, 157187 ) recently examined the bronchial anatomy of the left lung in patients (132 M, 84 W; mean age 47 yr) that underwent conventional thoracic computed tomography scans for various reasons. At the level of the segmen tal bronchus in the upper and lower lobes, a bifurcation occurred in the majority of patients. A trifurcation, however, was observed in 23% of the upper and 18% of the lower lobes. Other more unusual findings were also reported such as four bronchi arising from the left upper lobe bronchus. As described in Section 4.2.2.4 , deposition can be highly localized near the carinal ridge of bifurcations. The effect of a bifurcation versus other

December 2009 4-14branching patterns on airflow patterns and particle de position has not been described in the literature. Martonen et al. (1994, 000847 ) showed that a wide blunt carinal ridge shape dramatically affected the flow stream lines relative to a narrower and more rounded ridge shape. Specifically, there were high flow velocities across the entire area of the blunt carinal ridge versus a smoother division of the airstream in the case of the narrow rounded ridge shape. The implication may be that localized particle deposition on the carinal ridge would increase with ridge width. A similar situation might be expected for a trifurcation versus a bifurcation. These differences in branching patterns provide a clear example of anatomical variability among individuals that might affect both air flow patterns and sites of particle deposition.



## 4.2. 4.5. Resp iratory Tract Disease

The presence of respiratory tract disease can affect airway structure and ventilatory parameters, thus altering deposition compared to that occurring in healthy individuals. The effect of airway diseases on deposition has been studied extensively, as described in the 1996 and 2004 PM AQCD (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ). Studies described therein showed that people with chronic obstructive pulmonary dis ease (COPD) had very heterogeneous deposition patterns and differences in regional deposition compared to healthy individuals. People with obstructive pulmonary diseases tended to have gr eater deposition in the TB region than did healthy people. Furthermore, there tended to be an inverse relationship between bronchoconstriction and the extent of deposition in the alveolar region, wh ereas total respiratory tract deposition generally increased with increasing degrees of airway obstruction. The vast majority of deposition studies in i ndividuals with respiratory disease have been performed during controlled breathing, i.e., all subjects breathed with the same V T and f. However, although resting V T is similar or elevated in people with COPD compared to healthy individuals, the former tend to breathe at a faster rate, resulting in higher than normal tidal peak flow and resting minute ventilation. Thus, given that breathing patterns differ between healthy and obstructed individuals, particle deposition data for controlled breathing may not be appropriate for estimating respiratory doses from ambient PM exposures. Bennett et al. (1997, 078839 ) measured the fractional deposition of insoluble 2- μ m particles in moderate-to-severe COPD patients (n = 13; mean ag e 62 yr) and healthy older adults (n = 11; mean age 67 yr) during natural resting breathing. COPD patients had about a 50% greater deposition fraction and a 50% increase in resting minute vent ilation relative to the healthy adults. As a result, the patients had an average deposition rate of abou t 2.5 times that of healthy adults. Similar to previously reviewed studies (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ), these investigators observed an increase in deposition with an increase in airway resistance, suggesting that deposition increased with the severity of airway disease. Brown et al. (2002, 043216 ) measured the deposition of an UF aerosol (CMD = 0.033 μ m) during natural resting breathing in 10 patients with moderate-to-se vere COPD (mean age 61 yr) and 9 healthy adults (mean age 53 yr). The COPD gro up consisted of 7 patients with chronic bronchitis and 3 patients with emphysema. The total deposition fraction in the bronchitic patients (0.67) was significantly (p < 0.02) greater than in either the patients with emphysema (0.48) or the healthy subjects (0.54). Minute ventilation increased with disease severity (healthy, 5.8 L/min; chronic bronchitic, 6.9 L/min; emphysema, 11 L/min). Relative to the healthy subjects, the average dose rate was significantly (p < 0.05) increased by 1.5 ti mes in the COPD patients, whereas the average deposition fraction only tended to be increased by 1. 1 times. These data further demonstrate the need to consider dose rates (which depend on minute ve ntilation) rather than just deposition fractions when evaluating the effect of respiratory disease on particle deposition and dose.

December 2009 4-15Source:Data from Tu and Knutson (1984, 072870 ).



### Figure 4-6. Total deposition of hygroscopic sodium chloride and hydrophobic aluminosilicate aerosols during oral breathing (V T 

### = 1.0 L; f = 15 min -1 

### ).



## 4.2. 4.6. Hygr oscopicity of Aerosols

Experimental and modeling studies of hygroscopi c aerosol growth and deposition in the lung were extensively discussed in Section 10.4. 3.1 of the 1996 PM AQCD (U.S. EPA, 1996, 079380 ). Hygroscopic ambient aerosols include sulfates, nitrates, some organics, and aerosols laden with sodium or potassium. The high relative humidity in the lungs contributes to rapid growth of hygroscopic particles and dramatically alters the deposition characteristics of ambient hygroscopic aerosols relative to nonhygroscopic aerosols. Nonh ygroscopic particles in the range of 0.3 μ m have minimal intrinsic mobility and low tota l deposition in the lungs. However, a 0.3 μ m salt particle (dry) will grow in vivo to nearly 2 μ m and deposit to a far greater extent (Anselm et al., 1990, 156217 ). The hygroscopic growth of particles in the re spiratory tract decreases diffusive deposition and increases aerodynamic deposition as illustrated in Figure 4-6

.



## 4.2.5. Summary

Particle deposition in the respiratory tract occurs predominantly by diffusion, impaction, and sedimentation. Deposition is minimal for partic le diameters in the range of 0.1 to 1.0 μ m, where particles are small enough to have minimal sedimentat ion or impaction and sufficiently large so as to have minimal diffusive deposition. In humans, total respiratory tract deposition approaches 100% for particles of roughly 0.01 μ m (physical diameter) due to diffusive deposition and for particles of around 10 μ m d

ae due to the efficiency of sedimentation and impaction. The first line of defense for protecting the lowe r respiratory tract from i nhaled particles is the nose and mouth. Nasal deposition approach es 100% in the average human for 10 μ m d

ae particles. Experimental studies show lower nasal particle deposition in children than adults. Relative to adults, children also tend to breathe more through their mouth which is less efficient for removing inhaled particles than the nose. These findings suggest that the lower respiratory tract of children may receive a higher dose of ambient PM compared to adults. Since children breathe at higher minute ventilations relative to their lung volumes, the rate of particle deposition normalized to lung surface area may be further increased relative to adults. People with COPD generally have greater total deposition and more heterogeneous deposition patterns compared to healthy individuals. The observed increase in deposition correlates with increases in airway resistance, suggesting that de position increases with the severity of airway disease. COPD patients also have an increased re sting minute ventilation relative to the healthy adults. This demonstrates the need to consider d ose rates (which depend on minute ventilation)

December 2009 4-16rather than just deposition fractions when evaluating the effect of respiratory disease on particle deposition and dose. Modeling studies indicate that, for particles greater than ~0.01 μ m, some cells located near the carinal ridge of bronchial bifurcations may receive hundreds to thousands times the average dose (particles per unit surface area) of the parent an d daughter airways. The inertial impaction of particles ≥ 1 μ m d

ae at the carinal ridge of large bronchi in creases with increasing inspiratory flows. Airway constriction can further augment the overa ll deposition efficiency of coarse particles at downstream bifurcations. These findings suggest that substantial doses of particles ( ≥ 1 μ m d

ae ) may be justified for in vitro studies using tracheobronchial epithelial cell cultures. Our ability to extrapolate between species has not generally changed since the 2004 PM AQCD (U.S. EPA, 2004, 056905 ). However, some considerations rela ted to coarse particles warrant comment. The inhalability of particles having d

ae of 2.5, 5, and 10 μ m is 80, 65, and 44% in rats, respectively, whereas it remains near 100% for a d

ae of 10 μ m in humans. In most laboratory animal species (rat, mouse, hamster, guinea pig, and dogs) , deposition in the extrathoracic region is near 100% for particles greater than 5 μ m d

ae . By contrast, in humans nasal deposition approaches 100% for 10 μ m d

ae . Oronasal breathing versus obligate nasal breathing further contributes to greater penetration of coarse particles into the lowe r respiratory tract of humans than rodents.



# 4.3. Clearance of Poorly Soluble Particles

This section discusses the clearance and translocation of poorly soluble particles that have deposited in the respiratory tract. The term “clearance” is used here to refer to the processes by which deposited particles are removed by mucocili ary action or phagocytosis from the respiratory tract. “Translocation” is used here mainly to re fer to the movement of free particles across cell membranes and to extrapulmonary sites. In the lite rature, translocation may also refer to the extraand intracellular dissolution of particles and the subsequent transfer of dissociated material to the blood through extra- and intracellular fluids and across the various cell membranes and lung tissues. The clearance and distribution of soluble particle s and soluble constituents of particles are discussed in Section 4.4

. A basic overview of biological mechanisms and clearance pathways from various regions of the respiratory tract are presented in the following sections. Then regional kinetics of particle clearance are addressed. Subsequently, an update on interspecies patterns and rates of particle clearance is provided. The translocation of UFPs is also discussed. Finally, information on biological factors that may modulate clearance is presented.



## 4.3.1. Clearance Mechanisms and Kinetics

For any given particle size, the deposition patte rn of poorly soluble particles influences clearance by partitioning deposited material between lung regions. Tracheobronchial clearance of poorly soluble particles in humans, with some exce ptions, is thought (in general) to be complete within 24-48 h through the action of the mucociliary escalator. Clearance of poorly soluble particles from the alveolar region is a much slower proce ss which may continue from months to years.



## 4.3. 1.1. Extr athoracic Region

Particles deposited in either the nasal or or al passages are cleared by several mechanisms. Particles depositing in the mouth may generally be assumed to be swallowed or removed by expectoration. Particles deposited in the posterio r portions of the nasal passages are moved via mucociliary transport towards the nasopharynx and swallowed. Mucus flow in the most anterior portion of the nasal passages is forward, toward the vestibular region where removal occurs by sneezing, wiping, or nose blowing.

December 2009 4-17

## 4.3. 1.2. Trac heobronchial Region

Mucociliary clearance in the TB region has gene rally been considered to be a rapid process that is relatively complete by 24-48 h post-inhalati on in humans. Mucociliary clearance is frequently modeled as a series of “escalators” moving material proximally from one generation to the next. As such, the removal rate of particles from an airway generation increases with increasing tracheal mucus velocity. Assuming continuity in the amount of mucus between airway generations, mucus velocities decrease and transit times within an ai rway generation increase with distal progression. Although clearance from the TB region is generally ra pid, experimental evidence discussed in the 1996 and 2004 PM AQCD (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ) showed that a fraction of material deposited in the TB region is retained much longer. The slow-cleared TB fraction (i.e., the fraction of particles deposited in the TB region that are subject to slow clearance) was thought to increase with decreasing particle size. For instance, Roth et al. (1993, 156928 ) showed approximately 93% retention of UFPs (30 nm median diameter) thought to be deposited in the TB region at 24 h post-inhalation. The slow phase clearance of these UFPs continued with an estimated half-time (t 1/2 ) of around 40 days. Using a technique to target inhaled particles (monodisperse 4.2 μm MMAD) to the conducting airways, Möller et al. (2004, 155987 ) observed that 49 ± 9% of particles cleared rapidly (t 1/2 of 3.0 ± 1.6 h), whereas the remaining fraction cleared considerably slower (t 1/2 of 109 ± 78 days). The ICRP (1995, 006988 ) human respiratory tract model assumes particles ≤ 2.5 μm (physical diameter) to have a slow-cleared TB fraction of 50%. The slow-cleared fraction assumed by the ICRP (1995, 006988 ) decreases with increasing particle size to <1% for 9 μm particles. Considering the UF data of Roth et al. (1993, 156928 ) in addition to data considered by the ICRP (1995, 006988 ), Bailey et al. (1995, 190057 ) estimated a slow-cleared TB fraction of 75% for UF Ps. At that time, they (Bailey et al. 1995) also estimated the slow-cleared fraction to decrease w ith increasing particle size to 0% for particles ≥ 6 μm. Recent experimental evidence fro m the same group (Smith et al., 2008, 190037 ) showed no difference in TB clearance among humans for particles with geometric sizes of 1.2 versus 5 μm, bu the same d t

s,

s

an expected. ae (5 μm) so as to deposit similarly in the TB airways. For at least micron-sized particle these recent findings do not support the particle size dependence of a slow-cleared TB fraction. A discussed further below, much of the apparent slow-cleared TB fraction may be accounted for by differences in deposition patterns, i.e., greater de position in the alveolar region th A portion of the slow cleared fraction from the TB region appears to be associated with small bronchioles. For large particles (d

ae = 6.2 μ m) inhaled at a very slow rate to theoretically deposit mainly in small ciliated airways, 50% had cleared by 24 h post-inhalation. Of the remaining particles, 20% cleared with a t 1/2 of 2.0 days and 80% with a t 1/2 of 50 days (Falk et al., 1997, 086080 ). Using the same techniques, Svartengren et al. (2005, 157034 ) also reported the existence of long-term clearance in humans from the small airways. It should be noted that the clearance rates for the slow-cleared TB fraction still exceeds the cl earance rate of the alveolar region in humans. Kreyling et al. (1999, 039175 ) targeted inhaled particle (d

ae = 2.2 and 2.5 μ m) deposition to the TB airways of adult beagle dogs and subsequently quan tified particle retention using scintigraphic and morphometric analyses. Despite the use of shal low aerosol bolus inhalation to a volumetric lung depth of less than the anatomic dead space, 2.5-25% of inhaled particles depo sited in alveoli. At 24 and 96 h post-inhalation, more than 50% of the reta ined particles were in alveoli. However, 40% of particles present at 24 and 96 h were localized to small TB airways of between 0.3 and 1 mm in diameter. Collectively, these studies suggest that although mucociliary clearance is fast and effective in healthy large airways, it is less effective and sites of longer retention exist in the smaller TB airways. The underlying sites and mechanisms of long-term TB retention in the smaller airways remain largely unknown. Several factors may contribute to the existence or experimental artifact of slow clearance from the smaller TB airways. Even when inhaled to very shallow lung volumes, some particles reach the alveolar region (Kreyling et al., 1999, 039175 ). Therefore, experiments utilizing bolus techniques to target inhaled particle deposition to the TB airways may have had some deposition in the alveolar region. This may occur due to variability in path length and the number of generations to the alveoli (Asgharian et al., 2001, 017025 ) and/or differences in regional ventilation (Brown and Bennett, 2004, 190032 ). Nonetheless, the experimentally measured clearance rates measured for the slow cleared TB fraction are faster than that of the alveolar region in both humans and canines. Thus, although experimental artifacts li kely occur, they do not discount the existence of a slow cleared TB fraction. To some extent, it is possible that the slow cleared TB fraction may be due to bronchioles that do not have a continuous cili ated epithelium as in the larger bronchi. Neither

December 2009 4-18path length, ventilation distribution, nor a disc ontinuous ciliated epithelium explains an apparently slow cleared TB fraction with decreasing particle size below 0.1 μ m. As discussed in Section 4.3.3 on Particle Translocation, UFPs cross cell memb ranes by mechanisms different from larger (~1 μ m) particles. Based on that body of literature, particle s smaller than a micron may enter epithelial cells resulting in their prolonged retention, particularly in the bronchioles where the residence time is longer and distances necessary to reach the epithelium are shorter compared to that in the bronchi.



## 4.3. 1.3. Alve olar Region

The primary alveolar clearance mechanism is macrophage phagocytosis and migration to terminal bronchioles where the cells are cleared by the mucociliary escalator . Alveolar macrophages originate from bone marrow, circulate briefly as monocytes in the blood, and then become pulmonary interstitial macrophages before migr ating to the luminal surfaces. Under normal conditions, a small fraction of ingested particles may also be cleared through the lymphatic system. This may occur by transepithelial migration of alve olar macrophage following particle ingestion or free particle translocation with subsequent uptake by interstitial macrophages. Snipes et al. (1997, 156092 ) have also demonstrated the importance of ne utrophil phagocytosis in clearance of particles from the alveolar region. Rates of alveolar clearance of poorly soluble particles vary between species and are briefly discussed in Section 4.3.2 . The translocation of particles from their site of deposition is discussed in Section 4.3.3

. The efficiency of macrophage phagocytosis is t hought to be greatest for particles between 1.5 and 3 μ m (Oberdörster, 1988, 006857 ). The decreased efficiency of alveolar macrophage for engulfing UFPs increases the time av ailable for these particles to be taken up by epithelial cells and moved into the interstitium (Ferin et al., 1992, 044401 ). Consistent with this supposition (i.e., translocation increases with time), an increase in titanium dioxide (TiO 2 ) particle transport to lymph nodes has been reported following inhalation of a cytotoxin to macrophages (Greenspan et al., 1988, 045031 ). Interestingly, the long-term clearance kinetics of the poorly soluble UF (15-20 nm CMD) iridium (Ir) particles were found to be simila r to the kinetics reported in the literature for micrometer-sized particles (Semmler et al., 2004, 055641 ; Semmler-Behnke et al., 2007, 156080 ). Semmler-Behnke et al. (2007, 156080 ) concluded that UF Ir particles are less phagocytized by alveolar macrophage than larger particles, but ar e effectively removed from the airway surface into the interstitium. Particles are then engulfed by in terstitial macrophages which then migrate to the airway lumen and are removed by mucociliary clearance to the larynx. The major role of macrophage-mediated clearance was supported by la vage of relatively few free particles versus predominantly phagocytized particles at time-points of up to 6 mo. It is also possible that some free particles as well as particle-laden macrophages were carried from interstitial sites via the lymph flow to bronchial and bronchiolar sites, including bronchial-associated lymphatic tissue, where they were excreted again into the airway lumen.



## 4.3.2. Interspecies Patterns of Clearance and Retention

There are differences between species in both th e rates of particle clearance from the lung and manner in which particles are retained in the l ung. For instance, based on models of mucociliary clearance from un-diseased airways, >95% of par ticles deposited in the tracheobronchial airways of rats are predicted to be cleared by 5 h post deposi tion, whereas it takes nearly 40 h for comparable clearance in humans (Hofmann and Asgharian, 2003, 055579 ). As noted in Section 4.3.1.2 , however, there is considerable evidence that a sizeable fracti on of particles deposited at the bronchiolar level of the ciliated airways in humans (as well as cani nes) are cleared at a far slower rate. The slow cleared TB fraction appears to increase with decreasing particle size.

December 2009 4-190.10 1.00

0 100 200 300 400

Da y s after inhalationFraction Retained

F-344 rat Dog

Human

L-E rat

Hamster

HTM rat Guinea Pig

Source: Data from Kreyling and Scheuch (2000, 056281 ).



### Figure 4-7. Retention of poorly soluble particles (0.5-5 μm) in the alveolar region of the lung over time in various mammalian species.

Figure 4-7 illustrates rates of alveolar clearance for 0.5-5 μm particles in various mammalian species. The alveolar clearance rate of particles smal ler than 0.1 μm and larger than 5 μm is slower than that of particles in the 0.5-5 μm range. From interspecies comparisons of alveolar clearance, the path length from alveoli to ciliated terminal br onchioles may affect the particle transport rate (Kreyling and Scheuch, 2000, 056281 ). The average path length from alveoli to ciliated terminal bronchioli is longer in humans, monkeys, and d ogs, than in sheep, rats, hamsters, and mice. Transport time and hence retention times may increase with path length. This hypothesis fits with all species in this comparison, except guinea pigs, whic h have a short path length yet particle retention that is nearly as long as in humans, monkeys, a nd dogs. However, sheep have a short path length and particle transport as fast as rodents. In general, alveolar clearance rates appear to increase with increasing path length from the alveoli to ciliated airways. There are also distinct differences in the sites of particle retention between species. Large mammals retain particles in interstitial tissues under normal conditions, whereas rats retain particles in alveolar macrophages (Snipes, 1996, 076041 ). In rats with chronic high doses there is a shift in the pattern of dust accumulation and response from that observed at lower doses in the lungs (Snipes, 1996, 076041 ; Vincent and Donaldson, 1990, 002462 ). Rats chronically exposed to high concentrations of insoluble particles experience a reduction in their alveolar clearance rates and an accumulation of interstitial particle burden (Bermudez et al., 2002, 055578 ; Bermudez et al., 2004, 056707 ; Ferin et al., 1992, 044401 ; Oberdörster et al., 1994, 046203 ; Oberdörster et al., 1994, 056285 ; Warheit et al., 1997, 086055 ). The influence of exposure concentration on the pattern of particle retention in rats (exposed to diesel soot) and humans (exposed to coal dust) was examined by Nikula et al. (2001, 016641 ). In rats, the diesel particles were found to be primarily in the lumens of the alveolar duct and alveoli; whereas in humans, retained dust was found primarily in the interstitial tissue within the respiratory acini.



## 4.3.3. Particle Translocation

Mucociliary and macrophage mediated clearan ce of poorly soluble particles from the respiratory tract was discussed in Section 4.3.1 . There is evidence that particles may cross cell membranes and move from their site of deposition by other mechanisms. The following subsections discuss the movement of particles from the luminal surfaces of the alveolar region and from the olfactory mucosa. The clearance and distribution of soluble particles and soluble constituents of particles are discussed in Section 4.4

.

December 2009 4-20

## 4.3. 3.1. Alve olar Region

Numerous studies have examined the translocation of UFPs from their site of deposition in the lung. Traditionally viewed as a relatively inert particle type, UF TiO 2 has received the most study. At the time the 2004 PM AQCD was released, there were conflicting results regarding the rate and magnitude of UF carbon translocation from the huma n lung. Since that time, it has become wellestablished that the transport of UF carbon particles from the human lung is far slower than that of soluble materials. However, it has also been shown in animal studies (primarily of rats) that UFPs cross cell membranes by mechanisms different from larger (~1 μ m) particles and that a small fraction of these particles enter capillaries and may distribute systemically. Described in brief below, details of selected new studies investigating th e disposition of poorly soluble particles are provided in Annex B. There has been some contention regarding abi lity of UF carbon particles to rapidly diffuse from the lungs into the systemic circulation. Base d on their study of 5 healthy volunteers, Nemmar et al. (2002, 024914 ) suggested that UF carbon particles (<100 nm) pass rapidly into the systemic circulation. However, Brown et al. (2002, 043216 ) found that the majority of UF carbon particles (CMD, 33 ± 2 nm) were still in the lungs of healthy human adult volunteers (n = 9; aged 40-67 yr) and COPD patients (n = 10; 45-70 yr) at 24 h post-inhalation. Brown et al. (2002, 043216 ) and Burch (2002, 056754 ) contended that the findings reported by Nemmar et al. (2002, 024914 ) were consistent with soluble pertechnetate clearance, but not insoluble UF carbon particles. Highly soluble in normal saline, pertechnetate clears rapidly from the lung with a half-time of ~10 min and accumulates most notably in the bladder, stomach, t hyroid, and salivary glands. Three recent studies have confirmed that the majority (>95%) of UF carbon particles deposited in the lungs of human volunteers are retained at 24 h post-inhalation (Mills et al., 2006, 088770 ; Wiebert et al., 2006, 157146 ; Wiebert et al., 2006, 156154 ). Wiebert et al. (2006, 157146 ) modified their aerosol generation system to reduce leaching of the 99m Tc radiolabel from carbon particles. Except for a small amount of radiotracer leaching from particle s (1.0 ± 0.6% of initially deposited activity in urine by 24 h), these investigators found negligible radiolabel and associated particle clearance from the lungs by 70 h. The available data show that there is not a rapid or significant amount of UF carbon particle migration into circulation (Brown et al., 2002, 043216 ; Burch, 2002, 056754 ; Mills et al., 2006, 088770 ; Möller et al., 2008, 156771 ; Wiebert et al., 2006, 157146 ; Wiebert et al., 2006, 156154 ). Although human studies show that the vast majority of UF carbon particles are retained in the lungs until at least 24 h post-inhalation, both in vitro and in vivo studies support the rapid ( ≤ 1 h) translocation of free UF TiO 2 particles across pulmonary cell membranes (Churg et al., 1998, 085815 ; Ferin et al., 1992, 044401 ; Geiser et al., 2005, 087362 ). Peculiar to TiO 2 aerosols, there is evidence that particle aggregates may disassocia te once deposited in the lungs. This disassociation makes inhaled aggregate size the determinant of de position amount and site, but primary particle size the determinant of subsequent clearance (Bermudez et al., 2002, 055578 ; Ferin et al., 1992, 044401 ; Takenaka et al., 1986, 046210 ). Following disaggregation, the UF TiO 2 particles are cleared more slowly and cause a greater inflammatory response (neutrophil influx) than fine TiO 2 particles (Bermudez et al., 2002, 055578 ; Ferin et al., 1992, 044401 ; Oberdörster et al., 1994, 046203 ; Oberdörster et al., 1994, 056285 ; Oberdorster et al., 2000, 039014 ). The differences in inflammatory effects and possibly lymph burdens between fine and UF TiO 2 in many studies appear related to lung burden in terms of particle surface area and not particle mass or number (Oberdorster, 1996, 039852 ; Oberdörster et al., 1992, 045110 ; Oberdorster et al., 2000, 039014 ; Tran et al., 2000, 013071 ). More recently, others have noted that particle surface ar ea is not an appropriate metric across all particle types (Warheit et al., 2006, 088436 ). Surface characteristics such as roughness can also affect protein binding and potentially clearance kinetics, with smoother TiO 2 surfaces being more hydrophobic (Sousa et al., 2004, 089866 ). Geiser et al. (2005, 087362 ) conducted a detailed examination of the disposition of inhaled UF TiO 2 in 20 healthy adult rats. They found that distributions of particles among lung tissue compartments appeared to follow the volume frac tion of the tissues and did not significantly differ between 1 and 24 h post-inhalation. Averaging 1- and 24-h data, 79.3 ± 7.6% of particles were on the luminal side of the airway surfaces, 4.6 ± 2.6% we re in epithelial or endothelial cells, 4.8 ± 4.5% were in connective tissues, and 11.3 ± 3.9% were wi thin capillaries. Particles within cells were not membrane bound. It is not clear why the fraction of particles identified in compartments such as the capillaries did not differ between 1 and 24 h post -inhalation. These findings were consistent with the smaller study of 5 rats by Kapp et al. (2004, 156624 ) who reported identifying TiO 2 aggregates in a

December 2009 4-21type II pneumocyte; a capillary close to the endothelial cells; and within the surface-lining layer close to the alveolar epithelium immediately fo llowing a 1-h exposure. These studies effectively demonstrate that some inhaled UF TiO 2 particles, once deposited on the pulmonary surfaces, can rapidly ( ≤ 1 h) translocate beyond the epithelium and potentially into the vasculature. Extrapulmonary translocation has also been described for poorly soluble UF gold and Ir particles. In male Wistar-Kyoto rats exposed to UF gold particles (5-8 nm), Takenaka et al. (2006, 156110 ) reported a low but significant fraction (0.03 to 0.06% of lung concentration) of gold in the blood from 1 to 7 days post-inhalation. Semmler et al. (2004, 055641 ) also found small but detectable amounts of poorly soluble Ir particle (1 5 and 20 nm CMD) translocation from the lungs of male Wistar-Kyoto rats to secondary target organs like the liver, spleen, brai n, and kidneys. Each of these organs contained about 0.2% of deposited Ir. The peak levels in these organs were found 7 days post-inhalation. The translocated particles were largely cleared from extrapulmonary organs by 20 days and Ir levels were near background at 60 days post-inhalation. Particles may have been distributed systemically via the gastrointestinal tract. Immediately after the 6-h inhalation exposure, 18 ± 5% of the deposited Ir particles had already cleared into the gastrointestinal tract. After 3 wk, 31 ± 5% of the deposited particles were retained in the lung. By 2 and 6 mo post-inhalation, lung retention was 17 ± 3 and 7 ± 1%, respectively. The particles appeared to be cleared predominantly from the peripheral lung via the mucociliary escalator into the GI tract and were found in feces. A few recent studies have characterized difference s in the behavior of fine and UF particles in vitro. Geiser et al. (2005, 087362 ) found that both UF and fine (0.025 μm gold, 0.078 μm TiO 2 , and 0.2 μm TiO 2 ) particles cross cellular membranes by non-endo cytic (i.e., involving vesicle formation) mechanisms such as adhesive interactions and diffusion, whereas the phagocytosis of larger 1 μm TiO 2 particles is ligand-receptor mediated. Edetsberger et al. (2005, 155759 ) found that UFPs (0.020 μ m polystyrene) translocated into cells by first measurement (~1 min after particle application). Intracellular agglomerates of 88-117 nm were seen by 15-20 min and of 253-675 nm by 50-60 min after particle application. These intrace llular aggregates were thought to result from particle incorporation into endosomes or similar stru ctures since Genistein or Cytochalasin treatment generally blocked aggregate formation. Interestingl y, particles did not translocate into dead cells, rather they attached to the outside of the ce ll membrane. Amine- or carboxyl-modified surfaces (46 nm polystyrene) did not affect translocation acr oss cultures of human bronchial epithelial cells with about 6% regardless of the surface characteristics (Geys et al., 2006, 155789 ).



## 4.3. 3.2. Olfa ctory Region

Numerous studies have demonstrated the transloc ation of soluble and poorly soluble particles from the olfactory mucosa via the axons to the olfact ory bulb of the brain. The vast majority of these studies were conducted in rodents. However, DeLorenzo (1970, 156391 ) observed the rapid (within 30-60 min) movement of 50 nm silver-coated colloidal gold particles instilled on the olfactory mucosa into the olfactory bulb of squirrel monkeys. The specifics of this and other key studies that have investigated the translocation of particles to the olfactory bulb are provided in Annex B. Two recent studies reported the movement of UFPs deposited in the olfactory region of the nose along the olfactory nerve and into the olfactory bulb of the brain in rats. Oberdörster et al. (2004, 055639 ) exposed rats to UF carbon particles (36 nm CMD, 1.7 σ g) containing 13 C in a wholebody chamber for 6 h. The distribution of 13 C was followed for 7 days postexposure. There was a significant increase in 13 C in the olfactory bulb on Day 1 with persistent and continued increase through Day 7. Elder et al. (2006, 089253 ) exposed rats to manganese (Mn) oxide (~30 nm equivalent sphere with 3-8 nm primary particle s) via whole-body inhalation exposure for 12 days (6 h/day, 5 days/wk) with both nares open or Mn oxide for 2 days (6 h/day) with right nostril blocked. After the 12 days exposure via both nostrils, Mn in the olfactory bulb increased 3.5-fold. After the 2-day exposure with the right nostril blocked, Mn wa s found mainly in the left olfactory bulb (2.4- fold increase). These studies suggest the neuronal upt ake and translocation of UFPs without particle dissolution and in the absence of mucosal injury. Elder et al. (2006, 089253 ) also addressed the issue of whether solubilization of particles was requisite for translocation along the olfactory nerv e and into the brain. Similar amounts of soluble manganese chloride (MnCl 2 ) and poorly soluble Mn oxide were instilled onto the left naris of anesthetized rats. At 24 h post-instillation, similar amounts of Mn were found in the left olfactory bulb of rats instilled with MnCl 2 (8.2 ± 3.6% of instilled) and Mn oxide (8.2 ± 0.7% of instilled). If solubilization were required for translocation, then a lower amount of Mn oxide than MnCl 2 should

December 2009 4-22have reached the olfactory bulb. Following 14 c onsecutive days of aerosol exposure, Dorman et al. (2001, 055433 ) demonstrated that more soluble Mn sulfate reaches the olfactory bulb and striatum of rat brains than the poorly soluble form of Mn tetroxid e. Nonetheless, the Mn levels were statistically increased in both the olfactory bulb and striatum following exposure to Mn tetroxide relative to filtered air. In a subsequent 13-wk exposure study, Dorman et al. (2004, 155752 ) also demonstrated that more soluble manganese sulfate (MnSO 4 ) reached the olfactory bulb than was observed for the less soluble Mn form (hureaulite). Both the soluble and less soluble forms of Mn resulted in statistically increased levels of Mn in the olfactory bulb relative to air exposed controls. The soluble MnSO 4 was also observed to reach the striatum and cerebellum. In addition, Yu et al. (2003, 156171 ) demonstrated increased Mn levels in the brains of rats exposed to welding-fumes for 60 days, however, the role of transport via the blood is less clear in this study. The translocation of zinc (Zn) and TiO 2 to the olfactory bulb has also been reported in the literature. Persson et al. (2003, 051846 ) observed the translocation of Zn to the olfactory bulbs following instillation in both rats and freshwater pike. Wang et al. (2007, 156146 ) reported the translocation of both fine (155 nm) and UF (21 and 71 nm) TiO 2 particles in mice. Interestingly, a qualitative analysis of the data showed that more of the fine TiO 2 than UF TiO 2 reached the olfactory bulb. Wang et al. (2007, 156146 ) suggested that a strong hydrophilic character and propensity for aggregation reduced the translocation of the UF TiO

. 2 The importance of particle translocation to the brain is not yet understood. Translocation via the axon to the olfactory bulb has been observed for numerous compounds of varying composition, particle size, and solubility. Although the rate of tr anslocation is rapid, perhaps less than an hour, the magnitude of transport remains poorly characterized . With regard to the magnitude of transport, Elder et al. (2006, 089253 ) found that as much as 8% of both soluble and insoluble forms of Mn were translocated to the olfactory bulb in rats follow ing intranasal instillation. It is also still unclear to what extent translocation to the olfactory bul b and other brain regions may vary between species. The olfactory mucosa covers approximately 50% of the nasal epithelium in rodents versus only about 5% in primates (Aschner et al., 2005, 155663 ). Additionally, a greater portion of inhaled air passes through the olfactory region of rats relative to primates (Kimbell, 2006, 155902 ). These differences may predispose rats, more so than huma ns, to deposition of particles in the olfactory region with subsequent particle translocation to the olfactory bulb.



## 4.3.4. Factors Modulating Clearance

4.3. 4.1. Age

It was previously concluded that there appeared to be no clear evidence for any age-related differences in clearance from the lung or total respiratory tract, either from child to adult, or young adult to elderly (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ). Studies showed either no change or some slowing in mucus clearance with age after maturity. Although some differences in alveolar macrophage function were reported betw een mature and senescent mice, no age-related decline in macrophage function had been observed in humans. A comprehensive review of the recent and older literature supports a decrease in mu cociliary clearance with increasing age beyond adulthood in humans and animals. Limited animal data also suggest macrophage-mediated alveolar clearance may also decrease with age. Studies addressing the effects of age on respiratory tract clearance are provided in Annex B. Ho et al. (2001, 156549 ) demonstrated that nasal mucociliary clearance rates were about 40% lower in old (age >40-90 yr) versus young (age 11-40 yr) men and women. Tracheal mucus velocities in elderly (or aged) humans and beagle dogs are about 50% that of young adults (Goodman et al., 1978, 071130 ; Whaley et al., 1987, 156153 ). Several human studies have demonstrated decreasing rates of mucociliary particle clearance from the large and small bronchial airways with increasing age (Puchelle et al., 1979, 006863 ; Svartengren et al., 2005, 157034 ; Vastag et al., 1985, 157088 ). Linear fits to the data show that rapid clearance (within 1 h) from large bronchi and prolonged clearance (between 1-21 days) from the small bronchioles in an 80-year-old is only about 50% of that in a 20-year-old (Svartengren et al., 2005, 157034 ; Vastag et al., 1985, 157088 ). One study reported that alveolar particle clearance rates decreased by near ly 40% in old versus young rats (Muhle et al., 1990, 006853 ). Another study has reported that older rats have an increased susceptibility to

December 2009 4-23pulmonary infection due to altered alveolar macr ophage function and slowed bacterial clearance (Antonini et al., 2001, 156219 ). Although data are somewhat limited, they consistently show a depression of clearance throughout the respiratory tract with increasing age from young adulthood in humans and laboratory animals.



## 4.3. 4.2. Gend er

Gender was not found to affect clearance rates in prior reviews (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ). Studies not included in those reviews also show that human males and females have similar nasal mucus clearance rates (Ho et al., 2001, 156549 ), tracheal mucus velocities (Yeates et al., 1981, 095391 ), and large bronchial airway clearance rates (Vastag et al., 1985, 157088 ).



## 4.3. 4.3. Resp iratory Tract Disease

At the time of the last two reviews (U.S. EPA, 1996, 079380 ; U.S. EPA, 2004, 056905 ), it was well recognized that obstructive airways disease may influence both the site of initial deposition and the rate of mucociliary clearance from the ai rways. When deposition patterns are matched, mucociliary clearance rates are reduced in patients with COPD relative to healthy controls. The effects of acute bacterial/viral infections a nd cough on mucociliary clearance were briefly summarized in Section 10.4.2.5 (EPA, 1996, 079380 ) and Section 6.3.4.4 (EPA, 2004, 056905 ) of past reviews. While cough is generally a reaction to some inhaled stimulus, in some cases, especially respiratory disease, it can also serve to clear the upper bronchial airways of deposited substances by dislodging mucus from the airway surface. One of the difficulties in assessing effects on infection on mucociliary clearance is that spontaneous coughing increases during acute infections. Cough has been shown to supplement mucociliary clearance of secretions, especially in patients with obstructive lung disease and primary ciliary dyskinesia. Using a bolus technique to target specific lung regions, Möller et al. (2008, 156771 ) examined particle clearance from the ciliated airways and al veolar region of healthy subjects, smokers, and patients with COPD. Airway retention after 1.5 hours was significantly lower in healthy subjects (89 ± 6%) than smokers (97 ± 3%) or COPD patient s (96 ± 6%). At 24 and 48 h, retention remained significantly higher in COPD patients (86 ± 6% and 82 ± 6%, respectively) than healthy subjects (75 ± 10% and 70 ± 9%, respectively). However, these findings are confounded by the more central pattern of deposition in the healthy subjects than in the smokers and COPD patients. Alveolar retention of particles was similar betw een the groups at 48 h post-inhalation. The effect of asthma on lung clearance of par ticles may depend on disease status. Lay et al. (2009, 190060 ) found significantly (p < 0.01) more rapid particle (0.22 μm) mucociliary clearance over a 2-h period post-inhalation in mild asthmatics than in healthy volunteers. Although the pattern of deposition tended to be more central in the as thmatics, there was not a statistically significant difference from healthy controls. In vivo uptake by airway macrophages in mild asthmatics was also enhanced relative to healthy volunteers (p < 0.01). In an ex vivo study, airway macrophages from individuals with more severe asthma had impair ed phagocytic capacity relative to less severely affected asthmatics and healthy volunteers (Alexis et al., 2001, 190013 ). Lay et al. (2009, 190060 ) concluded that enhanced uptake and processing of particulate antigens could contribute to the pathogenesis and progression of allergic airways disease in asthmatics and may contribute to an increased risk of exacerbations with particulate exposure. Chen et al. (2006, 147267 ) investigated the effect of endotoxin on the disposition of particles. Healthy rats and those pretreated with endotoxin (12 h before particle instillati on) were instilled with UF (56.4 nm) or fine (202 nm) particles. In hea lthy rats, there were no marked differences in lung retention or systemic distribution between the UF and fine particles. In healthy animals, UFPs were primarily retained in lungs (72 ± 10% at 0.5-2 h; 65 ± 1% at 1 day; 62 ± 5% at 5 days). Particles were also detected in the blood (2 ± 1% at 0.5-2 h; 0.1 ± 0.1% at 5 days) and liver (3 ± 2% at 0.5-2 h; 1 ± 0.1% at 5 days) of the healthy animals. At 1 day post-instillation, about 13% of the particles were excreted in the urine or feces of the healthy animals. In rats pretreated with endotoxin, by 2 h post-instillation, the UFPs accessed the blood (5 versus 2%) and liver (11 versus 4%) to a significantly greater extent than fine particles. The endotoxin-treated rats also had significantly greater amounts of UFPs in the blood (5% versus 2%) and liver (11% versus 3%) relative to the

December 2009 4-24healthy control rats. This study demonstrates that acute pulmonary inflammation caused by endotoxin increases the migration of UFPs into systemic circulation. Adamson and Preditis (1995, 189982 ) investigated the possibility that particle deposition into an already injured lung might affect particle reten tion and enhance the toxicity of “inert” particles. Bleomycin was instilled into mice to induce epith elial necrosis and subsequent pulmonary fibrosis. Instilled 3 days following bleomycin treatment, wh ile epithelial permeability was compromised, carbon black particles in treated mice were transl ocated to the interstitium and showed increased pulmonary retention relative to untreated mice. Wh en instilled 4 wk post-bleomycin treatment, after epithelial integrity was restored, carbon black pa rticle retention was similar between treated and untreated mice with minimal translocation to the inte rstitium. The instillation of carbon particles did not appear to increase lung injury in the bleomyci n treated mice at either time point. This study shows that integrity of the epithelium affects particle retention and translocation into interstitial tissues.



## 4.3. 4.4. Part icle Overload

Unlike other laboratory animals, rats appear susceptible to “particle overload” effects due to impaired macrophage-mediated alveolar clear ance. Numerous reviews have discussed this phenomenon and the difficulties it poses for the ex trapolation of chronic effects in rats to humans (International, 2000, 002892 ; Miller, 2000, 011822 ; Morrow, 1994, 006850 ; Oberdorster, 1995, 046596 ; Oberdorster, 2002, 021111 ). Large mammals have slow pulmonary particle clearance and retain particles in interstitial tissues under norma l conditions, whereas rats have rapid pulmonary clearance and retain particles in alveolar macrophages (Snipes, 1996, 076041 ). With chronic high doses of PM there is a shift in the pattern of dust accumulation and response from that observed at lower doses in rat lungs (Snipes, 1996, 076041 ; Vincent and Donaldson, 1990, 002462 ). Rats chronically exposed to high concentrations of insol uble particles experience a reduction in their alveolar clearance rates and an accumulation of interstitial par ticle burden (Bermudez et al., 2002, 055578 ; Bermudez et al., 2004, 056707 ; Ferin et al., 1992, 044401 ; Oberdörster et al., 1994, 046203 ; Oberdörster et al., 1994, 056285 ; Warheit et al., 1997, 086055 ). With continued exposure, some rats eventually develop pulmonary fibrosis and both benign and malignant tumors(Lee et al., 1985, 067628 ; Lee et al., 1986, 067629 ; Warheit et al., 1997, 086055 ). Oberdörster (1996, 039852 ; 2002, 021111 ) proposed that high-dose effects observed in rats may be associated with two thresholds. The first threshold is the pulmonary dose that results in a reduction in macrophage-mediated clearance. The second threshold, occurring at a higher dose than the first, is the dose at which antioxidant defenses are overwhelmed and pulmonary tumors develop. Intrapulmonary tumors following TiO 2 exposures are exclusive to rats and are not f ound in mice or hamsters (Mauderly, 1997, 084631 ). Moreover, Lee et al. (1985, 067628 ) noted that the squamous cell carcinomas observed with prolonged high concentration TiO 2 exposures developed from the alveol ar lining cells adjacent to the alveolar ducts, whereas squamous cell carcinoma s in humans which are generally linked with cigarette smoking are thought to arise from basal cells of the bronchial epithelium. Quoting Lee et al. (1986, 067629 ), “Since the lung tumors were a unique t ype of experimentally induced tumor under exaggerated exposure conditions and have not usually b een seen in man or animals, their relevance to man in questionable.”



## 4.3.5. Summary

For any given particle size, the pattern of poorly soluble particle deposition influences clearance by partitioning deposited material betwee n regions of the respiratory tract. Particles depositing in the mouth may generally be assumed to be swallowed or removed by expectoration. Particles deposited in the posterior portions of the nasal passages or the TB airways are moved via mucociliary transport towards the nasopharynx and swallowed. Although clearance from the TB region is generally rapid, there appears to be fra ction of material deposited in the TB region of humans that is retained much longer. The underlying sites and mechanisms of long-term TB retention are not known. The primary alveolar cl earance mechanism is macrophage phagocytosis and migration to terminal bronchioles where the cell s are cleared by the mucoc iliary escalator. Clearance from both the TB and alveolar region is more ra pid in rodents than humans. Mucociliary and macrophage-mediated clearance decreases with age beyond adulthood.

December 2009 4-25Human data show that there is not a rapid or significant amount of UF carbon particle migration into circulation. However, both in vitro and in vivo animal studies support the rapid ( ≤ 1 h) translocation of free UF TiO 2 particles across pulmonary cell membranes. Extrapulmonary translocation has also been described in rats for po orly soluble UF gold and Ir particles. A low, but statistically significant, fraction (0.03-0.06% of lung concentration) of UF gold particles has been observed in the blood of rats from 1 to 7 days post-inhalation. The translocation in detectable amounts (<1% of deposited material) of poorly soluble Ir particles (15 and 20 nm CMD) from the lungs of rats to secondary target organs like th e liver, spleen, brain, and kidneys has also been reported. However, the systemic distribution of particles may have occu rred via normal clearance from the lungs to the gastrointestinal tract. Although the importance of particle translo cation to the brain is not yet understood, translocation from the olfactory mucosa via the axon to the olfactory bulb has been reported in primates, rodents, and freshwater pike for nu merous compounds of varyi ng composition, particle size, and solubility. The rate of translocation is rapi d, perhaps less than an hour. In rats, as much as 8% of material may become translocated to the olf actory bulb following intranasal instillation. It is unclear to what extent translocation to the olf actory bulb and other brain regions may vary between species. Interspecies differences may predispose ra ts, more so than humans, to the deposition of particles in the olfactory region with subsequent translocation to the olfactory bulb.



# 4.4. Clearance of Soluble Materials

Soluble particles and soluble constituents of pa rticles may be absorbed through the epithelium and distributed systemically or retained in the l ung. The rate of dissolution depends on a number of factors, including particle surface area and chemi cal structure. Some dissolved materials bind to proteins or other components in th e airway surface liquid layer. In the ciliated airways, solutes are cleared by mucociliary transport and diffuse into underlying tissues and the blood. In the alveolar regions, the thin barrier between the air and blood al lows for rapid transport of solutes into the blood. The movement of soluble materials depends on the site of deposition in the lung, the rate of material dissolution from particles, and the molecular wei ght of the solute. The rate of soluble material clearance from the lungs depends on epithelial permeability which may be affected by age, respiratory disease, and concurrent exposures. While enhanced clearance of insoluble particles acts to reduce dose to airway tissue, increased tra nsport of soluble matter into the blood stream may enhance effects on extra-pulmonary organs.



## 4.4.1. Clearance Mechanisms and Kinetics

The rate of absorption across the epithelium for materials that dissolve in the airway or alveolar lining fluid is fairly rapid (minutes to hours) and is a function of their molecular size and their water or lipid solubility (Enna and Schanker, 1972, 155767 ; Huchon et al., 1987, 024923 ; Oberdörster, 1988, 006857 ; Schanker et al., 1986, 005100 ). Huchon et al. (1987, 024923 ) studied the clearance of a variety of aerosolized solutes from the lungs of dogs. Solute clearance was inversely related to molecular weight. Neglig ible clearance of the largest molecular weight solute (transferrin mol wt ~76,000 daltons) in their study was found over a 30-min observation period. At the other extreme, free pertechnetate (mol wt ~163 daltons) ha d a clearance rate of 6% per min. Clearance of hydrophilic solutes is diffusion limited by pore size s associated with intercellular tight junctions (estimated at 0.6-1.5 nm). Absorption of li pophilic compounds that pass easily through cell membranes is perfusion limited and thus generally o ccurs very rapidly. However, if lipophilic materials are adsorbed onto insoluble particles their retention in the lung may be prolonged (Creasia et al., 1976, 059713 ). In addition to diffusion through interce llular junctions, transcellular transport of large solutes by pinocytosis into epithelial cells has also been observed (Chinard, 1980, 156341 ). A portion of poorly soluble particles may become dissolved with subsequent solute clearance. More rapid dissolution of poorly soluble nano- or UF particles relative to micro-sized particles occurs due to an increasing surface-to-volume ratio with decreasing particle size. Kreyling et al. (2002, 037332 ) examined the dissolution of poorly soluble UF Ir particle agglomerates (15-80 nm CMD) composed of 5 nm primary particles. After 7 days, <1% of the particles were dissolved inbuffered saline, whereas 6% dissolved in 1 N hydrochloric acid after 1 day. Thus, the high surfaceDecember 2009 4-26to-volume ratio of UFPs should not be misconstrued to imply rapid dissolution of poorly soluble particles following deposition in the respiratory tract. However, poorly soluble particles that become phagocytosed may slowly dissolve in the acidic (p H of 4.3-5.3) environment of the phagolysosome to be released in their solubilized form from the cell and potential move across the epithelium into the bloodstream. The dissolution rate is inversely re lated to particle size and directly related to specific surface area (Kreyling and Scheuch, 2000, 056281 ) and facilitated by the acidic environment of the macrophage (Kreyling, 1992, 067243 ). There is considerable evidence that soluble particles depositing in the bronchial airways are cleared by dual mechanisms (Bennett and Ilowite, 1989, 000835 ; Lay et al., 2003, 155920 ; Matsui et al., 1998, 040405 ; Sakagami et al., 2002, 156936 ; Wagner and Foster, 2001, 156143 ). The relative contribution of their removal by transepithelial abso rption versus mucociliary clearance is likely a function of both the molecular size and water or lip id solubility of the material (Enna and Schanker, 1972, 155767 ; Huchon et al., 1987, 024923 ; Oberdörster, 1988, 006857 ; Sakagami et al., 2002, 156936 ). Furthermore, the rate of mucociliary transpor t for soluble particles may be less than that of insoluble particles (Lay et al., 2003, 155920 ). Consequently, non-permeating hydrophilic solutes may remain in contact with the airway epithelium fo r a longer period than insoluble particles. This may be due to diffusion of a greater portion of the solute into the periciliary sol layer which may be transported less efficiently than the mucus layer during mucociliary clearance. Bronchial blood flow has also been shown to modulate airway retention of soluble particles (Wagner and Foster, 2001, 156143 ), i.e., decreasing blood flow increases airway retention of soluble particles. As an example of how transport of soluble components of PM may clear the lung by transepithelial absorption, Wallenborn et al. (2007, 156144 ) measured elemental content of lungs, plasma, heart, and liver of healthy male WKY ra ts (12-15 wk old) 4 or 24 h following a single intratracheal (IT) instillation of saline or 8.33 mg /kg of oil combustion PM containing a variety of transition metals with differing water and acid sol ubility. Metals with high water solubility and relatively high concentration in oil combustion PM were increased in extrapulmonary organs. Elements with low water or acid solubility, lik e silicon and aluminum, were not detected in extrapulmonary tissues despite decreased levels in the lung suggesting they cleared the lung primarily by mucociliary clearance. Thus, PM-ass ociated metals deposited in the lung may be released into systemic circulation at different rates depending on their water/acid solubility. The amount and type of water soluble or leachabl e metals associated with PM varies with location and by source. Furthermore some metals such as Zn, Cu, and Fe are essential to body function while others such as vanadium and nickel are nonessential metals. Consequently the body and the lung have different ways of dealing with excesses in inhaled soluble metals associated with PM. Bioavailability, and potentially the toxicity, of leachable metals may be altered by protein binding within the lung and blood as well as the affinities of these binding sites. For example Zn is tightly regulated by a variety of metal binding prot eins, including metallothionein and a family of Zn specific transporters. In the plasma, Zn binds to many proteins, including α 2-macroglobulin and albumin. Zn is an example of a common abundant water soluble metal in ambient air that may contribute to increased respiratory and cardiovascular disease risk associated with PM exposure. Wallenborn et al. (2009, 191172 ) recently showed that soluble Zn sulfate (in the form of 70 Zn, a rare isotope of Zn) introduced into the lungs by instillation not only reaches, but accumulates in extrapulmonary organs, including the heart and liver, following pulmonary exposure. However, the retention of greater than 50% of 70 Zn in the lung at 4 h post-instillation suggested that the transepithelial absorption of soluble Zn was indeed slowed by binding to proteins in the lungs. While it could not be ascertained if 70 Zn measured in the heart was replacing endogenous Zn pools, any accumulation in cardiac Zn levels could lead to mitochondrial dysfunction and ion channel disruption, possibly explaining adverse cardiac effects from inhalation of Zn-rich PM. Effects of Zn instillation on epithelial integrity were not evaluated.



## 4.4.2. Factors Modulating Clearance

A number of studies have evaluated the epithel ial permeability by measuring the clearance of

99m Tc-diethylenetriaminepentaacetic acid ( 99m Tc-DTPA), a small hydrophilic solute (492 daltons, 0.57 nm). These studies are the basis for much of the discussion in this section.

December 2009 4-27

## 4.4. 2.1. Age

In humans, the clearance of water-soluble particles ( 99m Tc-DTPA) from the alveolar epithelium generally slows with increasing age (Braga et al., 1996, 156289 ; Pigorini et al., 1988, 156027 ). However, Tankersley et al. (2003, 096363 ) recently showed enhanced permeability of soluble particles ( 99m Tc-DTPA) in terminally senescent mice just before death, suggesting that a disintegration of the epithelial barrier may be a f eature of lung homeostatic loss during this period of terminal senescence.



## 4.4. 2.2. Phys ical Activity

The transepithelial transport rates of soluble particles, 99m Tc-DTPA, have also been found to increase during exercise (Hanel et al., 2003, 155826 ; Lorino et al., 1989, 155946 ; Meignan et al., 1986, 156752 ). This enhancement was linked to increases in V T associated with exercise (Lorino et al., 1989, 155946 ). Regionally, this effect was dominated by increased apical lung clearance and attributed to an increase in apical blood flow (Meignan et al., 1986, 156752 ). The increased permeability with exercise appears to resolve to baseline after a short period post exercise, i.e., within a couple hours (Hanel et al., 2003, 155826 ).



## 4.4. 2.3. Dise ase

Because the integrity of the epithelial surface lining of the lungs may be damaged from lung disease, particles (either insoluble or soluble) may gain greater access to the interstitium, lymph, and blood stream. Damage to the epithelial barrier is most likely to acutely affect transepithelial transport rates of soluble particles. From bron chial biopsies, Laitinen et al. (1985, 037521 ) found various degrees of epithelial damage, from loosening of tight junctions to complete denudation of the airway epithelium, in asthmatics. Consistent with these findings, Ilowite et al. (1989, 156584 ) found that asthmatics had increased permeability of the bronchial mucosa to the hydrophilic solute

99m Tc-DTPA. On the other hand, a more recent study in a sheep model showed that the presence of bronchial edema could slow the uptake of soluble DTPA into the blood and enhanced retention in the airways, likely within the expanded inters titial barrier (Foster and Wagner, 2001, 155778 ). Both a leaky epithelial barrier and expanded interstitial barrier associated with asthma may result in enhanced exposure of submucosal immune and smooth muscle cells to xenobiotic substances. Alveolar epithelial permeability was also shown to be affected by the presence of lung inflammation. The most common finding has been a clear increase in alveolar permeability induced by cigarette smoking (Jones et al., 1980, 155883 ). This effect appears to be dependent on the recent cigarette smoke exposure as indexed by carboxyhaemoglobin (Jones et al., 1983, 155884 ) and is rapidly reversible within a week of smoking cessation (Mason et al., 1983, 013169 ). In fact, Huchon et al. (1984, 156576 ) demonstrated that COPD patients who have stopped smoking have normal clearance of 99m Tc-DTPA. In general, increased alveolar permeability to 99m Tc-DTPA has been found to be associated with any lung syndrome characterized by pulmonary edema. While the trans-alveolar transport of a small solute like DTPA is very sensitive to even mild acute lung injury (such as that associated with even mild cigarette smoking), increased transport rates of larger molecules (>100K daltons) across the alveolar epithelium require more severe damage like that seen in adult respiratory distress syndrome (ARDS) (Braude et al., 1986, 155701 ; Peterson et al., 1989, 024922 ). Interstitial lung disease and pulmonary fibrosis are also character ized by increased alveolar permeability (Antoniou et al., 2006, 156220 ; Bodolay et al., 2005, 156280 ; Watanabe et al., 2007, 157115 ). Interestingly, these recent studies have also shown that the increased permeability in these patients could be corrected with immunosuppressive/stero id treatments (Bodolay et al., 2005, 156280 ; Watanabe et al., 2007, 157115 ). Furthermore, studies of DTPA clearan ce in bleomycin injured dogs, a model of pulmonary fibrosis, suggest that the enhanced permeab ility is associated with the initial acute phase of the lung damage, with clearance rates returning to normal as chronic fibrosis developed over time (Suga et al., 2003, 157024 ). Finally, as evidence of lung complications associated with non-insulin dependent diabetes (type 2), Lin et al. (2002, 155932 ) found impairment of alveolar integrity as shown by increased transport rates of both hydrophilic and lipophilic solutes from the lungs in these patients. By contrast, a number of other studies have found epit helial permeability reduced, i.e., slower transport

December 2009 4-28rates, in diabetes (Caner et al., 1994, 156320 ; Mousa et al., 2000, 156786 ; Ozsahin et al., 2006, 156833 ) that may be related to disease duration an d metabolic control (Ozsahin et al., 2006, 156833 ) . These findings are consistent with thickening of alveolar basement membrane detected in autopsies of diabetes patients (Weynand et al., 1999, 157140 ).



## 4.4. 2.4. Conc urrent Exposures

The integrity of the alveolar epithelium may be disrupted by co-pollutants such that soluble components of inhaled particles can more easily en ter the interstitium and blood stream. Like active cigarette smoking discussed previously, Beadsmoore et al. (2007, 156259 ) showed clearance halftimes in healthy passive smokers to be shorter compared with healthy non-smokers but still longer than in healthy smokers. These findings show a progressive increase in epithelial permeability with exposure to cigarette smoke. Similarly, acute expo sure of humans to 0.4 ppm ozone for 2 h with intermittent exercise has been shown to alter ep ithelial integrity and increase clearance of soluble hydrophilic particles from the alveolar surfa ces of the lung (Kehrl et al., 1987, 040824 ). This effect persists to at least 24 h post-exposure even following lower exposure levels (0.24 ppm average for 130 min) of ozone (Foster and Stetkiewicz, 1996, 079920 ). Similarly, 0.8 ppm O 3 exposure for 2 h in rats caused increased permeability to macromolecules at all levels of the respiratory tract (Bhalla et al., 1986, 040407 ) that persisted in the alveolar regi on beyond 24 h post-exposure. Cohen et al. (1997, 009213 ) may have best illustrated the competing effects of mucociliary and transepithelial transport by showing that coexposure to ozone affected the retention of inhaled chromium in rats differently depending on its solubility. In its sol uble potassium chromate form, ozone decreased the retention of chromium, but when chromium was inha led as insoluble barium chromate, its retention in the lung was increased by ozone coexposure. Si milarly, a study that showed decreased clearance of insoluble cesium oxide particles following in fluenza infection also showed a virus-induced enhancement of clearance for a soluble cesium chloride (Lundgren et al., 1978, 155950 ). Chang et al. (2005, 097776 ) also recently showed that UF carbon black acts through a reactive oxygen species (ROS) dependent pathway to increase epithelial permeability in mice. Chronic exposure to other particulate or gaseous pollutants does not always led to increased epithelial permeability. Studying subjects with a va riety of occupational exposures, Kaya et al. (2006, 156632 ) showed that nonsmoking welders actually have decreased epithelial permeability relative to nonsmoking control subjects, and occupational exposure of painters to isocyanates has no effect on bronchoalveolar epithelial permeability (Kaya et al., 2003, 156631 ).



## 4.4.3. Summary

The healthy airway and alveolar epithelium is generally impermeable to very large insoluble macromolecules and particles. Water and acid sol uble particles may more rapidly move through the epithelium as they dissolve on the airway surface or within the phagolysomes of macrophages. The presence of airway inflammation in a variety of airway diseases (e.g., asthma, fibrosis, ARDS, pulmonary edema, inflammation from smoking) alte rs epithelial integrity to allow more rapid movement of these solutes into the bloodstream. Wh ile diabetics are another group recently shown to have increased susceptibility to particulate air pollution (Zanobetti and Schwartz, 2002, 034821 ), it is unclear whether transport of soluble particles acr oss the epithelium is affected in these patients. In general, it appears that coexposure to irritant pollu tants results in a disruption of epithelial integrity and macrophage function which, on the one hand, re tards mucociliary and alveolar clearance, but also allows for a more rapid movement of soluble constituents across the epithelial surface into the interstitium and blood stream. Alterations in epithelial permeability by disease, pollutant exposure, or infection may partially explain increased suscepti bility to PM associated with these co-conditions.

December 2009 4-29

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