18 Models for Testing the Pulmonary Toxicity of Particles: Lung BioassayScreening Studies in Male Rats with aNew Formulation of TiO 2 Particulates David B. Warheit, Kenneth L. Reed, and Christie M. Sayes DuPont Haskell Laboratory for Health andEnvironmental Sciences CONTENTS 18.1 Introduction 317 18.2 Methods 319 18.2.1 General Experimental Design 319 18.2.2 Animals 320 18.2.3 Particle Types 320 18.2.4 Lung Cell Proliferation Studies 320 18.2.5 Bronchoalveolar Lavage Methods 321 18.2.6 Lung HistopathologyStudies 321 18.2.7 Statistical Analyses 321 18.3 Results 321 18.3.1 Lung Weights 321 18.3.2 Lung Cell Proliferation Results 321 18.3.3 Bronchoalveolar Lavage FluidResults 323 18.3.3.1 Pulmonary Inflammation 323 18.3.3.2 Bronchoalveolar Lavage (BAL) Fluid Parameters 323 18.3.3.3 Pulmonary Histopathological Evaluations 325 18.4 Discussion 325 Acknowledgments 329 References 329 18.1 INTRODUCTION Formulation changesinthe form of altered surface treatments are known to frequently occur for a variety of commercialized particle-types. The R-100 formulation of rutile-type titanium dioxide is a well-knownlow-toxicity particulate.Thisstudywas designed as apulmonary screening tool to determinewhether PigmentATiO 2 particles(TiO 2 particles that have been substantially 317 © 2007 by Taylor & Francis Group, LLC encapsulated with pyrogenically deposited amorphoussilica) may impart significant toxicity in the lungs of rats; and moreimportantly, how the activityofthis TiO 2 formulation compares with other reference particulate materials, including standardized TiO 2 particulates. Thus, the objective of this study wastoevaluateinthe lungs of rats—usingawell-developed, short-term pulmonary bioassay—the acutepulmonary toxicity effects of intratracheally instilled Pigment ATiO 2 particle samplesand to comparethe pulmonary toxicity of thesesamples with twoother lowtoxicity particulate-types (reference negative controls), alongwithacytotoxicparticulate (reference positive control)sample. Another aim was to bridge the results obtained herein with data previously generated from inhalation studies with crystalline silica-quartz particles and with carbonyl iron particulates as the inhalation/instillation bridgematerials. Bridging studies can be useful in providing an inexpensive safety screen when assessing the hazards of new developmental compounds or when making small modifications to an existing commercial particle-type,suchassurface treatments. Thestrengthofthe bridging strategyis dependent upon having good inhalation toxicity dataonone of thebridgingcompounds. The particle-type for which inhalation data exists can then be used as areference control material for an intratracheal instillation bridgingstudy (see Figure 18.1). The basicidea for the bridging concept is that the effects of the instilled material serveasacontrol (known reference) material and then are “bridged”onthe one hand to the inhalation toxicitydata for that material, as well as to the new materials being tested. Theresults of bridging studies in rats are then useful as pulmonary toxicity screening (i.e., hazard) data, because consistency in the response of the inhaled and instilled control material serves to validatethe responses with the newly tested particulate matter. Numerous studies have investigated the pulmonary toxicological impactsofsurfaces treat- ments on titanium dioxide particles. In some cases, the surface coatingshad little or no impact on the lung toxicity of TiO 2 particles in rats [5,8–10].Inanother study, in evaluating six different formulations of commercial TiO 2 products, we reported that surface treatments might modify the lung inflammatory response following exposures in rats to fine-sized TiO 2 particle-types [2]. The Pulmonary bioassay bridging study Inhalation studies Carbonyl iron particles α -quartz particles Intratracheal instillation studies PBS sham α -quartz particles Carbonyl iron particles R-100 & Pigment A TiO 2 particles FIGURE 18.1 Schematic demonstrating the strategy for conducting pulmonary bioassay bridging studies. Bridging studies can have utility in providing an inexpensive preliminary safety screen when evaluating the hazards of new developmental compounds. The basic idea for the bridging concept is that the effects of the instilled material serve as acontrol (known) material and are then “bridged” to the inhalation toxicity data for that material and to the new materials being tested. Particle Toxicology318 © 2007 by Taylor & Francis Group, LLC current study describes amodelorpulmonary bioassay method for assessing the pulmonary hazard potentialofintratracheally instilled particle-types.The chapter alsodiscussesthe relevance of this screeningmethodology as apossiblesurrogate forinhalationstudies with low-solubility particle-types. 18.2 METHODS 18.2.1 G ENERAL E XPERIMENTAL D ESIGN The fundamental features of this pulmonary bioassay are dose-response evaluation, time-course assessments, and reference particle-types (positive and negative). The time-course studies are used to assess the sustainability of the observed affect. Themajor endpoints of this study were the (1) time-course and dose/response intensity of pulmonary inflammation and cytotoxicity (bronchoal- veolar lavage (BAL)parameters),(2) airway andlungparenchymal cell proliferation,and (3) histopathological evaluation of lung tissue (see Figure 18.2). The lungs of rats were exposed via intratrachealinstillation with single doses of 1or5mg/kg crystalline silica ( a -quartz) particles, carbonylironparticles, R-100fine-TiO 2 particles, or to Pigment Afine-TiO 2 particlescoated with amorphous SiO 2 .The intratrachealinstillation route of entry technique is not asubstitute for the more physiologically relevantinhalation method of exposure. However, the intratracheal instillation method of exposure can be aqualitatively reliable screen for assessing the pulmonary toxicity of particles [1,2].All particles were prepared in a volume of phosphate-buffered saline(PBS) solutionand subjectedtoprobe sonicationfor at least 15 minutes. Groups of PBS-instilled rats served as controls. The lungs of PBS and particle- exposed rats were evaluatedbyBAL fluid analysesat24h,1week, 1month,and 3months postexposure (pe).For lung cell proliferation andhistopathology studies, additional groups of animals were instilled with the particle-types listed above as well as aPBS solution. Forthe lung tissue studies,additionalgroups of animals (4 rats/group) wereinstilled with the particle-types listedabove plus the vehicle control, i.e., PBS. These studies were dedicated to lung tissue analyses, but only the high-dose groups (5 mg/kg) and PBS controls were utilized in the morphology studies. These studies consisted of cell proliferation assessments and histo- pathologicalevaluations of thelower respiratorytract.Similar to theBAL fluid studies,the intratracheal instillation exposure period was followed by 24-h, 1-week, 1-month, and 3-month recovery periods. Exposure groups • • PBS (vehicle control) Particle-types (1 and 5mg/kg) oR-100 fine-TiO 2 oPigment A-fine-TiO 2 coated with amorphous SiO 2 o α -Quartz particles (positive control) oCarbonyl iron (negative control) Instillation exposure Postexposure (pe) evaluation via BAL and lung tissue 24 h1week 1month 3months FIGURE 18.2 Experimental design for bridging case study. Models for Testing the Pulmonary Toxicity of Particles 319 © 2007 by Taylor & Francis Group, LLC 18.2.2 A NIMALS Groups of male Crl:CD w (SD)IGSBRrats(Charles RiverLaboratories,Inc., Raleigh, North Carolina) were used in this study. The rats were approximately8weeks old at studystart (mean weightsinthe range of 240–255 g). All proceduresusing animals were reviewed and approved by the Institutional Animal Care and Use Committee and the animal program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). 18.2.3 P ARTICLE T YPES The R-100 fine-titanium dioxide particles ( w 99 wt.% titanium dioxide, w 1wt.% alumina) posses- sing an average particle diameter of w 300 nm and an average BET surface area of w 6m 2 /g were obtained from the DuPontCompany. Apatented chloride process produced Pigment Afine-tita- nium dioxide particles with an amorphous SiO 2 surfacecoating(w 96 wt.% titanium dioxide, w 1wt.%alumina, w 3wt.%amorphous silica (particleencapsulating))possessing an average particle diameter of w 290 nm and an average BET surface area of w 7.9m 2 /g were also obtained from the DuPontCompany (Table 18.1). Both of the DuPontTiO 2 particle samples were in the rutile crystal phase and hydrophilic in nature. Crystallinesilicaparticles ( a -quartz, Min-U-Sil5)ranging in size from 0.3 to 3 m mwere obtained from the US Silica Company. Carbonyl iron (CI) particlesranging in size from 0.8 to 3.0 m mwereobtained from GAFCorporation. 18.2.4 L UNG C ELL P ROLIFERATION S TUDIES Groups of particulate-exposed rats and corresponding controls were pulsed 24 hafter instillation, as wellas1week, 1month, and3months postexposure,with an intraperitoneal injection of 5-bromo-2 0 deoxyuridine(BrdU)dissolved in a0.5 Nsodiumbicarbonate buffersolutionata dose of 100 mg/kg body weight. The animals were euthanized 6hlater by pentobarbital injection. Following cessation of spontaneous respiration, the lungs were infused with aneutral buffered formalinfixative at apressure of 21 cm H 2 O. After 20 min of fixation, the trachea was clamped, and the heart and lungs werecarefully removedenbloc and immersion-fixed in formalin.In addition,a1cmpiece of duodenum (which servedasapositivecontrol)was removedand stored in formaldehyde.Subsequently, parasagittal sectionsfromthe rightcranial andcaudal lobesand regions of the leftlung lobesaswellasthe duodenal sections weredehydrated in 70% ethanol andsectionedfor histology. Thesections were embeddedinparaffin, cut, and mounted on glass slides. Theslideswerestained with an anti-BrdU antibody, with an AEC (3-amino-9-ethylcarbazole) marker, andcounter-stainedwith aqueoushematoxylin.A minimum of 1000 cells/animal were counted in terminal bronchiolar and alveolarregions. For each treatment group, immunostained nuclei in airways (i.e., terminal bronchiolar epithelial cells) TABLE 18.1 Characterization of R-100 and PigmentAfine-TiO 2 ,asWell as Carbonyl Iron, and a -Quartz Particulates Particle Crystallinity Surface Area(m 2 /g) Average Particle Diameter (nm) R-100 fine-TiO 2 Rutile 6 w 300 Pigment Acoated with amorph SiO 2 Rutile 8 w 290 Carbonyl iron —Not determined w 1500 Crystalline silica a -quartz 5 w 480 Particle Toxicology320 © 2007 by Taylor & Francis Group, LLC or lung parenchyma(i.e.,epithelial,interstitialcells, or macrophages) were countedbylight microscopy at 1000! magnification [3,4]. 18.2.5 B RONCHOALVEOLAR L AVAGE M ETHODS Thelungs of sham andparticulate-exposed ratswerelavaged with awarmed PBSsolution as described previously. Methodologies for cell counts, differentials, and pulmonary biomarkers in lavaged fluids were conducted as previously described [3,4].Briefly, the first 12 mL of lavaged fluids recovered from the lungs of PBS or particulate-exposed rats was centrifuged at 700 g, and 2mLofthe supernatantwas removedfor biochemical studies. Allbiochemicalassays were performed on BALfluids usingaRocheDiagnostics (BMC)/Hitachi w 717clinicalchemistry analyzer using Roche Diagnostics (BMC)/Hitachi w reagents. Lactatedehydrogenase (LDH), alka- line phosphatase(ALP), andlavagefluidprotein were measured usingRoche Diagnostics (BMC)/Hitachi w reagents.LDH is acytoplasmicenzymeand is used as an indicatorofcell injury. ALP activity is ameasure of Type II alveolarepithelial cell secretory activity,and increased ALP activity in BAL fluids is considered to be an indicator of Type II lung epithelial cell toxicity. Increases in BAL fluid micro protein(MTP) concentrations generally are consistent with enhanced permeability of vascular proteins into the alveolar regions, indicating abreakdown in the integrity of the alveolar-capillary barrier. 18.2.6 L UNG H ISTOPATHOLOGY S TUDIES The lungs of rats exposedtoparticulates or PBS controlswere prepared for light microscopy by tracheobronchial airwayinfusion under pressure (21 cm H 2 O) at time periods of 24 hours,1week, 1 month, and 3months postexposure. Sagittal sections of the left and right lungs weremade using a razor blade. Tissue blockswere dissected from left, right upper, and right lower regions of the lung and were subsequently prepared for light microscopy (paraffin embedded, sectioned, and hematox- ylin–eosin stained) [3,4]. 18.2.7 S TA TISTICAL A NALYSES For analyses,each of the experimental values were compared to their corresponding sham control values for each time point. Aone-way analysis of variance(ANOVA) and Bartlett’s test were calculated for each sampling time. When the Ftest from ANOVA was determined to be significant, the Dunnett’s test was utilizedtocompare means from the control group and each of the groups exposedtoparticulates. Significance was judgedatthe 0.05 probability level. 18.3 RESULTS 18.3.1 L UNG W EIGHTS Lung weights of rats were enhanced with increasing age on the study (i.e., increased postexposure time periods followingintratracheal instillation exposures). Lung weights in high-dose quartz- exposedrats were slightly increased relative to controls at 1week, and at 1monthand 3months postexposure (data not shown). 18.3.2 L UNG C ELL P ROLIFERATION R ESULTS Tracheobronchial cell proliferationrates (percentage of immunostainedcells with BrdU) were measured only in high-dose(5mg/kg)particulate-exposed rats andcorrespondingcontrols at 24 h, 1week,1month, and3months postexposure(pe). Although increases in cell labeling Models for Testing the Pulmonary Toxicity of Particles 321 © 2007 by Taylor & Francis Group, LLC indices were measured in R-100 fine-TiO 2 as well as a -quartz-exposed animalsat24hpostexpo- sure,theseeffects were not sustained (data not shown). Lung parenchymal cellproliferation rates (percentage of immunostained cells with BrdU)were measured only in high-dose (5 mg/kg) particulate-exposed rats and corresponding controls at 24 h, 1week, 1month, and 3months postexposure (pe). Small but significant transient increases in lung cell proliferationindices were measured in carbonylironparticle or in PigmentAfine-TiO 2 particle-exposed rats at 24 h, but these effects were notsustained at any other postexposure time points. Significantly larger increases in cellproliferation indices were measured in the lungs of a -quartzexposed rats measured from24hpostexposure through 3months postexposure (data not shown). To summarize, exposures to 5mg/kg quartz particlesproduced increased tracheobronchial cell proliferation comparedtoPBS controls, butincreases werestatistically significant only at 24 h postexposure. In contrast to tracheobronchial cell labeling indices, exposures to 5mg/kg quartzparticlesproduced substantiallygreaterlung parenchymal cellproliferationrates at all time pointspostexposure,suggesting agreater likelihood to result in lung cellgenotoxicity or otheradverse pulmonary effects over time with continued exposures. Total cells in BAL fluids of rats exposed to pigment ATiO 2 particles and other particulates 0.00E+00 5.00E+06 1.00E+07 1.50E+07 2.00E+07 2.50E+07 Control 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Carbonyl iron R-100 Pigment A TiO 2 Quartz Exposure groups Mean number total cells in BAL fluids 3month 1month 1week 24 h FIGURE 18.3 Total number of cells in BAL fluids recovered from particulate-exposed rats and controls as evidenced by %neutrophils (PMN) in BAL fluids at 24 h, 1week, 1month, and 3months postexposure (pe). Values given are meansG S.D. The numbers of BAL cells recovered from the lungs of high-dose quartz groups were substantially higher than any other groups for all postexposure time periods. Particle Toxicology322 © 2007 by Taylor & Francis Group, LLC 18.3.3 B RONCHOALVEOLAR L AVAGE F LUID R ESULTS 18.3.3.1 Pulmonary Inflammation The numbers of cells recovered by BAL from the lungs of high-dose a -quartz-exposed (5 mg/kg) groups were substantiallyhigher than any of the other groups for all postexposure time periods (Figure 18.3). Intratracheal instillation exposures of virtually all particle-types produced ashort- term pulmonary inflammatory response, as evidencedbyanincrease in the percentages/numbers of BAL-recovered neutrophils,measuredat24hpostexposure.However, only theexposures to a -quartzparticles (1 and5mg/kg) produced sustainedpulmonary inflammatory responses, as measured through 3months postexposure (Figure18.4). 18.3.3.2 Bronchoalveolar Lavage(BAL) Fluid Parameters Transient and reversibleincreases in (BAL) fluid LDH values, as an indicator of general cyto- toxicity, weremeasured in the lung fluids of high-dose (5 mg/kg) R-100 fine-TiO 2 -exposed rats at 1 week postexposure, but werenot sustained through the otherpostexposure time periods. In contrast, exposures to high-dose (5 mg/kg) a -quartz particles produced apersistent enhancement in BAL fluid LDHvaluesthroughoutthe 3-monthpost-instillationexposure period (Figure18.5A). Percent neutrophils in BAL fluids of rats exposed to pigment ATiO 2 particles and other particulates 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% Control 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Carbonyl iron R-100 Pigment A TiO 2 Quartz Exposure groups Mean %PMNs 3month 1month 1week 24 h * * * * * * * * FIGURE 18.4 Pulmonary inflammation in particulate-exposed rats and controls as evidenced by %neutro- phils (PMN) in BAL fluids at 24 h, 1week, 1month, and 3months postexposure (pe). Values given are meansG S.D. Intratracheal instillation exposures of several particle-types produced ashort-term, pulmonary inflammatory response, as evidenced by an increase in the percentages/numbers of BAL-recovered neutro- phils, measured at 24 hpostexposure. However,onlythe exposures to quartz particles(1and 5mg/kg) produced sustainedpulmonary inflammatory responses,asmeasured through3months postexposure. * p ! 0.05. Models for Testing the Pulmonary Toxicity of Particles 323 © 2007 by Taylor & Francis Group, LLC Control 0.00 50.00 100.00 150.00 200.00 250.00 u/L LDH MTP Alkaline phosphatase 300.00 350.00 400.00 450.00 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Carbonyl iron R-100 Pigment A TiO 2Q uartz Exposure groups 3month 1month 1week 24 h * * * * * Control 0.00 10.00 20.00 30.00 40.00 50.00 mg/dL 60.00 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Carbonyl iron R-100 Pigment A TiO 2Q uartz Exposure groups 3month 1month 1week 24 h * * * * * * * Control 0.00 20.00 40.00 60.00 80.00 100.00 u/L 120.00 140.00 160.00 180.00 200 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg 1mg/kg 5mg/kg PBS Carbonyl iron R-100 Pigment A TiO 2Q uartz Exposure groups 3month 1month 1week 24 h A B C FIGURE 18.5 (See facing page.) Particle Toxicology324 © 2007 by Taylor & Francis Group, LLC Similarly, transient increases in BAL fluid microprotein (MTP) values were measured in the lung fluids of high-dose (5 mg/kg) R-100 fine-TiO 2 -exposed rats at 24 hpostexposure, but were not different from control values at 1week postexposure. In contrast, exposures to 5mg/kg a -quartz particlesproduced persistent increases in BAL fluid microprotein values at 24 h, 1week, 1month, and 3months postexposure (Figure 18.5B). Transientincreases in BAL fluid ALP values were measured only in the lungs of R-100 fine-TiO 2 -exposed rats at 1weekpostexposure (5 mg/kg),but substantial increases in BAL fluid ALP values were measured at 1week through 3months post- exposure in rats exposedto5mg/kg a -quartz particles(Figure 18.5C). To summarize the results from BAL fluid biomarker studies, intratracheal instillation exposures to a -quartz particles resulted in sustained, dose-dependent,lung inflammatory responses, associ- ated with cytotoxicand lung permeability effects,measured from24hthrough3months postexposure. Exposures to high-dose R-100 fine-TiO 2 particles (5 mg/kg)produced small but transient pulmonary inflammatory responses, and these effects were not sustained. Exposures to carbonyliron particlesortoPigment Afine-TiO 2 particles produced abrief neutrophilic response at 24 hpostexposure; however,this was in large part related to the bolus dose associated with the intratracheal instillation exposure methodology. 18.3.3.3 Pulmonary Histopathological Evaluations Histopathological analyses of lung tissues revealed that pulmonary exposures to carbonyl iron, to R-100 fine-TiO 2 particles, or to Pigment Afine-TiO 2 particlesinrats produced no significant adverse effectswhencomparedtoPBS-exposed controls,asevidenced by thenormallung architecture observed in the exposedanimals at postinstillation exposure time periods ranging from 24 hto3months (Figure18.6Aand B). Histopathologicalanalyses of lung tissues at several time points postexposure demonstrated no differencesbetween the R-100 fine-TiO 2 -exposed rats and those exposed to Pigment Afine-TiO 2 particles (Figure18.6B). Alight micrograph of alung tissue section of arat instilled with 5mg/kg carbonyl iron particlesat1month postexposure demonstrated appropriatealveolar macrophage phagocytic responses and normal lung architec- ture. Lung tissue sections from rats instilled with 5mg/kg R-100fine-TiO 2 particles appeared very similar histologically to the lung tissue sections recoveredfrom the Pigment Afine-TiO 2 particle-exposed rats at each postexposuretime period, anddemonstrated normal pulmonary architecture. Histopathological analyses of lung tissues in rats exposedto a -quartz particulates in rats revealedthat pulmonary exposures produced dose-dependent pulmonary inflammatory responses characterized by neutrophils and accumulations of foamy (lipid-containing) alveolar macrophage. In addition, lung tissue thickening as aprerequisite to the development of fibrosis was observed and progressive over postexposure time periods (Figure 18.6Cand Figure 18.6D). 18.4 DISCUSSION This chapter presents acase studyand methodology designed to investigate the hazard potential of changing the formulation (i.e., surface coating)ofaTiO 2 particle-type. More importantly, the study assessed how the activityofthis TiO 2 formulation, identified as Pigment Afine-TiO 2 particles, FIGURE 18.5 BAL fluid analyses for particulate-exposed rats and corresponding controls at 24 h, 1week, 1month, and 3months postexposure (pe). (A) LDH (lactate dehydrogenase), (B) MTP (microprotein values), and (C) alkaline phosphatase. Values given are meansG S.D. Transient and reversible increases in BAL fluid values were measured in the lungs of high-dose (5 mg/kg) R-100 fine-TiO 2 -exposed rats at 1week postexpo- sure. In contrast, exposures to 5mg/kg a -quartz particles produced sustained increases in BAL fluid LDH values through the 3-month postexposure period. * p ! 0.05. Models for Testing the Pulmonary Toxicity of Particles 325 © 2007 by Taylor & Francis Group, LLC compared with othernegative control,reference particle-types such as R-100 fine-TiO 2 particlesor carbonylironparticles. ThecombinationofBAL and lungtissuestudies concomitant with an experimental designconsisting of dose-response, time-course evaluations, and the inclusionof reference particle-types provides apowerful tool for assessing the acute pulmonary toxicity of this new particle-type. Thebioassay described herein provides evidence that toxicological information on aparticle’s various surface treatments can be assessed in aroutine systematic manner. Abeneficial featureof the bioassay is the ability to compare, via bridging strategies, the effects of inhaled versus instilled particulate materials. In this regard, pulmonary bridgingstudies can generateimportant preliminary hazard data whenassessing the safety of new developmental or commercial compounds or when making modifications to existing chemical products, such as surface coatingsonparticulates. The strengthofthe bridging strategyisdependent upon having good inhalation toxicity data for comparisons to the instillation data.The materials for which there is inhalation data can then be used as controlparticle-types forcomparingwithanintratracheal instillationbridging study (Figure 18.1). The basic idea for the bridging concept is that the effects of the instilled material serveasacontrol (known) material and then are “bridged”tothe inhalation toxicity data for that material, as well as to the new materials beingtested.The results of bridgingstudies in rats are then useful as preliminary pulmonary toxicity screening (i.e., hazard) data, because consistency in the response of the inhaled and instilled control material serves to validate the responses with the newly TB AD AD TB AD AD A C B D FIGURE 18.6 Light micrographs of lung tissue from arat exposed to (A) carbonyl iron particles (5 mg/kg) at 1month postexposure and (B) Pigment Afine-TiO 2 particles (5 mg/kg) at 3months postinstillation exposure (magnificationZ 100! ). Light micrographs of lung tissue from arat exposed to a -quartz particles (5 mg/kg) at 3months postinstillation exposure using (C) low magnification (40! )and (D) high magnification (200! ). For (A) and (B), these micrographs illustrate the terminal bronchial (TB) and corresponding alveolar ducts (AD) and demonstrate the lung architecture and normal macrophage phagocytosis of each particle (arrows). For (C) and (D), the arrows demonstrate accumulation of foamy alveolar macrophagesinthe alveolar regions of quartz-exposed rats. Particle Toxicology326 © 2007 by Taylor & Francis Group, LLC [...]... pulmonary toxicity potential of fine-titanium dioxide particles made hydrophobic by surface application of octyltriethoxysilane (OTES) was assessed [5] In similar-type pulmonary bioassay studies, at higher doses (2 and 10 mg/kg), the toxicity of OTES-coated TiO2 particles was not significantly different from the hydrophilic R-100 fine-TiO2 particles R-100 fine-TiO2 has a mean particle diameter of 300 nm and... Toxicity of Particles 327 tested particle- type It should be noted, however, that these pulmonary screening types of studies are not to be used as substitutes for longer-term, more substantive inhalation toxicity studies, such as 90-day inhalation studies or 2-year inhalation bioassay studies Based upon the data developed from this pulmonary bioassay, it was concluded that Pigment A fine-sized TiO2 particles... hydrophilic and hydrophobiccoated titanium dioxide particles As discussed above, we have previously evaluated in rats the pulmonary toxicity of instilled hydrophilic versus hydrophobic fine-TiO2 particles, using a pulmonary bioassay methodology The results demonstrated that only the high-dose (10 mg/kg) hydrophilic fine-TiO2 particles and those with particle- types containing a surfactant, Tween 80, produced... instillation exposure to ultrafine hydrophobic-coated titanium dioxide particles (T-805 sample) resulted in unexpected mortality of exposed rats [7,8] In their study, two rats treated with 6 mg hydrophobic ultrafine-TiO2 particles (T-805) demonstrated immediate symptoms of respiratory damage when compared to animals treated with other dusts, and the rats survived less than one-half hour; 3 mg also induced a fatal... produced by R-100 fine-TiO2 particles In a recent pulmonary bioassay study in rats, we evaluated the pulmonary toxicity of inhaled as well as intratracheally instilled TiO2 particle- types with various surface coatings, ranging from 0 to 6% alumina (Al2O3) and/or 0 to 11% amorphous silica (SiO2) The effects from exposures to the coatings on TiO2 particles were compared to animals exposed to base TiO2 particles... or ultrafineTiO2 particles ¨ In another study comparing effects of surface treatments, Oberdorster [9] exposed rats via intratracheal instillation to two different types of aggregated ultrafine-TiO2 particle samples (particle size of both types reported to be w20 nm) at doses of 50 or 500 mg One surface treatment was silane-coated, making the particle surface hydrophobic, while the other particle sample... reference particle- types In addition, we have combined BAL-based investigations concomitant with lung tissue evaluations Accordingly, based on the findings in the case study described herein, intratracheally instilled Pigment A TiO2 particles with amorphous silica surface treatment exhibited no significant differences in pulmonary toxicity responses when compared to R-100 TiO2 particles or carbonyl iron particles... hydrophilic, fine-TiO2 particles or carbonyl iron particles in rats produces low pulmonary toxicity and induces adverse inflammatory effects only at substantial particle overload concentrations [4,11–15] In the study reported herein, the highest dose of instilled hydrophilic, R-100 fine-TiO2 particles coated with amorphous SiO2 (5 mg/kg) or carbonyl iron particles produced only a minor, transient lung... hydrophobic-coated, ultrafine-TiO2 particles produced a reduced pulmonary inflammatory response at 24 h postexposure when compared to ¨ identical doses of the uncoated, hydrophilic TiO2 particles Oberdorster reported that his findings appear to conflict with an earlier report by Pott and coworkers [6,7] The results of numerous studies demonstrate that inhalation exposure to hydrophilic, fine-TiO2 particles... potential toxicity of 1% Tween, which was selectively added as a detergent to the T-805 sample but not to the P-25 sample Thus, it was conceivable that the detergent significantly contributed to the toxic effects observed in the T-805-exposed rats © 2007 by Taylor & Francis Group, LLC 328 Particle Toxicology The results reported by Pott and colleagues have not been confirmed by a variety of other investigators . was followed by 24-h, 1-week, 1-month, and 3-month recovery periods. Exposure groups • • PBS (vehicle control) Particle- types (1 and 5mg/kg) oR-100 fine-TiO 2 oPigment A-fine-TiO 2 coated with. fine-sized TiO 2 particle- types [2]. The Pulmonary bioassay bridging study Inhalation studies Carbonyl iron particles α -quartz particles Intratracheal instillation studies PBS sham α -quartz particles Carbonyl iron particles R-100. instilled particle- types.The chapter alsodiscussesthe relevance of this screeningmethodology as apossiblesurrogate forinhalationstudies with low-solubility particle- types. 18. 2 METHODS 18. 2.1 G