DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, Delaware
1 To whom correspondence should be addressed at DuPont Haskell Lab, 1090 Elkton Rd., PO Box 50, Newark, DE 19714-0050. Fax: (302) 366-5207. E-mail: david.b.warheit{at}usa.dupont.com.
Received July 12, 2005; accepted September 14, 2005
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ABSTRACT |
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Key Words: titanium dioxide particles; pulmonary toxicity; surface treatments on particles; particle coatings.
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INTRODUCTION |
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Pulmonary bridging studies can serve to provide relevant and accurate screening hazard data when assessing the safety of compounds in commercial development or when making modifications to existing products, such as surface treatments on particulates. The strength of the bridging strategy is dependent upon having good inhalation toxicity data for comparisons to data developed from instillation studies. The materials for which there is inhalation data can then be used as reference particle-types for comparison to the pulmonary bioassay (i.e., instillation) results (see Fig. 2). Thus, in describing the basic bridging concept, the effects of the instilled materials serve as a reference (known) particle-type and then are "bridged" to the inhalation toxicity data for that particle-type, concomitant with the new materials being tested (compared). The results of bridging studies 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 tested particle-type.
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MATERIALS AND METHODS |
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Animals.
Groups of male Crl:CD(SD)IGS BR rats (Charles River Laboratories, Inc., Wilmington, MA [inhalation] and Raleigh, NC [instillation]) were utilized in both the inhalation and instillation studies (CDIGS is the Charles River designation for SD, Sprague Dawley rats). The rats were approximately 8 weeks old at study start (body weights in the range of 240255 g). All procedures using 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).
Particle types.
Several rutile-type titanium dioxide formulations were tested in the two studies. The compositions of the nearly identical TiO2 formulations for both inhalation and intratracheal instillation studies are presented in Table 1. Particle sizes and corresponding surface areas (as measured by the BET nitrogen method; Brunauer et al., 1938) of the various TiO2 formulations are presented in Table 2. It should be noted that Base TiO2 and TiO2-I formulations are used commercially as a white pigment in plastics. TiO2 formulation II is used as a white pigment for interior paints. TiO2 formulations III and IV are utilized in exterior paints; and TiO2 formulation V is utilized in a wide variety of painting applications, including automotive paints. All TiO2 particle formulations (rutile type) were obtained from the DuPont Company (Wilmington, DE).
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Chamber atmospheric concentrations of TiO2 were determined using gravimetric methods. Particle sizes in the form of aerodynamic mass median diameter of the dust particles were determined using a cascade impactor.
Immediately following the last exposure, as well as 14 days, 3, 6, and 12 months postexposure, 5 rats from each of the control and test groups were sacrificed by chloroform anesthesia and exsanguinations and subjected to a histopathological evaluation. Body and lung weights were determined from all rats necropsied.
Pulmonary bioassay instillation study (see Fig. 4).
The primary features of this pulmonary bioassay are (1) dose response assessments, and (2) time course evaluations to gauge the persistence of any observed effect. Thus, the major endpoints of this study were the following: (1) time course and dose/response intensity of pulmonary inflammation and cytotoxicity; and (2) cell proliferation and histopathological assessments of particle-exposed lung tissues.
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Bronchoalveolar lavage.
The lungs of sham and TiO2-exposed rats were washed via bronchoalveolar lavage (BAL) with a phosphate-buffered saline (PBS) solution. Procedures for cell counts, differentials and pulmonary biomarkers in BAL fluids were implemented as previously reported (Warheit et al., 1991). The first 12 ml of BAL fluids recovered from the lungs of saline- or- TiO2-exposed rats were centrifuged at 700 x g, and 2 ml of the supernatant BAL fluid was removed for biochemical studies. All biochemical assays were performed on BAL fluids using a Roche Diagnostics (BMC)/Hitachi 717 clinical chemistry analyzer using Roche Diagnostics (BMC)/Hitachi reagents. Lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and lavage fluid microprotein were measured using Roche Diagnostics (BMC)/Hitachi reagents. Lactate dehydrogenase is a cytoplasmic enzyme and is used as an indicator of cell injury. Alkaline phosphatase activity is a measure of Type II epithelial cell secretory activity and increased ALP activity in BAL fluids is considered to be an indicator of Type II cell toxicity. Increases in BAL fluid microprotein (MTP) concentrations generally are consistent with enhanced permeability of vascular proteins into alveolar regions, and may also be reflective of dead cells and secretions.
Morphological studies.
The lungs of rats exposed to TiO2 particles or saline were prepared for microscopy by airway infusion of a 10% formalin solution under pressure (21 cm H2O) at 24 h, 1 week, 1 and 3 months postexposure. Sagittal sections of the left and right lungs were made with a razor blade. Tissue blocks were dissected from left, right upper, and right lower regions of the lung and were subsequently prepared for light microscopy (paraffin embedded, sectioned, and hematoxylin-eosin stained), as described previously (Warheit et al., 1991, 1997
).
Statistical analyses.
For analysis, each of the experimental values were compared to their corresponding sham control values for each time point. A one-way ANOVA and Bartlett's test were calculated for each sampling time. When the F test from ANOVA was significant, the Dunnett test was used to compare means from the control group and each of the groups exposed to titanium dioxide. Significance was judged at the 0.05 probability level.
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RESULTS |
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The greater lung weights were evident at the end of the 4-week exposure period as well as throughout the 12-month recovery period (see Table 4).
Histopathology.
By the end of the 4-week exposure period, the inhaled dust particles for all TiO2 formulations were heavily concentrated within the bronchioles, alveolar ducts, and alveoli where the dust particles had been mostly phagocytized by the alveolar macrophages. In addition, dust cell (particle-containing macrophages) accumulation and alveolar cell hyperplasia were observed in all TiO2-exposed rats at both the 14-day and 3-month postexposure evaluations. For all TiO2 formulations, there was a marked reduction in dust cells and alveolar cell proliferation but particle-containing macrophages were still evident at the 6 and 12-month postexposure time periods.
At the 3-month postexposure evaluation period, rats that had been exposed to TiO2 formulations III and V exhibited hyperplasia of the alveolar Type II epithelial cells and disintegration of dust cells which released numerous dust particles into the alveoli. Alveolar cell hyperplasia, observed at the 3-month postexposure time point, was also evident at the 6 and 12-month postexposure evaluations in rats exposed to TiO2 formulations III and V. In addition to slight collagen deposition, which represents a fibrogenic response in the dust-accumulated areas of the alveoli of rats exposed to TiO2 formulation III, some alveoli were filled with an eosinophilic proteinaceous material.
At the 1-year postexposure evaluation period, macrophages containing particle aggregates were observed mainly in the alveolar ducts adjacent to alveoli. However, no significant adverse lung tissue reactions to particle deposition were observed in rats exposed to base TiO2 particles (Figs. 5a and 5b), or to TiO2 formulations I, II, and IV. Most adverse lung tissue reactions observed in rats exposed to TiO2 formulation V were resolved by 1-year postexposure. Histopathological observations of the lungs of rats exposed for 4 weeks to TiO2 formulation III revealed diffuse, particle-containing macrophage accumulation in the alveolar duct regions (Figs. 6a and 6b) which was often associated with alveolar epithelial cell hyperplasia (Figure 6b) and occasional collagen deposition in the alveolar duct regions (Figure 6b). In particular, the micrograph in Figure 6b demonstrates the thickened alveolar walls enclosing particle-containing macrophages, in association with hyperplasia of alveolar epithelial cells and proliferation of fibroblasts.
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Pulmonary Bioassay Study (Intratracheal Instillation Exposures)
Pulmonary inflammation.
The numbers of cells recovered by bronchoalveolar lavage from the lungs of any of the TiO2-exposed groups were not significantly different from saline-instilled controls at any postexposure time point (data not shown). Intratracheal instillation exposures at 2 mg/kg dose for TiO2-III and V formulations; and at 10 mg/kg for all of the TiO2 formulations tested produced a short-term pulmonary inflammatory response, as evidenced by an increase in the numbers and percentages of BAL neutrophils at 24 h postexposure. At 1 week and subsequent evaluations, there were no significant differences between saline control animals and TiO2treated animals, indicating a transient inflammatory effect (Fig. 7). However, exposures to 10 mg/kg TiO2-III or TiO2 V formulations produced a more sustained lung inflammatory response, with the responses to TiO2-V and TiO2-III returning to control levels at 1 month (TiO2-V) or 3 months (TiO2-III) postexposure, respectively (Fig. 7).
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Similarly, no significant increases compared with saline-instilled controls were measured in BrdU lung cell proliferation rates in lung parenchymal cells (Fig. 10) or tracheobronchial epithelial cells (data not shown) of groups exposed to TiO2 coatings formulations at 24 h, 1 week, 1 month, and 3 months postexposure.
Lung morphology.
Histopathological analyses of lung tissues revealed no significant morphological differences among the exposed TiO2 formulation groups. Most of the lung tissue sections demonstrated particle-laden macrophages at bronchoalveolar junctions following instillation of particles (Fig. 11). Assessments of lung tissue sections following exposures to all of the TiO2 particle formulations demonstrated a normal architecture at all time periods postexposure.
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DISCUSSION |
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Histological evaluation of the particle-exposed lungs revealed that, overall, the pulmonary responses, as evidenced by dust clearance and Alveolar Type II epithelial cell hyperplasia, were similar for the different TiO2 formulations. However, the degree of dust clearance and proliferation response of the alveolar Type II epithelial cells were slightly different for the different TiO2 formulations. Additionally, slight collagen deposition was observed in rats exposed to TiO2 formulation III but no significant collagen deposition was observed in rats exposed to the other TiO2 formulations.
The TiO2-related histopathological effects observed in the inhalation study clearly were due to particle overload. In this regard, we have previously reported that aerosols exposures to base TiO2 particles in rats for 4 weeks at concentrations of 250 mg/m3 produced lung burdens of 11 mg TiO2/lung, with clearance half-times of 330 days, concomitant with sustained pulmonary inflammation, enhanced proliferation of pulmonary cells, impairment of particle clearance, macrophage functional deficits, and macrophage accumulation at sites of particle deposition (Warheit et al., 1997). In the current study, which was designed as a preliminary hazard screen, the aerosol exposure concentrations were approximately 5-fold greater than the aforementioned study. Thus, if exposure concentrations of 250 mg/m3 produced
11 mg/lung dust burden and particle overload pulmonary effects, then exposures of 1300 mg/m3 would likely produce estimated lung burdens of
4050 mg TiO2/lung, along with corresponding particle overload effects. In this regard, it is remarkable that high dose exposures did not produce lung fibrosis in any of the TiO2 formulations other than TiO2 formulation III.
Exposures of rats to TiO2 formulations with higher contents of Al2O3 and/or amorphous SiO2 (TiO2 formulations III and IV) resulted in loose aggregation of dust cells and proliferation of the cells lining the alveoli. Whether the presence of Al2O3 in the TiO2 formulations was causally related to these histological changes is unclear since Al2O3 was present in each of the TiO2 formulations and there were no Al2O3 concentration-dependent changes in histology. Additionally, the prolonged pulmonary effects in rats exposed to TiO2 formulations III and, to a lesser degree, TiO2-V are suggestive of a response induced by the presence of amorphous SiO2 in the formulations. However, in the absence of a similar response to TiO2 formulation IV which also contained amorphous SiO2, these data are not conclusive.
In the second study, groups of rats were intratracheally instilled with nearly identical TiO2 particle formulations to the inhalation study, at doses of 2 and 10 mg/kg. Following exposures, the lungs of saline-instilled and TiO2 formulation-exposed rats were evaluated using bronchoalveolar (BAL) biomarkers as well as histopathology/cell proliferation evaluation of lung tissues at 24 h, 1 week, 1 and 3 months postexposure. The results from the two studies were compared with their respective reference control materials and were also bridged (i.e., comparing inhalation results with instillation results). Remarkably, the results were quite similar for the two studies and demonstrated that only the TiO2 particle formulations, TiO2-V and TiO2-III which contained the largest concentrations of alumina and amorphous silica on their surfaces [85% TiO27% Al + 8% AMO- inhalation study; 82% TiO2 7% Al + 11% AMO-instillation study] produced mildly adverse pulmonary effects compared to base TiO2 control particle-types. It is also interesting to note that TiO2 formulation III does not have the smallest particle size (440 nm) but has the greatest surface area (27.8 m2/g) among all of the TiO2 formulations.
Bridging studies can have value in hazard evaluations when making a small modification to an existing chemical product, or when screening the safety of new developmental compounds. The reliability of accurate lung toxicity bridging studies is dependent upon having good inhalation toxicity data on the reference particulates. The reference particle-type for which there is inhalation toxicity data in rats then can be used as a control material for an intratracheal instillation bridging study. Accordingly, the basic bridging concept is that the effects of the instilled material serve as a reference material and then are "bridged" to the inhalation toxicity data for that material, and to the new test materials being evaluated. It should be noted however, that these pulmonary bioassay studies are effective screening tools, but do not substitute for more substantial inhalation toxicity studies, such as 4-week inhalation, 90-day inhalation studies, or 2-year inhalation bioassay studies.
Recently, utilizing a bridging study technique (i.e., comparing the effects of coated TiO2 particulates with base TiO2 particles), we reported the results of pulmonary toxicity studies in rats with commercial, hydrophobic-coated, pigment-grade TiO2 particles. In that study, we concluded that the OTES (triethoxyoctylsilanehydrophobic) surface treatment on the pigment-grade TiO2 particle did not cause significant pulmonary toxicity and the pulmonary effects measured were not significantly different from the effects produced by exposure to hydrophilic TiO2 base (reference) particle samples (Warheit et al., 2003).
Several investigators have considered the toxicological effects of coatings on TiO2 particles. Hohr and collaborators (2002) investigated the acute lung inflammatory responses following intratracheal instillation exposures of surface modified (hydrophilic and hydrophobic), fine (180 nm), or ultrafine (2030 nm) TiO2 particles in rats at equivalent mass doses (1 or 6 mg) or surface area doses (100, 500, 600, and 3000 cm2). These investigators concluded that BAL fluid biomarkers of inflammation were correlated with the administered surface dose delivered to the lungs. Exposures to hydrophobic-coated TiO2 particles produced less pulmonary inflammation, but these effects were not significantly different from those produced by hydrophilic-coated particulates. The investigators concluded that the surface area rather than the hydrophobic surface coating determines the acute lung inflammatory response induced by instillation of either fine or ultrafine TiO2 particle-types.
Rehn and coworkers (2003) assessed the pulmonary inflammatory and genotoxic effects of two types of ultrafine titanium dioxide particles in the lungs of female rats. Rats were exposed by intratracheal instillation to varying concentrations of uncoated (P-25) or hydrophobic-coated (T-805), ultrafine TiO2 particles and the effects were assessed at 3, 21, and 90 days postexposure. Crystalline silica, quartz particles (DQ12) and saline were utilized as positive reference and negative controls, respectively. Quartz exposure produced a persistent pulmonary inflammatory response, measured through 90 days postexposure. In contrast, rats exposed to both types of TiO2 produced no inflammation 90 days after exposure. These investigators concluded that neither the uncoated or hydrophobically coated TiO2 produced any significant pulmonary effects.
Oberdorster (2001) exposed rats via intratracheal instillation to two different types of aggregated ultrafine TiO2 particle-types (particle size of both types
20 nm) at doses of 50 and 500 µg/rat. One type was hydrophilic, while the other type was silane-coated, making the particle surface hydrophobic. Exposures to hydrophobic-coated, ultrafine TiO2 particles induced a much lower pulmonary inflammatory response at 24 h postexposure when compared to identical doses of the hydrophilic TiO2 particle-types. The author indicated that these results appear to contrast with an earlier report by Pott et al. (1998a
,b
), who reported that larger doses of instilled hydrophobic-coated, but not uncoated ultrafine TiO2 particles were acutely toxic and lethal.
With regard to the bridging study conducted herein, there is substantial evidence to suggest that inhalation of base pigment-grade TiO2 particles produces low pulmonary toxicity in exposed rats (Bermudez et al., 2002; Hext, 1994
; Muhle et al., 1991
; Vu, 1996
; Warheit et al., 1997
). In the current study, a large dose of intratracheally instilled base TiO2 particles (10 mg/kg) produced only a short-term, transient lung inflammatory response, measured at 24 h postexposure. These effects were reversible at one week postexposure. Thus, the inhalation data and the instillation toxicity results for base TiO2 particles in rats are consistenti.e., clearly demonstrating that pulmonary exposures produce few adverse effects. This is in contrast to the pulmonary effects measured following inhalation (Warheit et al., 1991
) or instillation exposures (Zhang et al., 2002
) to crystalline silica particles, which produced sustained pulmonary inflammation leading to the development of fibrosis, in both inhalation and instillation models.
Surface treatments form an important commercial component of titanium dioxide formulations and are utilized in numerous applications (e.g., paper, plastics, etc.). Most of the hydrophilic surface treatments on pigment-grade titanium dioxide particles contain varying concentrations of aluminum oxide or aluminum oxide and amorphous silicaranging from 111%. However, virtually all of the pigment-grade TiO2 samples that been tested in inhalation toxicity studies have been only slightly hydrophilic-coated TiO2 samples containing 1% aluminum oxide. Questions have been raised regarding the potential lung toxicity associated with commercial surface treatments on pigment-grade particles. The results of studies produced herein demonstrate that the bridging concept provides an effective screening tool for assessing the lung toxicity of TiO2 particle formulations containing various surface treatments.
In summary, a pulmonary bridging strategy has been presented to assess the toxicity of similar-sized particulates with small surface modifications. In this case, pulmonary toxicity comparisons were made of inhaled and instilled TiO2 formulations, and the results generated from the instilled base TiO2 particles were consistent with findings from an inhalation toxicity using TiO2 formulations with virtually identical surface treatments. Thus, three major conclusions can be drawn from these studies: (1) lung exposure in rats (the most sensitive species; Hext, 1994) to even exceedingly high concentrations of titanium dioxide particles produces low pulmonary toxicity; (2) surface treatments can influence the toxicity of TiO2 particles in the lung; TiO2 formulation III produced adverse lung effects compared to base TiO2 and other formulations, but even these impacts would be viewed as minor when compared to other dusts; and (3) the intratracheal instillation-derived, pulmonary bioassay methods represent an effective preliminary screening tool for gauging the safety of inhaled TiO2 formulations utilized in this study.
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NOTES |
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3 Present address: Newark, DE.
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ACKNOWLEDGMENTS |
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REFERENCES |
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