Elsevier

Toxicology

Volume 230, Issue 1, 25 January 2007, Pages 90-104
Toxicology

Pulmonary toxicity study in rats with three forms of ultrafine-TiO2 particles: Differential responses related to surface properties

https://doi.org/10.1016/j.tox.2006.11.002Get rights and content

Abstract

Surface properties are critical to assess effects of ultrafine-TiO2 particles. The aim of this study was to assess lung toxicity in rats of newly developed, well characterized, ultrafine-TiO2 particles and compare them to TiO2 samples in two different size ranges and surface modifications. Groups of rats were intratracheally instilled with doses of 1 or 5 mg/kg of either two ultrafine rutile TiO2 particles (uf-1 or uf-2); rutile R-100 fine-TiO2 (F-1); 80/20 anatase/rutile P25 ultrafine-TiO2 (uf-3); or α-quartz particles. Phosphate-buffered saline (PBS) solution instilled rats served as vehicle controls. Following exposures, the lungs of PBS and particle-exposed rats were evaluated for bronchoalveolar lavage (BAL) fluid inflammatory markers, cell proliferation, and by histopathology at post-instillation time points of 24 h, 1 week, 1 and 3 months.

The ranking of lung inflammation/cytotoxicity/cell proliferation and histopathological responses was quartz > uf-3 > F-1 = uf-1 = uf-2. Exposures to quartz and to a lesser degree, uf-3 anatase/rutile TiO2 particles produced pulmonary inflammation, cytotoxicity and adverse lung tissue effects. In contrast, exposures to F-1 fine-TiO2 particles or to uf-1/uf-2 ultrafine-TiO2 particle-types produced transient inflammation. We conclude that differences in responses to anatase/rutile uf-3 TiO2 particles versus the rutile uf-1 and uf-2 TiO2 particles could be related to crystal structure, inherent pH of the particles, or surface chemical reactivity. Thus, based on these results, inhaled rutile ultrafine-TiO2 particles are expected to have a low risk potential for producing adverse pulmonary health effects. Finally, the results demonstrate that exposures to ultrafine-TiO2 particle-types can produce differential pulmonary effects, based upon their composition, and crystal structure. Thus, the lung toxicity of anatase/rutile uf-3 should not be viewed as representative for all ultrafine-TiO2 particle-types.

Introduction

Titanium dioxide (TiO2) particles are regarded as poorly soluble particulates (PSP) by virtue of their low solubility and low toxicity (Hext, 1994, Hext et al., 2005, ILSI, 2000, Donaldson, 2000, Bermudez et al., 2002). TiO2 particles, in either the fine or ultrafine sizes, are widely used commercially as white pigments or in cosmetic applications, respectively. From a toxicology perspective, fine-sized titanium dioxide particulates are often utilized as negative control reference particle-types (Tran et al., 2000, Hext et al., 2005). However, following chronic exposures in rats to high particle concentrations resulting in substantial particle overload conditions (e.g., 250 mg/m3), inhaled pigment-grade TiO2 (fine- or F-1 TiO2) particles resulted in benign lung tumors, a response unique to that rodent species (Hext, 1994, ILSI, 2000, Warheit and Frame, 2006). Moreover, the limited toxicological data base demonstrates that P25 ultrafine anatase/rutile TiO2 particles (henceforth referred to as uf-3) are, on a mass basis, significantly more potent than fine-sized TiO2 particles in producing adverse lung effects (Bermudez et al., 2002, Bermudez et al., 2004, Warheit et al., 2006). In this regard, the results of 90-day and 2-year inhalation studies with uf-3 anatase/rutile ultrafine-TiO2 or F-1 rutile fine-TiO2 particles (average primary particle sizes ∼25 and ∼300 nm, respectively) have demonstrated that approximately 1/5 the inhaled mass concentrations of the ultrafine anatase/rutile (uf-3) TiO2 particles, when compared with the fine-TiO2 particles (F-1), produced equivalent numbers of pulmonary inflammation, fibrosis, or lung tumors in rats; effects occurring only at particle overload concentrations and unique to this species (Bermudez et al., 2002, Bermudez et al., 2004, Lee et al., 1985, Heinrich et al., 1995). The results of shorter-term pulmonary toxicity studies with ultrafine-TiO2 particles in rats have supported the notion of enhanced lung inflammatory potency of the ultrafine particles when compared to exposures of fine-sized particulates of similar composition. This was considered to be associated with a greater surface area of the ultrafine particles when compared to fine-sized particles (Bermudez et al., 2002, Bermudez et al., 2004).

However, a closer examination of the physicochemical properties of the two TiO2 particle-types indicates that the crystal structures of the fine-size, pigment-grade (100% rutile) and uf-3 ultrafine-TiO2 particles (80/20 anatase/rutile) are different. Accordingly, the two forms of TiO2 previously tested also represent different compositions as well as size characteristics. This would imply that the inherent differences are not simply particle size-related. Furthermore, recent in vitro studies suggest that TiO2 particles with different crystal structures (i.e., anatase or rutile) produce different toxicological responses. Indeed, nano-TiO2 particles in the anatase crystal phase were reported to be superior catalysts, better generators of reactive species, and more cytotoxic when compared to the rutile particle-type tested (Uchino et al., 2002, Sayes et al., 2006). These effects were considered to be due to differences inherent in the crystal structures of the two phases, and not due to differences in surface area. Thus, comparing the toxicological effects of anatase versus rutile TiO2 particle-types may be more analogous to comparisons of crystalline silica versus amorphous silica particles than simply examining particle size and surface area differences.

This study was designed to determine whether ultrafine-TiO2 particles impart significant toxicity in the lungs of rats, and more importantly, how the activity of different TiO2 formulations compares with other reference particulate materials, such as anatase/rutile ultrafine-TiO2 particles. Thus, the aim was to assess in rats, using a well-developed short-term pulmonary bioassay, the pulmonary toxicity effects of two intratracheally instilled, ultrafine-TiO2 particle samples and to compare the lung toxicity responses of these samples with (1) a low toxicity particulate-type (negative control, F-1 TiO2 particles); (2) a cytotoxic particulate-type (positive control, α-quartz particles); (3) an ultrafine-TiO2 particle reference sample in a similar agglomerated size range (uf-3 TiO2); (4) vehicle control (PBS).

Section snippets

Animals

Groups of male Crl:CD®(SD)IGS BR rats (Charles River Laboratories, Inc., Raleigh, North Carolina) were used in this study. The rats were approximately 8 weeks old at study start (mean weights in the range of 210–280 g). All procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee. The animal program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Physicochemical characterization of particles

The average particle size and size distributions in water and PBS, surface area measurements, crystal structures, pH in water and PBS, and chemical reactivity measurements for titanium dioxide test samples are presented in Fig. 1. The pH measurement for uf-3 shows it was much more acidic in deionized water than the other samples, but it was neutralized to approximately the same pH as the other samples in the phosphate-buffered saline solution.

The particle size distribution (PSD) results for all

Lung weights

Lung weights of rats were enhanced with increasing age on the study (i.e., increased postexposure time periods following instillation) (Fig. 6). Lung weights in high dose quartz-exposed rats were slightly increased versus controls at 1 and 3 months postexposure (Fig. 6).

Cell proliferation results

Tracheobronchial cell proliferation rates (% immunostained cells taking up BrdU) were measured in low dose (1 mg/kg) exposed rats at 24 h and 1 week and in high dose (5 mg/kg), particulate-exposed rats and corresponding controls at

Discussion

The objective of this study was to assess lung toxicity of intratracheally instilled, rutile-type ultrafine-TiO2 particles (uf-1 and uf-2) versus reference particle-types in rats. Using pulmonary bioassay methodology, the pulmonary toxicity of instilled ultrafine uf-1 and uf-2 particles were compared with a positive control particle-type (α-quartz), a negative control particle-type (F-1 fine-TiO2 particles), ultrafine uf-3 TiO2 particle control, and vehicle controls (PBS).

Ultrafine-TiO2 uf-1 or

Acknowledgments

This study was supported by DuPont Titanium Technologies. Denise Hoban, Elizabeth Wilkinson and William L. Batton conducted the BAL fluid biomarker assessments. Carolyn Lloyd, Lisa Lewis, John Barr prepared lung tissue sections and conducted the BrdU cell proliferation staining methods. Don Hildabrandt provided animal resource care. We thank Drs. Brian Coleman, Gerald Kennedy Jr., Scott Loveless, Gary Whiting, and Austin Reid Jr. for helpful comments on this manuscript.

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