Comparing fate and effects of three particles of different surface properties: Nano-TiO2, pigmentary TiO2 and quartz

Toxicology Letters 186 (2009) 152–159

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Comparing fate and effects of three particles of different surface properties: Nano-TiO2 , pigmentary TiO2 and quartz Ben van Ravenzwaay ∗ , Robert Landsiedel, Eric Fabian, Silke Burkhardt, Volker Strauss, Lan Ma-Hock BASF Product Safety, BASF SE, GV/T - Z470, D-67056 Ludwigshafen, Germany

a r t i c l e

i n f o

Article history: Available online 7 December 2008 Keywords: Nano-TiO2 Quartz DQ 12 Surface area Surface reactivity Distribution Toxic effects

a b s t r a c t The fate of nano-TiO2 particles in the body was investigated after inhalation exposure or intravenous (i.v.) injection, and compared with pigmentary TiO2 and quartz. For this purpose, a 5-day inhalation study (6 h/day, head/nose exposure) was carried out in male Wistar rats using nano-TiO2 (100 mg/m3 ), pigmentary TiO2 (250 mg/m3 ) and quartz dust DQ 12 (100 mg/m3 ). Deposition in the lung and tissue distribution was evaluated, and histological examination of the respiratory tract was performed upon termination of exposure, and 2 weeks after the last exposure. Broncho-alveolar lavage (BAL) was carried out 3 and 14 days after the last exposure. Rats were also injected with a single intravenous dose of a suspension of TiO2 in serum (5 mg/kg body weight), and tissue content of TiO2 was determined 1, 14 and 28 days later. The majority of the inhaled nano-TiO2 was deposited in the lung. Translocation to the mediastinal lymph nodes was also noted, although to smaller amounts than following inhalation of pigmentary TiO2 , but much higher amounts than after exposure to quartz. Systemically available nano-TiO2 , as simulated by the i.v. injection, was trapped mainly in the liver and spleen. The (agglomerate) particle size of lung deposited nano-TiO2 was virtually the same as in the test atmosphere. Changes in BAL fluid composition and histological examination indicated mild neutrophilic inflammation and activation of macrophages in the lung. The effects were reversible for nano- and pigmentary TiO2 , but progressive for quartz. The effects observed after 5-day inhalation exposure to nano-TiO2 were qualitatively similar to those reported in sub-chronic studies. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The increased use of nano-sized materials has compelled the scientific community to investigate the potential hazards of these unique and useful particles. When materials reach the nano-scale, they often display increased reactivity relative to the bulk compound. New approaches for testing and new ways of thinking about current materials are necessary to provide safe workplaces, products and environments. Whereas aerosols of atmospheric ultrafine particles formed by combustion (such as diesel exhaust) have been extensively studied for decades (Borm et al., 2006), little is known about aerosols from solid, manufactured nanomaterials. These are specially designed to have certain functional properties so that they have distinctively different characteristics (e.g. form and composition of the particles, surface chemistry and charge) than aerosols of anthropogenic origin. Due to their small sizes, primary nanoparticles do not follow the aerodynamic rule, but the rule of diffusion. Thus, these particles may persist in the air for a long time. There-

∗ Corresponding author. Tel.: +49 621 60 56419; fax: +49 621 60 58134. E-mail address: [email protected] (B. van Ravenzwaay). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.11.020

fore, the most important route of potential human exposure to these materials is considered to be by inhalation. The potential pulmonary toxicity of nanomaterials and nanoparticles may be assessed by both in vivo studies (e.g. inhalation, intratracheal instillation) and in vitro studies using cell culture systems (Oberdoerster et al., 2005; Sayes et al., 2007). A step-wise approach for human health hazard evaluation of nanoparticles was already proposed by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) during a workshop held in November 2005 (Borm et al., 2006). To ensure the product safety of nanomaterials, BASF initiated a multi-year project to establish a strategy for examining the potential inhalation toxicity of manufactured materials. The first part of this project covered technical preparation for the inhalation testing. Along the establishment and validation of technique for generation and characterization of test atmosphere, we observed that many powder materials agglomerate strongly. These agglomerates cannot be easily broken during the atmosphere generation, and effectively very little (<1% of total weight) of nano-sized particles were present in the atmosphere (Ma-Hock et al., 2007). Using our short-term inhalation test protocol, we aim to examine the influence of the surface area and surface reactivity on the depo-

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Fig. 1. Transmission electron micrographs of (a) nano-TiO2 , (b) pigmentary TiO2 , and (c) quartz DQ 12.

sition, translocation and toxic effects of the particles. Surface area and reactivity were discussed previously as possible determinant physicochemical characteristics for the biological activities of the particles (Bermudez et al., 2002, 2004; Oberdoerster et al., 2005; Warheit et al., 2006, 2007). For this purpose, we tested three materials in a short-term inhalation study: nano-TiO2 , pigmentary TiO2 and quartz dust DQ 12. In a technical trial, all of these three substances formed dust aerosols with very similar particle size distribution, indicated by the narrow range between the measured mass median aerodynamic diameters (MMADs). Nano- and pigmentary TiO2 are chemically identical substances with different surface area and slightly different surface chemistry. Their aerosols consisted of either agglomerates with large surface area or solid particles with small surface area. Pigmentary TiO2 and quartz dust are similar in particle size and surface area, but different in chemical reactivity of the surface. To address the concerns about the possible translocation ability of the nanomaterials (Oberdoerster et al., 2004; Nemmar et al., 2002), we determined the distribution of the inhaled materials in the body. To simulate a 100% bioavailability of the nano-TiO2 , the tissue distribution and the biological effect of the material was also examined following intravenous (i.v.) administration. To determine the state of the inhaled particles after the deposition, the lungs were examined by electron microscopy. Effects on the respiratory tract after inhalation were examined in the broncho-alveolar lavage fluid (BALF) and by light microscopy at two different time points. Possible systemic effects of nano-TiO2 were also examined in the animals treated by i.v. injection. 2. Materials and methods 2.1. Test materials The nano-titanium dioxide [TiO2 ; CAS No. 13463-67-7], consisted of both anatase and rutile forms (70/30) and had no surface coating. The TiO2 particles were in the size range 20–30 nm according to transmission electron microscopy (TEM, Philips CM 120; Fig. 1) and had a BET surface area (determined by the Brunauer–Emmett–Teller algorithm (Brunauer et al., 1938) of 48.6 m2 /g. Zeta potential was measured on a Zetasizer 300 HS (Malver, Germany) by a laser doppler electrophoresis method with a measurable range of pH 3–9. The isoelectrical point (IEP) of the nano-TiO2 was pH 7 in 10 mM KCl. Controls for the pulmonary bioassay study included pigmentary TiO2 particletypes in the rutile form (Kronos International) and quartz dust DQ 12 (Doerentrup Quarz GmbH, Germany). The pigmentary TiO2 had a purity of 99.4% and a median particle size of 200 nm in ethanol using dynamic light scattering measurement (Microtrac UPA 150). The average BET surface was 6 m2 /g. The quartz dust possessed a median particle size of 315 nm in ethanol and an average BET surface of 5.9 m2 /g was measured on a Zetasizer 300 HS (Malvern, Germany). In 10 mM KCl, the zeta potential of both pigmentary TiO2 and quartz dust were strongly ionic with IEP < pH 3. 2.2. Animals Male Wistar rats (strain Crl:WI (Han), 7 weeks of age) were obtained from Charles River Laboratories (Sandhofer Weg, Sulzfeld, Germany) and were allowed free access to mouse/rat laboratory diet (Provimi Kliba SA, Basle, Switzerland) and water. The

animals were housed singly in mesh floored cages in accommodation maintained at 20–24 ◦ C, with a relative humidity of 30–70%, a light/dark cycle of 06.00–18.00 h light and 18.00–06.00 h dark and were allowed to acclimatize to these conditions for approximately 2 weeks before commencement of the study. 2.3. Tissue distribution and toxicity after inhalation Groups of animals were head–nose exposed to dust aerosols for 6 h a day on 5 consecutive days. Following the exposure, the amount of the test materials in several organs was determined (organ burden), the respiratory tract was evaluated histopathologically, the lung was evaluated by electron microscopy and bronchoalveolar lavage (BAL) was performed. To enable a comparison of biological effects caused by nano-TiO2 , pigmentary TiO2 and quartz dust, high concentrations were chosen for all three test substances to produce toxicity. For nano-TiO2 , the targeted atmospheric concentration was 100 mg/m3 , which was 10 times as high as the concentration, which had caused increased incidence of benign squamous-cell tumour, adenocarcinoma and sqamous-cell carcinoma in rats after 18-month inhalation exposure to a similar material (Heinrich et al., 1995). This high concentration was justified by the short exposure time of 5 days. Pigmentary TiO2 was studied extensively in the past. After 2-year inhalation exposure of rats to 50 mg/m3 or 250 mg/m3 TiO2 several adverse effects (e.g. type II pneumocyte hyperplasia, alveolar proteinosis, alveolar bronchiolarisation and cholesterol granulomas) were observed (Lee et al., 1985). Adenoma and cystic keratinizing squamous-cell carcinomas were noted only in animals of the 250 mg/m3 group. Again, taken the short exposure time of 5 days into consideration, a high concentration of 250 mg/m3 was chosen. Quartz dust DQ 12 was considered to be fibrogenic and caused squamous-cell carcinoma after 2-year inhalation exposure (Muhle et al., 1991). Considering the time factor and to enable a comparison with nano-TiO2 , the same atmospheric concentration (100 mg/m3 ) as for nano-TiO2 was applied. To generate the test atmosphere of nano-TiO2 , the material was dispersed in highly de-ionised water at a concentration of 0.5% by weight. The suspension of the test material was nebulized by a two-component atomizer (stainless steel, Schlick mod. 970), fed at a constant rate by means of a piston metering pump (Sarstedt Desaga, Germany). The aerosols were generated with compressed air in a mixing stage, mixed with conditioned dilution air and passed via a cyclone into a head–nose inhalation system. The water evaporated very quickly resulting in dry dust aerosols in the test atmosphere. The pigmentary TiO2 and the quartz dust were dispersed by brush generators (developed by the Technical University of Karlsruhe in cooperation with BASF, Germany). The targeted atmospheric concentrations were 250 mg/m3 and 100 mg/m3 for TiO2 and quartz dust, respectively. Dust aerosols were produced by dry dispersion of powder pellets with a brush. The aerosols were generated with compressed air in a mixing stage, mixed with conditioned dilution air and passed via a cyclone (to separate large particles >3 ␮m) into a head–nose inhalation system. To reduce electrostatic charging, brushes made of stainless steel were used. The generator itself and all conducting tubes were grounded. The technical setups are presented in Table 1. The mass concentration of the test substance in the inhalation atmosphere was determined gravimetrically after sampling of the particles in the exposure atmosphere onto a filter. The particle size was determined gravimetrically after separation in an eight-stage Marple Personal Cascade Impactor (Sierra-Andersen), optically by an optical particle counter (OPC) PCS 2000 (Palas, Karlsruhe), and by their mobility using a scanning mobility particle sizer (SMPS), 3022A/3071A (TSI, USA). Technical details of the measurements were described elsewhere (Ma-Hock et al., 2007). To assess the tissue distribution of the test substance in the body, the amount of the test materials in the lung, mediastinal lymph nodes, liver, kidney, spleen and basal brain with olfactory bulb were determined immediately after the last exposure and after 14 days recovery. For this purpose, three animals per group and time point were used.

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Table 1 Substance feeding rate and air flows that were scheduled in the inhalation study. Test group

Test substance

0 1 2 3

Conditioned air Nano-TiO2 Pigmentary TiO2 Quartz DQ 12

Substance feeding rate (g/h) – 1.5–15 4.0–40 1.5–15

Supply air 1 conditioned (m3 /h) 6.0 4.5 4.5 4.5

± ± ± ±

0.3 0.3 0.3 0.3

The BAL was carried out in 5 rats per group and time point, either 3 days or 14 days after the last exposure. The animals were killed by exsanguination under Narcoren® anaesthesia and the lungs lavaged 2 times with 6 ml (22 ml/kg body weight) of 0.9% (w/v) saline to obtain the BALF. The two washes were combined (around 10 ml lavage fluid) and aliquots of the combined washes used for the determinations described below. Total BALF cell counts were determined with an Advia 120 (Siemens Diagnostics, Fernwald, Germany) haematology analyzer. Counts of macrophages, polymorphonuclear neutrophils, lymphocytes, eosinophils, monocytes and atypical cells were performed on Wright-stained cytocentrifuge slide preparations as described by Warheit and Hartsky (Warheit and Hartsky, 1993). Levels of BALF total protein (mg/l) and activities of lactate dehydrogenase (LDH, ␮kat/␮l), alkaline phosphatase (ALP, ␮kat/␮l), ␥-glutamyltransferase (GGT, nkat/␮l) and N-acetyl-␤-glucosaminidase (NAG, nkat/␮l) were determined with a Hitachi 917 (Roche Diagnostics, Mannheim, Germany) reaction rate analyzer. Histological examinations were carried out in 6 animals immediately after the exposure and after a recovery period of 14 days. The animals were killed as described above and underwent gross necropsy. The lung, mediastinal lymph nodes, liver, kidney, adrenals, thymus, spleen and brain were weighed. After the initial fixation (at least 24 h), the head was decalcified using formic acid/citric acid. After the decalcification, four transverse sections (posterior part of upper incisors, incisive papilla, second palatine crest, first molar teeth) were taken according to Young (1981). After weighing of the lungs, the lungs were slowly instilled with buffered formaldehyde solution (at a hydrostatic pressure of 30 cm) until formalin reached the edges of the lobes. After the instillation the proximal trachea were clamped and preserved in the same fixative. Paraffin sections were stained with haematoxylin and eosin and examined by light microscopy. Immediately after exposure and a 14-day recovery period, 3 animals per group and time point were subjected to deep anaesthesia with Isoflo® (Essex GmbH, München, Germany) and sacrificed and fixed by whole-body perfusion via left ventricle of the heart using caccodylate buffer as a rinsing solution, followed by perfusion of a 5% glutardialdehyde as fixation solution. For TEM analysis, the tissue samples of the lungs and mediastinal lymph nodes were refixed with 2% buffered osmium tetraoxide and embedded in Epon mixture (Polysciences Europe GmbH, Eppelheim, Germany). Semi-thin sections (500 nm thick) were stained with azure–methylene blue–basic-fuchsine (Ambf), and ultrathin sections (∼70 nm thick) were stained with uranyl acetate and lead citrate. 2.4. Tissue distribution and toxicity after intravenous treatment The application suspension, nano-TiO2 was dispersed in rat serum (0.5%). The TiO2 particle size distribution was determined by analytical ultracentrifugation of ∼500 ␮l of a test substance application preparation that was made in parallel with that used for the i.v. administration. At the acceleration of up to 300,000 × g used in analytical ultracentrifuge, solutes and nanoparticles sediment into fractions that are separated according to their size in the range 0.5–10,000 nm. We used a Beckman model XL ultracentrifuge modified for the online recording of sedimentation with turbidity, interference, and Schlieren or ultraviolet (UV) detection. A total of 12 male healthy Wistar rats, with a narrow range of body weights, were randomly assigned to three experimental groups, with one control and three treated animals in each group. The control animals were injected intravenously via the tail vein with ∼1 ml of sterile saline, and the treated animals were injected i.v. via the tail vein with the test substance application preparation which contained TiO2 at

Supply air 2 compressed (m3 /h)

Exhaust air 1 (m3 /h)

– 1.5 ± 0.3 1.5 ± 0.3 1.5 ± 0.3

5.4 5.4 5.4 5.4

± ± ± ±

0.3 0.3 0.3 0.3

a dose of 5 mg/kg body weight (∼1 ml of test substance preparation/kg of rat body weight). This dose was chosen because it was high enough to allow spectroscopic quantification of residue levels in organs of interest (assuming that the residues are distributed homogenously within the organism) and low enough to be acutely nontoxic (determined in a subgroup of animals by dosing with 5 mg/kg and observing no clinical signs of toxicity for 1 day). Moreover, this dose was in the same dose range as that after inhalation exposure, if the total lung burden would enter the blood circulation. The tails of lightly anesthetized animals were warmed in a beaker of warm water, and blood flow was stimulated by wiping with alcohol pads before being injected with either saline (controls) or the TiO2 application preparation (followed by ∼750 ␮l of saline to flush the TiO2 application preparation into the vein). Following the treatment, the animals were returned to their individual cages. At 1, 14, and 28 days post-i.v. injection of saline or the TiO2 application preparation, animals in groups 1, 2, and 3, respectively, were anesthetized with isoflurane and sacrificed by cervical dislocation. Blood samples were taken, lung, liver, kidney, spleen and brain were collected and weighed immediately after sacrifice of the animals. The amounts of the test materials in these organs as well as in blood cells, plasma and popliteal lymph nodes were determined. 2.5. Analyses of TiO2 and quartz in tissues To determine Ti content in the tissues, wet samples of the lung, lymph nodes, kidney, spleen and brain were digested in an automated digestion apparatus. After addition of an ammonium sulphate tablet, sulphuric acid was added and the mixture was heated. Then, a mixture of nitric, sulphuric, and perchloric acids (2:1:1, v/v/v) was added to the mixture, and this solution was heated again. Following oxidation, the mineral acids were evaporated, and the residue was taken up with hydrochloric acid. The Ti content in the obtained solution was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) with a Thermo Jarrell Ash “IRIS 1” spectrometer (calibration: external; nebulizer: Meinhard or Ultrasonic; plasma power: 1150 W; Ti wavelength: 338.376 nm). Samples of liver and lung were treated with sulphuric acid and heat in a platinum dish. After digestion, the acid was evaporated and the residue was ashed at 600 ◦ C in a muffle furnace. The ash was then mixed with a sodium/potassium carbonate–borax mixture and melted above a blowtorch flame. The resultant flux was cooled down, dissolved in water, and acidified with hydrochloric acid. The Ti content in the obtained solution was measured by ICPAES, with a VARIAN “Vista pro” (calibration: external; nebulizer: Meinhard; plasma power: 1200 W; Ti wavelength: 334.941 nm). To determine the Si content in the tissues, the wet samples were digested with sulphuric acid in the platinum dish by heating. Afterwards the digestion acid was evaporated. The residue was mixed with sodium/potassium carbonate–borax mixture and melted above a blowtorch flame. The flux was cooled down, dissolved in water and acidified with hydrochlorid acid. The obtained solution was measured by ICP-AES as described above, at 288.15 nm for silicon.

3. Results 3.1. Characterization of test atmospheres The data of the target inhalation atmosphere concentrations, the actual analyzed concentrations and the particle size distribution

Table 2 Particle size and concentration in the test atmospheres. Variable

Test substance

3

Target concentration (mg/m ) Measured concentration (mg/m3 ) MMAD (␮m)/GSD OPC (␮m): count median diameter (Q0 ) SMPS (␮m):count median diameter (Q0 ) Count concentration of particles in SMPS (number particle/cm3 ) Count concentration of particles <100 nm (number particle/cm3 ) Calculateda mass fraction measured <100 nm

Nano-TiO2

Pigmentary TiO2

Quartz DQ 12

100 88.0 ± 6.4 1.0/2.2 0.6 0.2 88 × 104 205,920 0.5%

250 274.0 ± 30.5 1.1/2.2 1.5 0.2 13 × 105 54,600 0.05%

100 96.0 ± 5.4 1.2/2.1 0.4 0.2 33 × 104 21,292 0.03%

a Assuming all particles are spherical particles with d = 100 nm, this mass fraction (%) was calculated by multiplying the volume of a particle with diameter 100 nm with particle count concentration (<100 nm) and physical density 4.2 g/cm3 for TiO2 and 2.65 g/cm3 for quartz dust DQ 12.

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Table 3 Organ distribution after 5-day inhalation exposure (mean values of 3 animals per group and time point in ␮g per organ). Organ

n-TiO2

Liver, kidney, spleen, basal brain with olfactory bulb (␮g) Lung (␮g) Lymph nodes (␮g) a b

p-TiO2

Quartz DQ 12

Day 5

Day 19

Day 5

Day 19

Day 5

Day 19

<0.5a 2025 2.2

<0.5a 1547 8.5

<0.5a 9182 8.2

<0.5a 7257 108

<11b 2190 19

<11b 1975 56

0.5 ␮g per organ was the detection limit by ICP-AES for TiO2 . 11 ␮g per organ was the detection limit by ICP-AES for quartz.

Table 4 TiO2 distribution in organs after i.v. injection of 5 mg nano-TiO2 /kg body weighta . Organ

Liver Spleen Lung Kidney a b c

TiO2 on day 1

TiO2 on day 14

Controlb

Treatedc

0.5 0.8 1.5
133.8 78.7 8.8 0.67

± ± ± ±

26.0 19.3 0.6 0.06

TiO2 on day 28

Controlb

Treatedc

Controlb

Treatedc

0.5 0.9 1.7
99.5 ± 55.8 48.8 ± 27.8 2.8 ± 1.4
0.5 0.7 1.5
111.3 ± 27.3 33.3 ± 28.8 2.3 ± 1.0
Values expressed as mean ± S.D. (␮g/g wet tissue). n = 1. n = 3.

measurements are presented in Table 2. The MMADs were between 1.0 ␮m and 1.2 ␮m, which is highly respirable for all three materials without any differences between those of the nanomaterial and the pigmentary materials. The calculated respirable fraction (MMAD < 3 ␮m) ranged from 81.7% to 93.1%. Only approximately 10% of the particles measured by the SMPS were smaller than 100 nm for all three exposure atmospheres (Table 2). The mass concentration of this fraction (<100 nm) was calculated (Table 2). For atmospheres with nano-TiO2 the number concentrations of particles <100 nm represented only 0.5% of the total particle mass.

3.2. Characterization of application suspension for the i.v. administration We used an analytical ultracentrifuge to measure both the highly concentrated and very turbid samples used for the i.v. injections and a sample that was diluted with foetal calf serum (FBS gold). The particle size of the TiO2 used for the i.v. injection was mostly in the fine fraction with components of up to 1 ␮m in size. Approximately 10 wt% of the particles were found in the nano-size range (<100 nm).

Fig. 2. Light microscopic pictures of the lungs of (a) untreated control animals, (b) animals treated with nano-TiO2 , (c) animals treated with pigmentary TiO2 , and (d) animals treated with quartz DQ 12 H&E.

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Fig. 3. Transmission electron micrographs from the lungs of rats exposed to aerosol of nano-scale TiO2 . Left: agglomerates located free in the alveolar space. Right: alveolar macrophages with agglomerates in the cytoplasm.

3.3. Tissue distribution

3.5. Biological effects

After the 5-day inhalation exposure, Ti or Si was only detectable in the lung and mediastinal lymph nodes of the exposed animals (Table 3). The limit of quantification for Ti was 0.3 ␮g per tissue sample, corresponding to 0.5 ␮g TiO2 . The detection limit for Si was 5 ␮g per tissue sample, corresponding to 11 ␮g SiO2 . After i.v. administration of nano-TiO2 , there were no detectable levels of TiO2 (<0.5 ␮g per sample) in blood cells, plasma, brain, or lymph nodes (mediastinal, mesenteric, popliteal) at any of the three time points tested. The average distributions of TiO2 in the liver, spleen, lung, and kidney are shown in Table 4. The TiO2 levels were highest in the liver, followed in decreasing order by the levels in the spleen, lung, and kidney (very low levels of <0.7 ␮g). In all four organs, TiO2 levels were highest on day 1. TiO2 levels remained relatively high throughout the 28-day duration of the experiment. In the spleen, the results were more variable than in the liver, but showed a trend to decreased TiO2 levels from day 1 to days 14 and 28. An early distribution of TiO2 to the lung on day 1 returned to near control levels by day 14. Similarly, in the kidney, there was an increase in TiO2 levels compared with control on day 1 and a return to control levels by day 14.

BAL is an established method to determine early changes in the lung. We examined several humoral and cytological parameters in the lavage fluid to determine the toxic effect in the lung after inhalation exposure. The treatment of male rats with nanoTiO2 , pigmentary TiO2 and quartz dust by inhalation for 6 h/day on 5 consecutive days led to significant increases in total cell count, with polymorphonuclear neutrophils being significantly increased in all groups (Fig. 4 a and b) in the lavage fluid. Moreover, slightly increased lymphocytes and monocytes values were also observed. Beside the cytological parameters, levels of total protein and activities of lactate dehydrogenase, alkaline phosphatase, ␥glutamyltransferase and N-acetyl-␤-glucosaminidase were found significantly increased in all test groups. Summarizing the changes, we noticed a very similar patter of changes in the BALF of animals exposed either to nano-TiO2 or pigmentary TiO2 or Quartz DQ 12. Whereas the increases in the Quartz DQ 12 group were more pronounced and not reversible, the increases of BALF parameters in the animals exposed to nano- and pigmentary TiO2 were partly reversible within the recovery period. The 5-day inhalation exposure to 88 mg/m3 nano-TiO2 resulted in a >30% increase in lung weight, which is a global indicator for the inflammatory processes induced by the deposited particles. Accordingly, diffuse histiocytosis and a mild neutrophilic inflammation was observed. In the mediastinal lymph nodes, lymphoreticulocelluar hyperplasia was observed. In single animals a few particles were observed intracellularly in the olfactory epithelium of the nasal cavity. After the 14-day recovery period the inflammatory response declined and only focal infiltrates of alveolar macrophages were observed, still containing particles in their cytoplasm. This was also reflected by the lung weight, which returned to control levels. In the mediastinal lymph nodes the activation ceased but the number of pigment-containing macrophages was still increased slightly. While the inhalation of nano-TiO2 apparently induced local toxic effects in the lung, systemically available TiO2 , as demonstrated by the i.v. treatment, did not cause any detectable toxic effect. To assess potential inflammatory responses and/or organ injury after i.v. exposure to nano-TiO2 , various cytokines and enzymes were measured in blood samples (a total of 67 parameters, for details see Fabian et al., 2007). None of these parameters showed definitive changes at days 1, 14, or 28 following TiO2 exposure, indi-

3.4. Deposition of inhaled nano-TiO2 In H&E stained sections (Fig. 2) pigmentary and nano-scale TiO2 could be observed mainly in alveolar macrophages. The number of alveolar macrophages was moderately increased and there were few numbers of neutrophils within the alveolar space. In animals treated with quartz dust DQ 12, an increased number of alveolar macrophages showing a vacuolated cytoplasm and an increased number of neutrophils were observed in the alveolar space. Electron microscopy was used to characterize the particles deposited in the tissues (Fig. 3). The particles from both the pigment and the nano-scale material were mainly located extracellularly in the lumen of the alveoli and bronchi. Moreover, particles were detected in the cytoplasm of alveolar macrophages. The particles from nano-scale material found in the lung, were mostly agglomerates of about the same size as found in the atmosphere; there were no signs of desagglomeration of the inhaled agglomerates.

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macrophages. This was also reflected by the lung weight, which was still 45% higher (absolute and relative) as compared to control animals. The mediastinal lymph nodes of all animals were activated; they had numerous macrophages within the sinus and in addition displayed granulomatous inflammation. 4. Discussion 4.1. General considerations The presented data are part of a complex, ongoing project, which was started to establish a test strategy for safety evaluation of designed ultrafine (nano)materials. In the incipient part of the project, methods for generation and characterization of the test atmospheres were established and it was apparent, that aerosols from nanomaterials consist of agglomerates in the micrometer range and only a smaller fraction of ultra fine particles with diameters below 100 nm (typically <1% (w/w) as observed with 10 nanomaterials) (Ma-Hock et al., 2007). The decisive factors for the biological activities of the nanoparticles are being discussed controversially. The current opinion focuses on the surface area and the surface activity of the particles. To understand the influence of these two factors, we compared the body distribution and effects of nano-TiO2 , pigmentary TiO2 and quartz dust after inhalation exposure. These three substances allowed for a pair-wise comparison of two factors: (1) nano- and pigmentary TiO2 which differ from each other mainly in surface area. Surface activity may play a role, as the nano-TiO2 consisted of anatase and rutile form, while pigmentary TiO2 consisted of rutile. (2) Pigmentary TiO2 and quartz dust which differ from each other in surface activity. To demonstrate the distribution and effect of systemically available nano-TiO2 , the nanomaterial was also administered by i.v. injection. 4.2. Fate Fig. 4. (a) Changes (fold of control) of the BALF parameters 3 days after the last exposure and 14 days (lower panel). (b) Changes (fold of control) of the BALF parameters 14 days after the last exposure.

cating that there was no detectable inflammatory response or organ toxicity. The 5-day inhalation exposure to 274 mg/m3 pigmentary TiO2 led to >30% increase of the lung weight. Again diffuse histiocytosis was noted, but without granulocytic infiltration. The mediastinal lymph nodes were activated (3 out of 6 animals), and pigmentloaded macrophages were found in 4 out of 6 animals. Single animals revealed very few particles on the surface or intracellularly in the olfactory epithelium of the nasal cavity. After the recovery period, the numbers of infiltrating histiocytes (only focal infiltrates present) as well as particle numbers decreased, which was reflected in a no longer significantly increased lung weight. The mediastinal lymph nodes of 5 out of 6 animals showed activation, and in the lymph nodes of all animals pigment-loaded macrophages were observed. The 5-day inhalation exposure to quartz DQ 12 led to a strong increase of the lung weight (45%). Beside the diffuse histiocytosis, multifocal granulocytic infiltration was noted. Although these particles could not be detected by light microscopy, via transmission electron microscopy they could be found intracytoplasmatically within alveolar macrophages. The mediastinal lymph nodes showed a minimal to moderate activation in all animals. After the recovery period histological findings in the lungs increased in severity: all animals showed a mild to moderate diffuse inflammation composed of alveolar macrophages, neutrophils and cell detritus, which most likely resulted from degenerated

As reported previously, nano-TiO2 agglomerated strongly in the atmosphere (Ma-Hock et al., 2007). By count, we found in the atmospheres around 20% of the particles as nano (<100 nm), corresponding to 0.5% by weight. After inhalation exposure, the tissue distribution analysis of the exposed animals demonstrated that the majority of the material in the form of Ti or Si was found in the lung. Only a small fraction was detected in the lymph nodes. Immediately after the 5-day exposure period, the lung burden was around 2.0 mg nano-TiO2 , approximately 9.2 mg pigmentary TiO2 and about 2.2 mg quartz per lung. Assuming a minute ventilation volume of 0.2 l per rat, using the measured mean concentration over the 5 exposure days, a 100% theoretical deposition could be calculated as 31.7 mg nano-TiO2 , 98.6 mg pigmentary TiO2 and 34.6 mg quartz. The resulting ratio of the measured deposited mass and the theoretical deposition were the deposition rates of 6.3% for the nano-TiO2 and quartz and 9.3% for the pigmentary TiO2 . These values were very close to each other and suggest that the deposition rate after the inhalation is mainly triggered by the MMAD of the aerosols in a similar way as described by Anjilvel and Asgharian (1995) for solid particles. Although the lung burden was higher than the overload dose of 1 mg lung (Ferin and Feldstein, 1978; Morrow, 1988), a certain clearance of the lung was observed: after 2 weeks recovery, the lung burden of the nano-TiO2 decreased substantially (−24%), followed by pigmentary TiO2 (−21%) while the clearance of quartz occurred only slowly (−10%). Instead of the expected slower clearance (Bermudez et al., 2002, 2004) of nano-TiO2 , we found a comparable clearance for the nano- and pigmentary TiO2 . This may be explained by the fact that nano-TiO2 formed agglomerates of sizes not dissimilar to that of pigmentary TiO2 , which may be recognized and

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phagocyted by macrophages to a similar degree as the pigmentary particles. This assumption was substantiated by the electron microscopic examination showing mainly agglomerates inside or outside the macrophage (Fig. 2). On the other hand, overload in the lung of animals exposed to pigmentary TiO2 , might have slowed down the phagocytic clearance in this group. In fact, we found that, compared to nano-TiO2 , more pigmentary TiO2 was translocated from the lung to the mediastinal lymph node (8.2 ␮g and 108 ␮g, respectively). This difference can be partly explained by the differences in lung burden (Snipes, 1989). It might be speculated that the interstitial clearance of the pigmentary material is some what higher than that of the nanomaterial. Moreover, under the current test conditions clearance into the lymphatic system may be different for the two materials. This difference may be the result of the different surface area, but may also have their root in the higher degree of lung overload in the animals exposed to the pigmentary TiO2 . Besides in the lung, inhaled material was only found in the mediastinal lymph nodes but not in other organs, indicating a low systemic availability of the tested materials including nano-TiO2 after inhalation exposure. It should be noted however, that the limit of detection of the analytical method used was 0.5 ␮g per organ sample for TiO2 , and 5 ␮g per organ sample for quartz. Thus, the translocation of smaller amounts of the test substances cannot be ruled out. The i.v. experiment, where rats were administered with an application preparation containing 10% (w/w) nanoparticles, provided 100% bioavailability. After i.v. injection, TiO2 (in the form of Ti) was found mainly in the liver followed by the kidney, spleen and lung. Retention of particles was observed in the liver and to a lesser extent in the spleen, however, without obvious signs of toxicity.

approximately 980 cm2 per lung some what higher than the pigmentary material with 550 cm2 , the overall toxicity of nano-TiO2 was only marginally higher: with respect to the changes of BALF parameters, the effects were completely comparable, with respect to the histological findings a mild neutrophilic inflammation was noted in the nano-TiO2 -exposed animals but not in those exposed to pigmentary TiO2 . Quartz dust DQ 12 exerted the highest biological response at the low deposited surface area of only approximately 107 cm2 per lung. These data thus suggest that, the surface area may have some influence on the biological effect of the particles, but more important contribution concerning the induction of toxicity of these materials is provided by surface reactivity. 5. Conclusion Due to comparable aerodynamic particle size distributions, the deposition rate of nano-, pigmentary TiO2 and quartz in the lung was very similar after inhalation exposure. Translocation to other organs or tissues other than the lung-draining lymph nodes was not observed for any of the tested materials. Systemically administered nano-TiO2 by i.v. injection was mainly found in the liver. Comparing similar doses of inhaled nano-TiO2 , pigmentary TiO2 and quartz, it appears that mainly surface chemistry (reactivity) influences the toxic effects: quartz dust which showed that the lowest deposited surface area in the lung exerted the highest toxicity. Agglomerates of nano-TiO2 with the highest deposited surface area had only marginally stronger effect than solid TiO2 pigment. Conflict of interest BASF SE produces products containing TiO2 .

4.3. Toxic effects References Another important aspect of our study was the toxicity of nanoTiO2 in comparison with the pigmentary material and quartz. In BALF, the pattern of changes was remarkably similar for all three substances (Fig. 3), the most prominent change being the increase of the neutrophilic granulocytes in the lavage fluid, indicating an inflammatory effect in the lung. The examination carried out 3 days after the last exposure showed such a close magnitude of changes in all the examined parameters, that the lines of nano- and pigmentary TiO2 in the spider diagram are congruent. More severe changes in the lavage fluid of the quartz-treated animals could be found, as indicated by the red line outside those of the two TiO2 materials. After a period of 14 days, recovery was noted in animals exposed to either of the two TiO2 materials, but not in those exposed to quartz. The recovery seemed to be even faster in animals exposed to nano-TiO2 than those exposed to pigmentary TiO2 . This phenomenon could possibly be explained by the higher lung burden of the pigmentary TiO2 -treated animals. In clinical pathology, distinct increases of neutrophils were noted in BALF for all three test materials, whereas only focal mild infiltration of neutrophils were observed in exposed animals at different time points in H&E stained slides: in animals exposed to nano-TiO2 , granulocytic infiltration was only noted immediately after the last exposure but disappeared after 14 days’ recovery. In animals exposed to pigmentary TiO2 , neutrophils appeared only after the recovery period of 14 days, probably due to overload-associated macrophage activation. The same effect was observed in the quartz-exposed animals, which progressed to diffuse inflammation 14 days after the last exposure. To examine the influence of surface area on toxicity, the surface area was calculated for the amounts of the three materials deposited in the lung by multiplying the BET surface of the respective material, which we assume to correspond to the biologically active surface, with the lung burden at the end of the exposure. Although the deposited surface area for nano-TiO2 was with

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