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Considerations for Characterizing the Potential Health Effects from Exposure to Nanomaterials. David B. Warheit, PhD. DuPont Haskell Laboratory Newark, Delaware, USA NNI-NIST Workshop Gaithersburg, MD September 13, 2007. Outline. Particle characterization as it relates to
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Considerations for Characterizing the Potential Health Effects from Exposure to Nanomaterials David B. Warheit, PhD. DuPont Haskell Laboratory Newark, Delaware, USA NNI-NIST Workshop Gaithersburg, MD September 13, 2007
Outline • Particle characterization as it relates to • particle deposition, macrophage interactions, particle translocation • Particle characterization for 5 studies • Fine/Ultrafine TiO2 particle types; • Fine/Nanoscale Quartz particle-types; • Summary - Recommendations
Rat Lung Tissue Dissected to Demonstrate the Junction of the Terminal Airway and Proximal Alveolar Region
Iron Particle Deposition at Bronchoalveolar Junction(Backscatter Image)
Alveolar Macrophage Clearance of Inhaled Iron Particles(Backscatter Image)
Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis
Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis(Backscatter Image)
Two Alveolar Macrophages (M) Sharing a Chrysotile Asbestos Fiber () with an Alveolar Epithelial Cell (E) M E M
TEM demonstrating pathways for possible translocation of particles
Translocation of chrysotile asbestos fibers from airspace to epithelium
1) Pulmonary Instillation Studies with Nanoscale TiO2 Rods and Dots in Rats: Toxicity is not dependent upon Particle Size and Surface Area. Toxicol Sci., 2006 • Material characterization employed in this study: • synthesis method • crystal structure • particle size • surface area • composition/surface coating • aggregation status • cryo TEM • crystallinity • purity (TGA)
2) Pulmonary bioassay studies with nanoscale and fine quartz particles in rats: Toxicity is not dependent upon particle size but on surface characteristics. Toxicol Sci. 2007 • Material characterization employed in this study: • synthesis method • crystal structure/crystallinity (XRD) • median particle size - particle size (range) • purity (% Fe content)– ICP-AES • surface area • TEM • aggregation status • purity • surface reactivity (erythrocyte hemolysis) • reactive oxygen species (ESR)
3) Pulmonary Toxicity Study in Rats with Three Forms of ultrafine-TiO2 Particles: Differential Responses related to Surface Properties Toxicology, 2007 • Material characterization employed in this study: • crystal phase • median particle size and size distribution in water and PBS • pH in water and PBS • surface area (BET) • TEM • aggregation status, • chemical (surface) reactivity – (Vitamin C assay) • surface coatings/composition, purity
4) Assessing toxicity of fine and nanoparticles: Comparing in vitro measurements to in vivo pulmonary toxicity profiles. Toxicol Sci. 2007. • Particle-types utilized in this study: • Fine-sized carbonyl iron • Fine-sized crystalline silica • Fine-sized amorphous silica • Nano ZnO • Fine ZnO • Particle characterizations conducted both in the “dry state” and “wet state” • Material characterization employed in this study: • Particle characterization in the dry state • particle size - surface area – density - crystallinity • calculated size in dry state (based on surface area determinations) • purity
4) Assessing toxicity of fine and nanoparticles: Comparing in vitro measurements to in vivo pulmonary toxicity profiles. Toxicol Sci. 2007. (cont) • Particle characterization in the wet state • particle size in solutions – PBS, culture media, water • average aggregated size in solutions, • % distribution • surface charge • aggregation status • Conversion and comparisons of in vitro and in vivo doses for dosimetric comparisons
5) Comparative Pulmonary Toxicity Assessments of C60 Water Suspensions in Rats: Few Differences in Fullerene Toxicity In Vivo in Contrast to In Vitro Profiles. Nano Lett. 2007. • Material characterization employed in this study: • particle size and size distribution • surface charge • crystallinity • TEM • composition • oxidative radical activity (ESR measurements) • surface reactivity (erythrocyte hemolytic potential)
Recommendations for Minimal Essential Material Characterization for Hazard Studies with Nanomaterials • Particle size and size distribution (wet state) and surface area (dry state) in the relevant media being utilized – depending upon the route of exposure; • Crystal structure/crystallinity; • Aggregation status in the relevant media; • Composition/surface coatings; • Surface reactivity; • Method of nanomaterial synthesis and/or preparation including post-synthetic modifications (e.g., neutralization of ultrafine TiO2 particle-types); • Purity of sample;
Pulmonary Toxicity Study in Rats with Three Forms of ultrafine-TiO2 Particles: Differential Responses related to Surface Properties Toxicology 230: 90-104, 2007
300 nm 300 nm 300 nm A uf-1 B uf-2 C uf-3 Characterization of Ultrafine TiO2 Particle-types - 1
Protocol for ultrafine TiO2 Pulmonary Bioassay Study • Exposure Groups • PBS (vehicle control) • Particle-types (1 and 5 mg/kg) • rutile-types uf-1 TiO2 • rutile-type uf-2 TiO2 • anatase/rutile-type uf-3 TiO2 • rutile-type F-1 fine TiO2 (negative control) • α-Quartz particles (positive control) Instillation Exposure Postexposure Evaluation via BAL and Lung Tissue 24 hr 1 wk 1 mo 3 mo
RESULTSBiomarkersPulmonary InflammationPulmonary CytotoxicityLung cell Proliferation
Lung Sections of Rats exposed to uf-1 (A); uf-2 (B); or F-1 (C)- 3 months pe
Lung Section of Rat exposed to Quartz particles - 3 months postexposure
Pulmonary Bioassay Studies with Nanoscale and Fine Quartz Particles in Rats: Toxicity is not Dependent upon Particle Size but on Surface Characteristics Toxicol Sci. 95:270-280, 2007
A B D C Lung Tissue Sections – Control (A); Min-U-Sil (B); NanoQ II (C); Fine Quartz (D).
The hemolytic potential of the four a-quartz samples used in the study. The samples, including: These samples show a similar trend as the inflammation, cytotoxicity, and cell proliferation data. Crystalline Silica (Min-U-Sil 5) 534 nm Fine Quartz 300 nm ABS @ 540 nm Nano Quartz I 50 nm Nano Quartz II 12 nm Concentration (mg/mL) Hemolytic Potential of a-Quartz Samples Hemolytic potential is a measure of surface reactivity. • Min-U-Sil • fine-quartz • nano-quartz I • nano-quartz II nano-quartz II = Min-U-Sil > fine-quartz > nano-quartz I
H O O H O H H O O H O H O H O H H O O H H O O H O H H O O H H O Fullerene Water Suspensions Characterization C60(OH)24 Nano-C60
200 150 Population 100 50 0 50 100 150 200 250 Size (nm) Fullerene Water Suspensions Characterization Nano-C60 characterization 200 nm
Recommendations for Minimal Essential Material Characterization for Hazard Studies with Nanomaterials • Particle size and size distribution (wet state) and surface area (dry state) in the relevant media being utilized – depending upon the route of exposure; • Crystal structure/crystallinity; • Aggregation status in the relevant media; • Composition/surface coatings; • Surface reactivity; • Method of nanomaterial synthesis and/or preparation including post-synthetic modifications (e.g., neutralization of ultrafine TiO2 particle-types); • Purity of sample;
Acknowledgments • This study was supported by DuPont Central Research and Development. • Tom Webb and Ken Reed provided the pulmonary toxicology technical expertise for the study. Dr. Christie Sayes – postdoctoral fellow. Denise Hoban, Elizabeth Wilkinson and Rachel Cushwa 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.