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Considerations for Characterizing the Potential Health Effects from Exposure to Nanomaterials

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

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  1. 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

  2. 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

  3. Rat Lung Microdissection

  4. Rat Lung Tissue Dissected to Demonstrate the Junction of the Terminal Airway and Proximal Alveolar Region

  5. Iron Particle Deposition at Bronchoalveolar Junction

  6. Iron Particle () Deposition in the Lungs of Exposed Rats

  7. Iron Particle Deposition at Bronchoalveolar Junction(Backscatter Image)

  8. Alveolar Macrophage Clearance of Inhaled Iron Particles

  9. Alveolar Macrophage Clearance of Inhaled Iron Particles(Backscatter Image)

  10. Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis

  11. Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis(Backscatter Image)

  12. Macrophage phagocytosis of TiO2 particles

  13. Two Alveolar Macrophages (M) Sharing a Chrysotile Asbestos Fiber () with an Alveolar Epithelial Cell (E) M E M

  14. TEM demonstrating pathways for possible translocation of particles

  15. Translocation of chrysotile asbestos fibers from airspace to epithelium

  16. 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)

  17. 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)

  18. 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

  19. 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

  20. 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

  21. 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)

  22. 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;

  23. Studies to Assess Pulmonary Hazards to Nanoparticulates

  24. Ultrafine TiO2 Studies

  25. Pulmonary Toxicity Study in Rats with Three Forms of ultrafine-TiO2 Particles: Differential Responses related to Surface Properties Toxicology 230: 90-104, 2007

  26. 300 nm 300 nm 300 nm A uf-1 B uf-2 C uf-3 Characterization of Ultrafine TiO2 Particle-types - 1

  27. Characterization of Ultrafine TiO2 Particle-types - 2

  28. 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

  29. RESULTSBiomarkersPulmonary InflammationPulmonary CytotoxicityLung cell Proliferation

  30. Pulmonary Inflammation

  31. BAL Fluid LDH Values (cytotoxicity)

  32. Pulmonary Cell Proliferation Rates

  33. Lung Sections of Rats exposed to uf-1 (A); uf-2 (B); or F-1 (C)- 3 months pe

  34. Lung Section of Rat exposed to uf-3 3 months postexposure

  35. Lung Section of Rat exposed to Quartz particles - 3 months postexposure

  36. Nanoscale Quartz

  37. 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

  38. Nanoscale Quartz Particles

  39. Characterization of Nanoscale Quartz Particles

  40. Pulmonary Inflammation – Nanoscale Quartz study

  41. BAL Fluid LDH Values – Nanoscale Quartz study

  42. Lung Parenchymal Cell Proliferation– Nanoscale Quartz study

  43. A B D C Lung Tissue Sections – Control (A); Min-U-Sil (B); NanoQ II (C); Fine Quartz (D).

  44. 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

  45. Summary of α-Quartz Results

  46. 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

  47. 200 150 Population 100 50 0 50 100 150 200 250 Size (nm) Fullerene Water Suspensions Characterization Nano-C60 characterization 200 nm

  48. 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;

  49. 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.

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