1. Potential for Bio-uptake and Bioaccumulation of Nanotechnology Particles: Impact of Carbon Nanotube Exposures in the Lung David B. Warheit, Ph.D. DuPont Haskell Laboratory IOM – Implications of Nanotechnology May 27, 2004

2. Outline Lung structure and particle deposition Pulmonary bioassay as a measure of bioaccumulation Pulmonary bioassay with single wall carbon nanotubes Preliminary results with Fine/Nano- scale quartz, Fine/Nanoscale TiO 2 dots and rods, Fine and Nano ZnO Summary

3. Definitions- Particle Size Nano = Ultrafine = < 100 nm Fine = 100 nm - 3  m Respirable (rat) = < 3  m (max = 5  m) Respirable (human) = < 5  m (max = 10  m) Inhalable (human) = ~ 10 - 50  m

4. Particle Scale Nanoparticles UltrafineRespirable PM 10 1 nm10 nm100 nm 1  m 10  m PM 2.5

5. Rat Lung Microdissection

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

7. Iron Particle Deposition at Bronchoalveolar Junction

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

9. Equivalent dia. ~2 x Settling velocity ~3-4 x Mechanical interlocking Capillary (surface tension) Van der Waals (cohesive force α 1/d**2) Chemical bonds Single particle Equivalent diameters of 10-1000x are common Interparticle Forces (Aggregates & Agglomerates)

10. Alveolar Macrophage Clearance of Inhaled Iron Particles

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

12. Alveolar Macrophage Migration to Iron Particle Deposition and Phagocytosis

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

14. Clearance of Iron Particles on the Airway Mucociliary Escalator

16. Morphometry at Bronchoalveolar Junctions

17. Cytocentrifuge Preparation of BAL – Recovered Cells From a Sham – Exposed Rat

18. Cytocentrifuge Preparation of BAL – Recovered Cells From a Quartz (Crystalline Silica) – Exposed Rat

19. External Perceptions on Pulmonary Toxicity of Nanoparticles Nanoparticles are more toxic (inflammogenic, tumorigenic) than fine- sized particles of identical composition. Concept generally based on 3 particle-types: –Titanium Dioxide particles –Carbon Black particles –Diesel Particles

20. Complications related to the Dogma of Nanoparticulate Toxicology Not all Nanoparticles are more toxic Surface coatings of particles –Coatings - passivated or dispersion Species Differences in Lung Responses –Rat is the most sensitive species Particle aggregation/disaggregation potential Fumed vs. precipitated Nanoparticles Surface charge of particles

21. Pulmonary Bioassay Studies Working hypothesis Four factors influence the development of pulmonary fibrosis 1)inhaled materials which cause cell/lung injury 2)inhaled materials which promote ongoing inflammation 3)inhaled materials which reduce alveolar macrophage function 4)inhaled materials which persist in the lung

22. Pulmonary Toxicity Screening Studies with Single Wall Carbon Nanotubes DB Warheit, BR Laurence, KL Reed, DH Roach*, GAM Reynolds*, G Joseph*, and TR Webb DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE and *DuPont Company, Wilmington, DE

23. Objective The objective of this pulmonary bioassay study was to evaluate, using a bridging methodology, the acute lung toxicity of intratracheally instilled single wall carbon nanotubes (CNT) in rats when compared to positive and negative particle controls. Nanoparticles are considered to be more hazardous when compared to fine-sized particles of identical chemical composition.

24. Transistors and diodes Field emitter for flat- panel displays Cellular-phone signal amplifier Ion storage for batteries Materials strengthener Source: Scientific American- Illustration: RICHARD E. SMALLEY, Rice University Some Possible Uses for Carbon Nanotubes

25. Pulmonary Bioassay Components Bronchoalveolar Lavage Assessments Lung Inflammation & Cytotoxicity Cell Differential Analysis BAL Fluid Lactate Dehydrogenase (cytotoxicity) BAL Fluid Alkaline Phosphatase (epithelial cell toxicity) BAL Fluid Protein (lung permeability) Lung Tissue Analysis Lung Weights Lung Cell Proliferation (BrdU)  Parenchymal  Airway Lung Histopathology

27. Parameter BALF Cells and Differentials BALF Lactate Dehydrogenase BALF Alkaline Phosphatase BALF Protein Lung Weights Macrophage phagocytosis Cell Proliferation Histopathology Use of Bronchoalveolar Lavage, Cell Proliferation, and Histopathology to Assess the Lung Toxicity of CNT samples Indicator Lung Inflammation Non-specific cytotoxicity Type 2 cell epithelial toxicity Permeability  of alveolar/ capillary barrier Pulmonary edema or fibrosis Lung clearance functions Inflammation/lung fibrosis and tumor potential Evaluation of lung tissue responses (BALF = Bronchoalveolar Lavage Fluid Analysis)

28. Protocol for Carbon Nanotube Bioassay Study Intratracheal Instillation Exposure Doses of 1 and 5 mg/kg Exposure Groups PBS (control) PBS-Tween 80 (control) Particulate Types (1 and 5 mg/kg) Carbon Nanotubes Quartz Particles (positive control) Carbonyl Iron Particles (negative control) Graphite (carbon particle control) Instillation Exposure 24 hr1 wk1 mo3 mo Postexposure Evaluation via BAL and Lung Tissue

29. Pulmonary Bioassay Bridging Studies Carbonyl Iron Particles Quartz Particles PBS Tween Sham Carbonyl Iron Particles Carbon Nanotube Particles Quartz Particles vs Inhalation Studies Intratracheal Instillation Studies

30. RESULTS

31. Light micrograph of lung tissue from a rat exposed to 5 mg/kg CNT (a few hours after exposure). The major airways are mechanically blocked by the CNT instillate. This lead to suffocation in 15% of the CNT- exposed rats and was not evidence of pulmonary toxicity of CNT.

33. Bronchoalveolar Lavage Fluid Results

34. Pulmonary inflammation in particulate-exposed rats and controls as evidenced by % neutrophils (PMN) in BAL fluids at 24 hrs, 1 week, 1 month and 3 months postexposure (pe). Instillation exposures resulted in transient inflammatory responses for nearly all groups at 24 hrs pe. However, exposures to Quartz particles at 1 and 5 mg/kg produced a sustained lung inflammatory response. p < 0.05

35. BAL fluid LDH values for particulate-exposed rats and corresponding controls at 24 hrs, 1 week, 1 month and 3 months postexposure (pe). Significant increases in BALF LDH vs. controls were measured in the CNT 5 mg/kg exposed group at 24 hrs pe and the 5 mg/kg Quartz- exposed animals at all 4 time periods pe. p < 0.05.

36. Lung Tissue Results Lung Histopathology Studies

37. Carbonyl Iron particles - 1 Month postexposure

38. Quartz particles - 1 Month postexposure

40. Quartz particles - 3 Months postexposure

41. CNT - 1 Week postexposure

42. CNT - 1 Month postexposure

44. Conclusions from Instillation Studies Intratracheal instillation exposures in rats to CNT (5 mg/kg) produced ~15% mortality. Further investigation of this finding demonstrated that following instillation of the dispersed CNT suspension (with PBS and Tween), the CNT agglomerated in the main airways and the rats died from suffocation – not CNT toxicity.

45. Conclusions from Bronchoalveolar Lavage Study Pulmonary exposures to Quartz particles produced a sustained, dose-dependent lung inflammatory response measured from 24 hrs  3M postexposure. Exposures to CNT (5 mg/kg) produced a transient pulmonary inflammatory response at 24 hrs but was not sustained. Exposures to Carbonyl Iron produced a brief neutrophilic lung response – related to the instillation exposure.

46. Observations on CNT- induced Lung Lesions Multifocal granulomas consisted of macrophage-like multinucleate giant cells – foreign body rxn Granulomatous lesions appeared to be associated with a black CNT bolus Distribution of lesions - not consistent in lung lobes No apparent dose response relationship Possible regression with time 1M  3M Not consistent with ADBF paradigm – unlike silica/asbestos or other dust-related lesions BAL results were not predictive of this lesion Likely affected by route of exposure? – instillation of CNT bolus?

47. Exposure to carbon nanotube material: Aerosol release during the handling of unrefined SWCNT- Andrew Maynard et al. Laboratory study and field-based study Field study – assessed airborne and dermal exposure to SWCNT while handling unrefined material. Lab studies – SWCNT can release fine particles with sufficient agitation. Field studies – concentrations generated while handling material were very low- always < 53  g/m 3.

48. Handling nanotube material Raw single walled nanotube material

49. Unique structures and morphologies Carbon Nanotubes

50. Case Study Health risk associated with carbon nanotube production

51. Characteristics of airborne ‘nanotube’ particle Expected Morphology Predominant Morphology (Field Samples)

52. Particle Scale Nanoparticles UltrafineRespirable PM 10 1 nm10 nm100 nm 1  m 10  m PM 2.5

53. DuPont exposure assessment study Monitored area where single-wall carbon nanotubes were handled. Exposure levels were below detectable limits.

54. Final Thoughts To reconcile the results discrepancies in this study, the pulmonary effects of CNT in rats must be evaluated by conducting an inhalation toxicity study of carbon nanotubes. Two exposure assessment studies of CNT in the workplace have been conducted – the results indicate very low aerosol exposures to respirable-sized Carbon Nanotube particulates.

55. Collaborative effort with Rice (NanoQuartz and NanoTitania) + Fine & Nano ZnO particle inhalation Pulmonary bioassay studies with Nanoquartz & TiO2 rods and TiO2 dots Vicki Colvin and Christie Sayes – Rice University Short-term inhalation studies in rats - Fine (< 1  m) vs. Nano ZnO (65 nm) particles

56. Nanoscale Quartz Particles

57. Preliminary Collaborative Studies with Rice University: SiO 2 Nano-SiO 2 is lower than Min-U-Sil

58. TiO2 Nanoscale Dots

59. TiO2 Nanoscale Rods

60. Preliminary Collaborative Studies with Rice University: TiO 2 Pigmentary & Nano-TiO 2 are not different

61. Preliminary Collaborative Studies with Rice University Comparing the toxicity of Nanoscale quartz particles vs. fine-sized quartz particles (IARC Category I – human carcinogen) –preliminary findings are that uncoated Nanoquartz particles are less toxic than fine- sized quartz. Comparing the toxicity of pigment-grade TiO2 particles (~300 nm) vs. TiO2 nanorods and TiO2 nanodots (30 nm). –prelim findings – no difference in lung toxicity

62. Inhalation Studies in Rats with Fine or Nano Zinc Oxide particles

63. Preliminary Studies with Fine or Nano Zinc Oxide particles

64. Summary Cannot assume that nanomaterials are the same as their bulk counterpart Each particle-type should be tested on a case-by-case

65. Acknowledgments This study was supported by DuPont Central Research and Development. Dr. David Roach and Dr. Gillian Reynolds supplied the nanotube and graphite materials. Tom Webb and Ken Reed provided the pulmonary toxicology technical expertise for the study. 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. Dr. Steven R. Frame provided histopathological evaluations of lung tissues. Don Hildabrandt provided animal resource care.