Betraying Paracelsus, Ignoring Newton: A Flaw in the Nanotoxicology Paradigm

1. Betraying Paracelsus, Ignoring Newton: A Flaw in the Nanotoxicology Paradigm Justin Teeguarden, PHD, DABT [Paul Hinderliter, Joel Pounds, Brian Thrall,, Galya Orr, Katrina Waters, Tom Weber, Barbara Tarasevich, Bobbie-Jo Webb Robertson] Funded by the Environmental Biomarkers Initiative, DOE

2. Nano, Nanomaterial, Nanotechnology Nano is 10-9. Nanotechnology is the understanding and control of matter at dimensions of roughly 1 to 100 nanometers, where unique phenomena enable novel applications. Nanomaterials are 1-100 nm in at least one dimension National Nanotechnology Initiative, 2002 TiO2 Agglomerates SARS Virus Avain Flu Virus 100 nm Sayes, Tox Sci 2006

3. Nanotoxicology is in its Infancy Do the unique material properties of nanomaterials equate to unique biological properties as well? Which of these properties are responsible for the biological effects? Are the effects limited by the traditional defintion of nanomaterial (1-100 nm)? Who should be studying the biocompatibility or toxicity of these new materials? What studies should be done? Where do we obtain high quality materials for testing?

4. Nanotoxicology is in its Infancy 236 January ‘08

5. What We Know Unknown Nanomaterial Human Health Risks and Risk Assessment Insufficient Data

6. In Vitro Dosimetry

7. The State of The Science (Ignorance?) Response ?

8. Major Challenges for Dose-Response Assessment • What is dose? • Size, shape, particle number, agglomeration state, surface chemistry, reactivity, surface area? Dose rate is also important. • Do researches have all the tools necessary to measure these characteristics? • What is dose for in vitro studies? • 67% of published NM toxicity studies use in vitro systems. • Mass concentrations are typically reported. • Cannot be compared to doses for in vivo studies. • How do you measure NM characteristics in liquid systems? • How do you extrapolate animal study doses to human doses • This extrapolation is required for all risk assessments based on animal studies

9. Cell Culture is a Standard Tool for Mechanistic Work and Hazard Screening Expose cells to selected concentrations of suspended particles Report dose-response on a nominal media concentration basis • The paradigm for chemicals is being used without consideration of the unique kinetic differences between chemicals and particles in solution: • Response is considered a function of the nominal media mass or surface area concentration (cm2/ml, µg/ml)

10. Dosimetry for Particles is Important! Response is proportional to concentration at the target site!

11. So What’s the Problem? In solution, particles settle and diffuse at rates which depend on: Size Density Shape Agglomeration state Surface area changes with size Number concentrations change with size While nominal media concentrations stay the same across density and size, DELIVERY RATES DO NOT!

12. Nominal Media Concentration is A Poor Metric of Dose Mass Concentration (ug/ml) Mass Concentration (ug/ml) SA Concentration (cm2/ml) # concentration (#/ml) 1 nm Particle Size 1000 nm 1 nm Particle Size 1000 nm Decreases as the cube of the radius Decreases as the square of the radius

13. Nominal Media Concentration is A Poor Metric of Dose Mass Concentration is Constant Diffusivity (Cm2/s) Settling Rate (cm/s) SA Concentration (cm2/ml) # concentration (#/ml) • Density 20 • (g/cm3) 1 Particle Size 1000 (nm)

14. Nominal Media Concentration is A Poor Metric of Dose Mass Concentration is Constant Diffusivity (Cm2/s) Settling Rate (cm/s) • Density 20 • (g/cm3)

15. Dose-Related Parameters are Not Constant Across Particle Types How Misleading can Nominal Media Concentrations Be?

16. Different Particles = Different Delivery Rates to Cells Slower Faster T = 23 Hours Post Mixing

17. From First Principles: TiO2 and Gold Nanoparticle Dosimetry

18. Nominal Media Concentration Obscures Underlying Dose-Response Behavior California EPA October 3, 2007

19. Compartative Potency is affected by the Choice of Dose Metric Particle Size (nm) Material

20. Delivered Dose Reveals Surface Area Relationship California EPA October 3, 2007

21. Is Any of this Real, or is it Just a Theory? Ceria Oxide Nano Particles 25-300 nm

22. Cellular Uptake is Diffusion Driven for Small Nanoparticles and Gravity Driven for Larger particles

23. Which Particle is Most Toxic?

24. Settling Impacts CNT Toxicity? Pellet Supernatant Rapid Settling Slow Settling

26. Add LD50 Table

27. The Evidence this is Nonsense

28. Delivery of These Particles is Driven by Diffusion Three Cell Types, Three Laboratories, Several Endpoints One Particle Size Dose Not Test The Hypothesis that Size and Density Affect Delivery

29. Response is proportional to Concentration! Wait, no its proportional to mass! Changing mass & concentration Changing concentration Changing mass Lison et al. 2008

30. Response is proportional to Concentration! Wait, no its proportional to mass! Changing mass & concentration Changing mass Changing concentration Lison et al. 2008

31. Cellular Dose is Related to Mass and Concentration Lison et al. 2008

32. Response Could be Related to Mass and Concentration if Agglomerates are Settling Out But we really need to know better what is going on in these systems!

33. Conclusions • In vitro, untested assumptions and a flawed paradigm are the norm • Biologically and kinetically relevant measures of dose are not being used, imperiling interpretation of many studies.. • What should the future be? • Research to improve our understanding of kinetics and dosimetry in vitro is essential, but not often appreciated • Direct measures of particle behavior in solution • Direct measures of cellular particle dose • Computational models of delivery • These studies will support interpretation as well as extrapolation (NRC Vision for Toxicology)

34. What is the Future of Nanomaterial Dosimetry at PNNL? • Establish a Paradigm for Rapid Development of Biokinetic Models From In Vitro Kinetics and Cellular Uptake Data • Complete development of predictive models of in vitro kinetics and cellular uptake (Macrophages, and other elements of the RES system) • Correlate material properties and serum protein binding to rates of uptake in tissues of the RES system • Scale models of cellular uptake to full tissues/in vivo and integrate within our PBPK models • Test and revise predictive models of in vivo dosimetry • This is best accomplished as one element of an integrated program in nanomaterial biocompatibility.

35. PBPK Model Development and In Vivo Rodent Kinetics Completes our Dosimetry Program 18 nm gold +, - and PEG 28 day blood, tissue kinetics in rats

36. Computational In Vitro Dosimetry Particle Settling Rate Equation: Galya’s Cellular Uptake Data Where Partial differential equations representing Navier-Stokes settling and Fickian diffusion are combined to form a single partial differential equation describing macroscopic particle transport. The solution to this equation is the basis for the computational model of nanoparticle solution dynamics and dosimetry in vitro (NanoDose). The model is written in MatLab. Parameters: n, number-density of particles at place x and time t; t, time; x, vertical distance from bottom of well; R, gas constant; T, temperature; N, Avogadro’s number; m, viscosity of liquid, a particle radius; g, gravitational acceleration; d, effective particle density (density of particle – liquid density). From Mason and Weaver (1924).