Application of ADME/PK Studies to Improve Safety Assessments for Food and Cosmetics
February 23, 2015 Consideration of ADME/PK in Safety Assessments for Engineered Nanomaterials: Example with Silver Nanoparticles
William K. Boyes, PhD. Office of Research and Development US Environmental Protection Agency Research Triangle Park, NC
Dr. Boyes has no financial conflicts of interest
The materials in this presentation do not represent endorsement of commercial products or official policies of the Environmental Protection Agency What is nanotechnology?
Nanotechnology is science, engineering, and “There is plenty of room at the technology conducted at the nanoscale bottom” (Richard Feynman) Nanoparticles are usually defined as having at least 1 dimension between 1 to 100 nanometers. 1 nm = 10-9 m 1 nm x 109 = 1 m 1 m x 107 = size of earth
90 nm size of HIV 200 nm = 2 x 10-7 m (limit visible microscope) 400-700 nm, wavelengths of visible light
10-9 10-7 10-5 10-3 10-1 101 103 105 107 109 1 nm 100 nm meters
Visible Earth (http://visibleearth.nasa.gov/) Nanotechnology is growing
President’s Council of Advisors on Science and Technology, 2014 Categories of commercial nanomaterials
Category # product Examples listings Carbon nanotubes 714 SWCNT, MWCNT
Fullerenes 136 Pure or functionalized
Graphene 38 Film, on substrate
Nanoparticles of elements 549 Metals (silver, gold etc.)
Binary compounds 750 metal oxides, salts, carbonates
Complex compounds 205 Doped metal oxides
Quantum Dots 183 Cadmium selenide
Biomedical Quantum Dots 205 Peg modified Qdots, Antibody coated
Nanowires 26 Copper, gold, indium
Nanofibers 30 Carbon
Non-carbon nanowires 1 Titania 6 http://www.nanowerk.com/phpscripts/n_dbsearch.php Accessed Aug 6, 2012 Health and Safety Issues for Engineered Nanomaterials (ENM)
High surface area / mass causes high reactivity High reactivity may lead to inadvertent toxicity Small size may enable them to distribute widely in biological systems Evaluating ENM risks will require methods and technology beyond traditional toxicological tools Rapid development and application exceeds capacity to test potential toxicity using conventional approaches Risk assessments will need to consider: – alternative testing strategies (ATS) – in vitro - in vivo extrapolation (IV-IVE) Transformative Science
Toxicity Testing in the 21st Century: A Vision and a Strategy (NRC, 2007) Growing number of ENM; can’t afford to test one-by-one Behavior of ENM depends on their inherent chemical and physical properties and how those properties interact with the environment and sensitive species in the environment Transformative science will understand the influence of ENM material properties and build predictive models so that each new material does not need to be fully tested Chemical Safety for Sustainability Objectives: Emerging Materials/ Nanomaterials
Developing test systems that are adequate for evaluation of nanomaterials Identifying critical parameters that influence their behavior in the environment Determining how the inherent properties influence behavior in biological systems and act in adverse outcome pathways Research Approach
10 Nano-silver (AgNP) Uses & Misuses
Anti-microbial properties (AgNP release Ag+ )
Fabric coatings “Homeopathic remedies” Surface coatings colloidal silver products Spray disinfectants – Dietary supplements – Inhalant formulations Children’s products – Skin products Electronics FDA (1999): Household appliances – NOT safe and effective Water disinfectants – Side effects include: Medical wrappings and Argyria (blue-gray skin) Poor absorption of drugs devices Possible kidney, liver, or Food packaging nervous system problems
Food supplements
Silver Nanomaterials
Release of Silver from Nanotechnology-Based Consumer Released from Products for Children Products • Examined bioavailable silver released from products for children • Among liquid media, sweat and urine caused the largest amount of silver to be released Fate Transport & • Fabrics, plush toys and spray products were most likely to Transformation release silver • Dissolution of silver particles to ionic form facilitated exposure • Overall, however, the level of exposure to children from consumer products was predicted to be low Health Effects
Ecological Effects
Comprehensive Analysis QUADROS, M. E., PIERSON, R., TULVE, N. S., WILLIS, R., ROGERS, K., THOMAS, T. A. & MARR, L. C. 2013. Environmental Science & 12 Technology, 47, 8894-8901. Silver Nanomaterials
Released from Silver speciation and release in commercial antimicrobial Products textiles as influenced by washing • The speciation of silver in commercial textiles as revealed by XANES, is complex • Silver nanoparticles are only one of several Ag species in Fate, Transport & commercial textiles (Ag(0), AgCl, Ag2S, Ag–phosphate, ionic Ag and other species) Transformation • Washing with two detergents resulted in significant changes in silver speciation (Ag-phosphates, nitrates and sulfates) • The complexity of Ag speciation in textiles complicates exposure assessments Health Effects
Ecological Effects
LOMBI, E., DONNER, E., SCHECKEL, K. G., SEKINE, R., LORENZ, C., GOETZ, N. V. & NOWACK, B. 2014.. Chemosphere, 111, 352-358. Comprehensive Analysis 13 Silver Nanomaterials
Released from Alterations in physical state of silver nanoparticles exposed Products to synthetic human stomach fluid
• Acidic conditions in synthetic stomach fluid altered the physical and chemical state of silver nanoparticles Fate, Transport & Transformation • Citrate-stabilized AgNPs agglomerate and form AgCl during exposure to simulated stomach fluid.
• Ingested AgNPs may be converted to a variety of aggregated and chemically modified particles in the stomach Health Effects
ROGERS, K. R., BRADHAM, K., TOLAYMAT, T., THOMAS, D. J., HARTMANN, T., MA, L. & WILLIAMS, A. 2012. Science of The Total Environment, 420, 334-339.
Ecological Effects
Comprehensive Analysis 15 Silver Nanomaterials
Released from Products Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: effect of size, surface coating, and intracellular uptake. Fate, Transport & Transformation • Silver nanoparticles and silver nitrate activate same cellular stress-response and inflammatory pathways
• Smaller nanoparticles are more potent than larger particles
Health Effects • Effects of silver nanoparticles likely mediated by silver ions
PRASAD, R. Y., MCGEE, J. K., KILLIUS, M. G., SUAREZ, D. A., BLACKMAN, C. F., DEMARINI, D. M. & SIMMONS, S. O. 2013. Toxicol In Ecological Effects Vitro, 27, 2013-21
Comprehensive Analysis Silver Nanomaterials
Released from Toxicogenomic Responses of Nanotoxicity in Daphnia Products magna Exposed to Silver Nitrate and Coated Silver Nanoparticles • Daphnia showed different genomic responses to silver nitrate and silver nanoparticles Fate, Transport & • Silver nanoparticles disrupted protein metabolism and signal Transformation transduction • Silver nitrate downregulated developmental processes, particularly in sensory systems
Health Effects
Ecological Effects
POYNTON, H. C., LAZORCHAK, J. M., IMPELLITTERI, C. A., BLALOCK, B. J., Comprehensive ROGERS, K., ALLEN, H. J., LOGUINOV, A., HECKMAN, J. L. & GOVINDASMAWY, S. Analysis 2012. Environmental Science & Technology, 46, 6288-6296. Silver Nanomaterials
Comprehensive Environmental Assessment (CEA) 2012 Released from Products • a framework for systematically organizing complex information
• a process of collective judgment to evaluate information and identify research gaps. Fate, Transport & Transformation
Health Effects
Ecological Effects
Comprehensive Analysis http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=241665 PBPK model for silver ion and silver NP Bachler et al 2013
Model Rat and human Ag+ and AgNP 3 routes of exposure Fit to data from literature
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771750/bin/ijn-8-3365Fig1.jpg Gerald Bachler, Natalie von Goetz, and Konrad Hungerbühler. A physiologically based pharmacokinetic model for ionic silver and silver nanoparticles. Int J Nanomedicine. 2013; 8: 3365–3382. PBPK model for silver ion and silver NP Bachler et al., 2013
Ionic Silver Nanoparticle Silver
MPS – mononuclear phagocyte system
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771750/bin/ijn-8-3365Fig2.jpg Gerald Bachler, Natalie von Goetz, and Konrad Hungerbühler. A physiologically based pharmacokinetic model for ionic silver and silver nanoparticles. Int J Nanomedicine. 2013; 8: 3365–3382. Simulations vs rat oral 28 day exposure
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771750/bin/ijn-8-3365Fig4.jpg Gerald Bachler, Natalie von Goetz, and Konrad Hungerbühler. A physiologically based pharmacokinetic model for ionic silver and silver nanoparticles. Int J Nanomedicine. 2013; 8: 3365-3382. Simulations vs human data
Model fit to data from: A. Deceased normal adults B. Burn patients treated with silver nitrate C. Occupational exposure with silver in air
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771750/bin/ijn-8-3365Fig6.jpg Gerald Bachler, Natalie von Goetz, and Konrad Hungerbühler. A physiologically based pharmacokinetic model for ionic silver and silver nanoparticles. Int J Nanomedicine. 2013; 8: 3365–3382. Conclusions from PBPK model
Particle size, surface charge and coating had a minor influence on distribution due to opsonization and corona stabilization More likely for AgNP to be stored as insoluble salt particles than dissolve into Ag+ in vivo. No significant dissolution in vivo Mononuclear phagocyte system plays a minor role at relevant exposure levels for human consumers
Gerald Bachler, Natalie von Goetz, and Konrad Hungerbühler. A physiologically based pharmacokinetic model for ionic silver and silver nanoparticles. Int J Nanomedicine. 2013; 8: 3365–3382.
In vitro nanotoxicology considerations
Physical/chemical Dose metric (e.g.) characterization of particles – Mass concentration (ug/ml, or Suspension / dispersion ppm) protocols – Mass / surface area of well (ug/m2) Stability of suspensions over time – # particles – Surface area of particles (m2/g) Agglomeration / transformation – Delivered dose to cells Assay measurements & (estimated) interference with luminance, – Delivered dose to cells fluorescence or colorimetric (measured) assays (e.g. MMT)
(in addition to normal considerations for in vitro – in vivo extrapolation (IV – IVE)) ToxCast HTS assay data to classify nanomaterials based biological activity
Problem – Many more ENM are being developed than can be tested with existing approaches – High Throughput Screening (HTS) is being developed for chemicals: will it work for ENM? Approach – Evaluate a variety of ENM using the ToxCast assays – Select nanomaterials that range across composition size and structure – Evaluate outcomes as they map to classes and types of ENM Deliverables – Data set has been collected and will be available – Analysis is complex and underway. Impact – Decisions and approaches to screening novel ENM – Ranking and classification of ENM by profile of outcomes – Prioritization for further assessment
2 4 Screening diverse classes of NMs in ToxCast
Screened 67 samples (62 unique) Endpoint types by platforms
nano micro ion Ag 5+2* 1 1 DNA • Transcription factor Asbestos 3 activation Au 1 RNA CNT 8 • Protein profile CeO 4 1 1 2 Protein Cu 4+2# 2+1# 2
SiO 5 1 • Cell growth kinetics 2 Function/ TiO 9 4 2 Phenotype • Toxicity phenotype ZnO 2 1 1 • Developmental * IAT NP and IAT NP infused with Ag ion malformation # purified sample with no/low ions Not listed: Dispersant of one of the nano-Ag (zebrafish)
25 Overview of ToxCast Screening of 62 Nano and Reference Materials Yellow: Less active Blue: More active Groups of assays
Bioactivity generally in the 1-100 ug/ml range Individual NM with Ag, Cu, Zn are more active than others particlesData are being analyzed NT & asbestos had different inflammatory response profiles
26 Further in vitro studies
Evaluate – The role of particle size and coating – Measures of cellular uptake & distribution – Measures of cellular dose Model – Human derived retinal pigment epithelial cells (APRE-19) – Suspend in cell culture medium (containing protein) – Treat cells for 24 hrs – Evaluate uptake of AgNP and cytotoxicity
Silver Samples Analyzed
10nm PVP 50nm PVP 75nm PVP
25 nm 50 nm 100 nm
10nm Citrate 50nm Citrate 75nm Citrate
10 nm 50 nm Silver nanoparticle cytotoxicity
• Cytotoxicity of silver nanoparticles evaluated in human derived retinal pigment epithelial cells (ARPE-19) • Small particles more toxic than large particles • PVP coating more toxic than citrate coating in larger sized particles
29 Silver nanoparticles in ARPE-19 cells in culture under dark field / fluorescence microscopy
Blue: DNA Green: Golgi White: AgNP Orange: Cell Membrane Blue: DNA Green: Golgi White: AgNP Orange: Cell Membrane Zucker et al., 2013 Flow-cytometry
Cells that incorporate reflective ENM will show – Increased side scatter (SSC) – Reduced forward scatter (FSC) SSC is a function of – ENM particle size – ENM particle number AgNP show increased far red fluorescence from Surface Plasmon Resonance Zucker et al., 2013 Target cell dosimetry in vitro
Particle deposition influenced by: • Size • Agglomeration media • Density • Media • Viscosity • Density • Temperature cell layer • Time • ISDD Model • Stokes Law (sedimentation) • Stokes-Einstein Equation (diffusion) Diffusion • Refs: Sedimentation • Teeguarden et al, 2007 • Hinderliter et al., 2010 • Cohen et al., 2103 1 10 100 1000 Size (nm) [Ag] in cells and 0 ug/ml No Cells ARPE-19 Cells 20 nm AG NP 3 ug/ml
media 10 ug/ml 0 ug/ml 30 ug/ml
3 ug/ml Into 2 flasks
Into 2 flasks APRE-19 cells in vitro 10 ug/ml
AgNP (citrate), control and 3 30 mg/ml Into 2 flasks concentrations 300 ul / flash for each flask After 24 hrs., separate cells from pellet
cells media 0.25 mls 0.25 mls
Media and Measure [Ag] in cells & media wash Cells
300 ul via ICP-MS supernat cells Compare flow side-scatter with cells ICP-MS Do with both 20 nm and 75 nm AgNP
ICP-MS Flow cytometry [Ag] in Media and Cells by ICP-MS
• After 24 hours, as much as 90% of silver remains in the media • Smaller ENM are the more likely to remain dispersed in media • Dose level (ug/ml) added to the culture can be very different from actual dose to the cell layer for adherent cell cultures Dose measures compared
• Measures of dose include: • mass • particle number • surface area • Vary greatly across particle size • In some cases are inversely correlated Flow Cytometry Side Scatter vs Dose
• Side scatter is linearly related to measures of absorbed dose • Relationship varies with particle size • Side scatter could be used as a rapid and inexpensive measure of cellular dose if pre-calibrated for particle size and composition
Conclusions
Expanding development and use of nanomaterials requires new approaches for safety and risk evaluations Alternative testing data with pharmacokinetic information promises to be increasingly important In vitro dose metrics and dosimetry models are critical for evaluating in vitro toxicity data AgNP vs Ag+ – Many (but perhaps not all) toxic actions of AgNP are related to Ag+ – AgNP may be accessible to pharmacokinetic compartments unavailable to Ag+ Unresolved: – in vitro: AgNP particle size and coating determine toxicity – In vivo: AgNP particle size and coating not important for distribution
Acknowledgements
Katlin Daniel Laura Degn Sarah Karafas Keith Houck Jayna Ortenzio Lila Thornton Amy Wang Robert Zucker
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References (cont.) NRC (2007). Toxicity Testing in the 21st Century: A Vision and a Strategy. National Research Council of the National Academies, Washington, DC. POYNTON, H. C., LAZORCHAK, J. M., IMPELLITTERI, C. A., BLALOCK, B. J., ROGERS, K., ALLEN, H. J., LOGUINOV, A., HECKMAN, J. L. & GOVINDASMAWY, S. 2012. Toxicogenomic Responses of Nanotoxicity in Daphnia magna Exposed to Silver Nitrate and Coated Silver Nanoparticles. Environmental Science & Technology, 46, 6288-6296. PRASAD, R. Y., MCGEE, J. K., KILLIUS, M. G., SUAREZ, D. A., BLACKMAN, C. F., DEMARINI, D. M. & SIMMONS, S. O. 2013. Investigating oxidative stress and inflammatory responses elicited by silver nanoparticles using high-throughput reporter genes in HepG2 cells: effect of size, surface coating, and intracellular uptake. Toxicol In Vitro, 27, 2013-21. President’s Council of Advisors on Science and Technology. Report to the President and Congress on the Fifth Assessment of the National Nanotechnology Initiative. October 2014. QUADROS, M. E., PIERSON, R., TULVE, N. S., WILLIS, R., ROGERS, K., THOMAS, T. A. & MARR, L. C. 2013. Release of Silver from Nanotechnology-Based Consumer Products for Children. Environmental Science & Technology, 47, 8894-8901. ROGERS, K. R., BRADHAM, K., TOLAYMAT, T., THOMAS, D. J., HARTMANN, T., MA, L. & WILLIAMS, A. 2012. Alterations in physical state of silver nanoparticles exposed to synthetic human stomach fluid. Science of The Total Environment, 420, 334-339. TEEGUARDEN, J. G., HINDERLITER, P. M., ORR, G., THRALL, B. D. & POUNDS, J. G. 2007. Particokinetics in vitro: dosimetry considerations for in vitro nanoparticle toxicity assessments. Toxicol Sci, 95, 300-12. U.S. EPA. Nanomaterial Case Study: Nanoscale Silver in Disinfectant Spray (Final Report). U.S. Environmental Protection Agency, Washington, DC, EPA/600/R-10/081F, 2012. U.S. Food and Drug Administration. Rules and regulations: over-the-counter drug products containing colloidal silver ingredients or silver salts. Final rule. Federal Register. 1999;64(158):44653–44658. ZUCKER, R. M., DANIEL, K. M., MASSARO, E. J., KARAFAS, S. J., DEGN, L. L. & BOYES, W. K. 2013. Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence. Cytometry Part A, 83, 962-972.