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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 technology conducted at the nanoscale

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

Visible Earth (http://visibleearth.nasa.gov/)

“There is plenty of room at the bottom” (Richard Feynman)

meters

10-9 10-7 10-5 10-3 10-1 101 103 105 107 109

1 nm 100 nm

90 nm size of HIV 200 nm = 2 x 10-7 m (limit visible microscope) 400-700 nm, wavelengths of visible light

Nanotechnology is growing

President’s Council of Advisors on Science and Technology, 2014

Categories of commercial nanomaterials

Category # product listings

Examples

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

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

Objectives: Emerging Materials/ Nanomaterials

Research Approach

10

Nano-silver (AgNP) Uses & Misuses

Fabric coatings Surface coatings Spray disinfectants Children’s products Electronics Household appliances Water disinfectants Medical wrappings and

devices Food packaging Food supplements

“Homeopathic remedies” colloidal silver products

– Dietary supplements – Inhalant formulations – Skin products

FDA (1999): – NOT safe and effective – Side effects include:

Argyria (blue-gray skin) Poor absorption of drugs Possible kidney, liver, or

nervous system problems

Anti-microbial properties (AgNP release Ag+ )

Silver Nanomaterials

12

Release of Silver from Nanotechnology-Based Consumer Products for Children • Examined bioavailable silver released from products for

children • Among liquid media, sweat and urine caused the largest

amount of silver to be released • Fabrics, plush toys and spray products were most likely to

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

Released from Products

Fate Transport & Transformation

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 & Technology, 47, 8894-8901.

13


Silver speciation and release in commercial antimicrobial 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

commercial textiles (Ag(0), AgCl, Ag2S, Ag–phosphate, ionic Ag and other species)

• 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


Fate, Transport & Transformation

Health Effects

Ecological Effects

Comprehensive Analysis

LOMBI, E., DONNER, E., SCHECKEL, K. G., SEKINE, R., LORENZ, C., GOETZ, N. V. & NOWACK, B. 2014.. Chemosphere, 111, 352-358.

15


Alterations in physical state of silver nanoparticles exposed to synthetic human stomach fluid • Acidic conditions in synthetic stomach fluid altered the

physical and chemical state of silver nanoparticles

• 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

Ecological 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.


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. • Silver nanoparticles and silver nitrate activate same cellular

stress-response and inflammatory pathways

• Smaller nanoparticles are more potent than larger particles

• Effects of silver nanoparticles likely mediated by silver ions



Health Effects

Ecological Effects


PRASAD, R. Y., MCGEE, J. K., KILLIUS, M. G., SUAREZ, D. A., BLACKMAN, C. F., DEMARINI, D. M. & SIMMONS, S. O. 2013. Toxicol In Vitro, 27, 2013-21




Health Effects

Ecological Effects


Toxicogenomic Responses of Nanotoxicity in Daphnia magna Exposed to Silver Nitrate and Coated Silver Nanoparticles • Daphnia showed different genomic responses to silver nitrate

and silver nanoparticles • Silver nanoparticles disrupted protein metabolism and signal

transduction • Silver nitrate downregulated developmental processes,

particularly in sensory systems

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. Environmental Science & Technology, 46, 6288-6296.

Silver Nanomaterials Comprehensive Environmental Assessment (CEA) 2012 • a framework for systematically organizing complex information

• a process of collective judgment to evaluate information and

identify research gaps.



Health Effects

Ecological Effects

Comprehensive Analysis http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=241665

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=241665�

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

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�

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-8-3365Fig4.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 rat oral 28 day exposure



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






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.

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

Conclusions from PBPK model

In vitro nanotoxicology considerations Physical/chemical

characterization of particles Suspension / dispersion

protocols Stability of suspensions over

time Agglomeration / transformation Assay measurements &

interference with luminance, fluorescence or colorimetric assays (e.g. MMT)

Dose metric (e.g.) – Mass concentration (ug/ml, or

ppm) – Mass / surface area of well

(ug/m2) – # particles – Surface area of particles (m2/g) – Delivered dose to cells

(estimated) – Delivered dose to cells

(measured)

(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

24

Screening diverse classes of NMs in ToxCast

* IAT NP and IAT NP infused with Ag ion # purified sample with no/low ions Not listed: Dispersant of one of the nano-Ag

DNA

RNA

Protein

Function/ Phenotype

Screened 67 samples (62 unique) Endpoint types by platforms

• Transcription factor activation

• Protein profile

• Cell growth kinetics

• Toxicity phenotype

• Developmental malformation (zebrafish)

nano micro ion Ag 5+2* 1 1 Asbestos 3 Au 1 CNT 8 CeO2 4 1 1 Cu 4+2# 2+1# 2 SiO2 5 1 TiO2 9 4 ZnO 2 1 1

25

Overview of ToxCast Screening of 62 Nano and Reference Materials

26

Individual particles

Yellow: Less active Blue: More active Groups of

assays

Bioactivity generally in the 1-100 ug/ml range NM with Ag, Cu, Zn are more active than others Data are being analyzed NT & asbestos had different inflammatory response profiles

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 75nm PVP 50nm PVP 10nm PVP

10nm Citrate 50nm Citrate

50 nm 10 nm

25 nm 50 nm 100 nm

75nm Citrate

Silver nanoparticle cytotoxicity

29

• 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

Silver nanoparticles in ARPE-19 cells in culture under dark field / fluorescence microscopy

Blue: DNA Green: Golgi White: AgNP Orange: Cell Membrane

Zucker et al., 2013

Blue: DNA Green: Golgi White: AgNP Orange: Cell Membrane

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

cell layer

media

Size (nm)

1 10 100 1000

Diffusion Sedimentation

Particle deposition influenced by: • Size • Agglomeration • Density • Media

• Viscosity • Density • Temperature

• Time • ISDD Model

• Stokes Law (sedimentation)

• Stokes-Einstein Equation (diffusion)

• Refs: • Teeguarden et al, 2007 • Hinderliter et al., 2010 • Cohen et al., 2103

ICP-MS Flow cytometry

No Cells

20 nm AG NP

0.25 mls

cells

cells

cells0.25 mls

0 ug/ml

3 ug/ml

30 ug/ml

ARPE-19 Cells

Cells

pellet

Into 2 flasks3 ug/ml

Into 2 flasks

Into 2 flasks10 ug/ml

0 ug/ml

30 mg/ml

10 ug/ml

300 ulsupernat

Media and wash

300 ul / flash for each flask

[Ag] in cells and media

APRE-19 cells in vitro AgNP (citrate), control and 3

concentrations After 24 hrs., separate cells from

media Measure [Ag] in cells & media

via ICP-MS Compare flow side-scatter with

ICP-MS Do with both 20 nm and 75 nm

AgNP

[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

References BACHLER, G., VON GOETZ, N. & HUNGERBUHLER, K. 2013. A physiologically based pharmacokinetic

model for ionic silver and silver nanoparticles. Int J Nanomedicine, 8, 3365-82. COHEN, J. M., TEEGUARDEN, J. G. & DEMOKRITOU, P. 2014. An integrated approach for the in vitro

dosimetry of engineered nanomaterials. Part Fibre Toxicol, 11, 20. COHEN, J., DELOID, G., PYRGIOTAKIS, G. & DEMOKRITOU, P. 2013. Interactions of engineered

nanomaterials in physiological media and implications for in vitro dosimetry. Nanotoxicology, 7, 417-31.

DAMOISEAUX, R., GEORGE, S., LI, M., POKHREL, S., JI, Z., FRANCE, B., XIA, T., SUAREZ, E., RALLO, R., MADLER, L., COHEN, Y., HOEK, E. M. & NEL, A. 2011. No time to lose--high throughput screening to assess nanomaterial safety. Nanoscale, 3, 1345-60.

HINDERLITER, P., MINARD, K., ORR, G., CHRISLER, W., THRALL, B., POUNDS, J. & TEEGUARDEN, J. 2010. ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies. Particle and Fibre Toxicology, 7, 36.

IMPELLITTERI, C. A., HARMON, S., SILVA, R. G., MILLER, B. W., SCHECKEL, K. G., LUXTON, T. P., SCHUPP, D. & PANGULURI, S. 2013. Transformation of silver nanoparticles in fresh, aged, and incinerated biosolids. Water Research, 47, 3878-3886.

LOMBI, E., DONNER, E., SCHECKEL, K. G., SEKINE, R., LORENZ, C., GOETZ, N. V. & NOWACK, B. 2014. Silver speciation and release in commercial antimicrobial textiles as influenced by washing. Chemosphere, 111, 352-358.

MEYER, D., CURRAN, M. & GONZALEZ, M. 2011. An examination of silver nanoparticles in socks using screening-level life cycle assessment. Journal of Nanoparticle Research, 13, 147-156.

NEL, A., XIA, T., MENG, H., WANG, X., LIN, S., JI, Z. & ZHANG, H. 2013. Nanomaterial toxicity testing in the 21st century: use of a predictive toxicological approach and high-throughput screening. Acc Chem Res, 46, 607-21.

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.

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